Effect of intra-articular hyaluronan on pressure-flow relation across synovium in anaesthetized rabbits.

1. Hyaluronan is the major polysaccharide of synovial fluid, responsible for its high viscosity. The effect of hyaluronan on fluid transport across the synovial lining of the joint was investigated. Rate of fluid absorption from the joint cavity (Qs) was measured at intra-articular pressures (Pj) of up to 24 cmH2O in knees of anaesthetized rabbits, in the presence or absence of hyaluronan in intra-articular infusates. 2. Viscometry studies in vitro showed that the commercial hyaluronan used had a molecular weight of 549,000-774,000, a radius of gyration of 48-99 nm and a critical concentration for molecular overlap of 1.3 g l-1. 3. With intra-articular Krebs solution (control) or subnormal, subcritical concentrations of hyaluronan (0.5 g l-1), flow increased with pressure. Hyaluronan reduced the fluid escape rate by reducing slope dQs/dPj by 32-64% relative to Krebs solution. 4. At normal to high hyaluronan concentrations (3-6 g l-1) and low pressures, hyaluronan again reduced slope dQs/dPj, by 39-64%. The reduction in slope was slight, however, when compared with the reduction in bulk fluidity (1/relative viscosity). Fluidity at high shear rates was reduced to 6% of control values by 6 g l-1 hyaluronan. The effect on slope did not correlate significantly with the effect on fluidity. 5. At pressures above approximately 12 cmH2O, 3-6 g l-1 hyaluronan altered the shape of the pressure-flow relation: a flow plateau developed. In some joints raising pressure even reduced trans-synovial flow slightly. The pressure required to drive unit trans-synovial flow (an index of outflow resistance) increased 2.5-fold between 5 and 25 cmH2O in the presence of hyaluronan. By contrast, in the absence of hyaluronan the outflow resistance fell as pressure was raised. 6. It is suggested that the increasing resistance to flow in the presence of hyaluronan may be caused by partial molecular sieving of hyaluronan by the small porosities of the synovial interstitial matrix, leading to accumulation of a resistive filter cake of hyaluronan chains at the tissue-cavity interface. Since hyaluronan impedes fluid escape when pressure is raised, it may serve to preserve synovial fluid volume in vivo, e.g. during sustained joint flexion.

Hyaluronan reduces fluid escape rate from rabbit knee joints disparately from its effect on fluidity.

The effect of hyaluronan, the major viscous component of synovial fluid, on trans-synovial flow was measured in rabbit knees. Hyaluronan reduced fluid escape rate by up to 45%. This was much less than the reduction in intra-articular fluidity (1/viscosity). Electron micrographs supported the view that hyaluronan might be partially reflected by synovium, creating a molecular filtercake at the surface.

Viscous and osmotically mediated changes in fluid movement across synovium in response to intraarticular albumin.

Fluid exchange in synovial joints involves both interstitial and transcapillary flow. Here the influence of intraarticular albumin, a normal component of intraarticular fluid, on net transsynovial flow was explored by infusing albumin solutions into the synovial cavity of rabbit knees and recording net flow across the synovial lining. The latter is a discontinuous cell layer possessing matrix-filled intercellular spaces and fenestrated capillaries. Albumin solutions (10-250 g.liter-1) of known oncotic pressure and viscosity were infused at a constant pressure between 3 and 18 cm H2O, either in vivo or post mortem. The rate of absorption (outflow) of Krebs solution from the cavity at the same pressure was measured as the control. Albumin reduced the net rate of absorption of fluid from the joint cavity (Qs). The reduction in Qs in vivo, expressed as a fraction of control, was greater than the fractional reduction in the fluidity of the albumin solution (i.e., 1/relative viscosity). At high albumin concentrations in vivo, the net flow reversed direction and became a net filtration into the joint cavity. The change in Qs per unit change in intraarticular oncotic pressure was then 29.7 x 10(-3) microliters.min-1.cm H2O-1. Even when there was continuous net outflow across the lining, however, as at lower albumin concentrations or higher joint pressures, the intraarticular albumin became diluted compared with the infusate. When synovial blood flow was stopped by killing the animal, albumin had less effect on net transsynovial outflow than that in vivo. Outflow was still reduced by albumin but the fractional reduction was now less than the fractional reduction in bulk fluidity. Also, intraarticular dilution was less marked postmortem than in vivo. The observations were compatible with the view that the net transsynovial flow in vivo comprised two opposing flows, namely, capillary (fenestral) filtration into the joint cavity and interstitial flow from cavity to subsynovium. These two flows were affected oppositely by albumin: filtration was enhanced by the oncotic pressure of albumin in the pericapillary interstitium, whereas interstitial flow was reduced by the effect of albumin on fluidity. The results also raised the issues of whether fenestral filtration reduces nearby interstitial albumin concentration and how interstitial and bulk-phase fluidities are related.

Effect of extravascular plasma protein on pressure-flow relations across synovium in anaesthetized rabbits.

1. The effect of extravascular plasma protein on fluid flux through interstitial matrix was investigated in vivo by studying the pressure-flow relation across synovium during intra-articular infusions of protein solutions (usually bovine serum albumin). Synovium is a sheet of non-epithelial cells separated by interstitium-filled gaps, beneath which are fenestrated capillaries: synovium regulates synovial fluid volume and composition. 2. Albumin solutions (10-150 g l-1) of measured oncotic pressure and viscosity were infused at known pressure into the synovial cavity of knees of anaesthetized rabbits. Flow across the synovial lining in the steady state (absorption rate Qs) was recorded at a series of joint pressures (Pj) to define the pressure-flow relation. Krebs solution was infused into the opposite knee as a control (26 animals). 3. Infusion of a low albumin concentration (10 g l-1, bovine or rabbit) or diluted rabbit serum revealed no specific effect of plasma protein on interstitial matrix permeability (cf. specific protein effect on capillary glycocalyx permeability). Physiological (22.5 g l-1) and higher concentrations reduced trans-synovial absorption rate. The slope of the pressure-flow relation was reduced and the pressure intercept displaced to the right (i.e. Pj at zero flow was raised). 4. Slope dQs/dPj correlated negatively with intra-articular viscosity (P = 0.001-0.04), in keeping with viscous interstitial flow. The reduction in normalized slope, however, did not equal the reduction in fluidity (1/viscosity) quantitatively. It is proposed that apparent fluidity within the interstitial matrix is higher than in the bulk phase due to steric exclusion of albumin (radius 3.55 nm) by the interstitial glycosaminoglycans. The latter form spaces of estimated mean hydraulic radius 14-18 nm in synovium. 5. The joint-pressure intercept at zero net trans-synovial flow was displaced 0.015 cmH2O per cmH2O intra-articular oncotic pressure (pi j; S.E.M. +/- 0.006). Thus large trans-synovial osmotic gradients were not maintained at physiological flow velocities. The 1.5% displacement of the Pj intercept by pi j was attributed principally to interstitial albumin exerting pericapillary oncotic pressure and enhancing net Starling filtration pressure. Indeed, net trans-synovial flow at low joint pressure sometimes reversed from absorption to filtration into the joint cavity at high intra-articular oncotic pressures. 6. The displacement of the trans-synovial flow intercept per unit change in intra-articular oncotic pressure, (dQs/d pi j)Pj = 0, was 18 +/- 3 nl min-1 cmH2O-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Physical and behavioral findings in a pseudohermaphrodite American bison.

A pseudohermaphrodite American bison (Bison bison) behaved like an adult male when interacting with adult females, but like an adult female when interacting with adult males. Its chromosomes were a normal 60, XX and it had a uterus; but it had bilateral testes rather than ovaries. Skull measurements were intermediate between a bull and a cow. Gonadotrophin levels were typical of ovariectomized domestic cows or steers.

Evidence for simultaneous bidirectional fluid flux across synovial lining in knee joints of anaesthetized rabbits.

A recent model predicted a local synovial circulation of extravascular fluid in which capillary filtrate entered a joint cavity and fluid simultaneously drained out of the cavity via intercellular pathways distant from capillaries. Here, solutions of albumin (10-250 g l-1) were infused continuously into the knee joint cavity in fifteen anaesthetized rabbits at 3-18 cmH2O pressure and steady-state net trans-synovial flow was recorded. The intra-articular fluid collected after 15-30 min was substantially diluted despite a sustained net efflux from the cavity. This supports the prediction that net efflux comprises an influx of low-protein plasma ultrafiltrate and a larger efflux of joint fluid.

Asymmetrical effects of albumin on transsynovial fluid movement.

The effect of intraarticular infusions of albumin solution on transsynovial flow was studied in healthy rabbit knee joints and compared with the effect of albumin solution perfused through the synovial microcirculation. Increasing intravascular albumin levels enhanced fluid absorption from the joint cavity, whereas increasing intraarticular albumin levels reduced the absorption rate. The slope of intraarticular pressure-versus-absorption rate plots was reduced by albumin in proportion to the reduction in fluidity (1/viscosity). When joint pressure was held constant, the transsynovial absorption rate was reduced by albumin in excess of the fluidity reduction and even reversed to filtration into the joint cavity. Thus intraarticular albumin acts by a dual mechanism, namely by increasing synovial interstitial fluid viscosity and by exerting a peri-capillary oncotic pressure. However, the latter effect was much less than that of intravascular albumin. Reasons for this are discussed.

Epidermolysis bullosa acquisita antigen and the carboxy terminus of type VII collagen have a common immunolocalization to anchoring fibrils and lamina densa of basement membrane.

In this study a variety of immunoelectron microscopic methods were used to define the precise ultrastructural binding site of epidermolysis bullosa acquisita antibodies (EBA-Ab). We used two EBA sera which immunoblotted with the same skin-extracted protein as that labelled by a monoclonal antibody (LH7.2) which is known to react with the carboxy terminus of type VII collagen. Gold-conjugated antibodies were used in two different immunoelectron microscopic procedures to compare the labelling characteristics of EBA-Ab and LH7.2 in normal human skin. Antibody incubations were performed using ultra-thin cryosections of unfixed skin and thin slices of fresh skin (en bloc technique) before conventional fixation and embedding in Epon. Both methods showed similar labelling features for both EBA-Ab and LH7.2. With ultra-thin cryosections there was labelling of the lamina densa and an undefined component of the sublamina densa region. With the en bloc technique, labelling of dermal ends of anchoring fibrils and of amorphous material recently defined as 'anchoring plaques' was evident. There was no labelling of the central banded portions of anchoring fibrils. We conclude that EBA-Ag is localized to the dermal ends of anchoring fibrils in addition to the lamina densa and possibly anchoring plaques, and thus has the same distribution as the carboxy terminus of type VII collagen.

Pressure induced deformation of the interstitial route across synovium and its relation to hydraulic conductance.

The ease with which fluid passes across the synovial lining (i.e., the lining's hydraulic conductance) is enhanced when intraarticular fluid pressure (IAP) is raised acutely to pathological levels in rabbit knees. A structural basis for this pathophysiological change was sought by morphometry of synovial sections from rabbit knees fixed in situ at less than or equal to 5 cm H2O and 25 cm H2O IAP. Light and electron microscopy showed that the main structural changes induced in areolar synovium by raising IAP to 25 cm H2O were (1) a reduction in synovial thickness to 56% control value; (2) an increase in the area of interstitium exposed at the synovial surface (3) an increased proximity of the synovial capillaries to the joint lumen, the mean distance of capillaries from the surface falling from 8.9 +/- 0.5 microns (n = 391) to 3.3 +/- 0.3 microns (n = 92: p less than 0.001). The capillary profiles showed slight compression under 25 cm H2O IAP, but no collapse. The ratio of interstitial area to thickness is the geometric factor governing hydraulic conductance. The maximum change in interstitial area/thickness was 6.8 times for the blood-joint barrier. A change of this magnitude accounts partly (but not fully) for the experimentally observed conductance changes; and it highlights the importance of capillary depth as a factor governing exchange in joints.

Common antigen expression between human periderm and other tissues identified by GB1-monoclonal antibody.

From 4 weeks estimated gestational age (EGA) until the end of the second trimester (24 weeks EGA) the fetal epidermis is covered by a specialised epithelium, the periderm. The origin and function of periderm remain speculative. We have demonstrated, using indirect immunofluorescence and immunoperoxidase staining, that periderm is recognised by a mouse IgM monoclonal antibody (Mab) GB1, which has been raised against a simple extract of human amnion. Immunoelectron microscopy localises GB1 to the amniotic surface of periderm, particularly in association with the microvilli, and also bordering cellular identations of the periderm cells. GB1 antigen (ag) is also expressed by the epithelium of fetal oesophagus, fetal and adult conjunctiva and cornea but is absent in a variety of other fetal and adult tissues including bladder, oral mucosa and thymus. The similar distribution of GB1 ag in both periderm and membranes possibly suggests a common origin and the shared expression with fetal oesophagus and fetal and adult eye may indicate a function related to the fluid environment. We therefore feel that GB1 Mab may be of use in further investigations into the origin, structure and function of human periderm.

Microfibrillar meshwork of the synovial lining and associated broad banded collagen: a clue to identity.

The surface layer of synovial interstitium lining the rabbit knee was studied by transmission electron microscopy. Over a distance of 2-3 microns normal to the surface the interstitium contained a network of fine microfibrils (diameter 9.3 (0.7) nm, mean (SEM] which was quite dense in places (fractional area of projection 0.189 (0.023], and stained with ruthenium red. Periodic collagen fibrils were relatively scanty and fine (diameter 32 (2) nm) in this surface layer. Broad cross-striated bundles occurred in association with the microfibrils and B cells. These fibrous long spacing bundles (FLS) had a single period of 92.8 (2.8) nm with a broad dark band (37.6) (1.8) nm--so called 'zebra collagen'. Both the periodicity of the FLS and the morphological characteristics of the microfibrils are typical of type VI collagen, a widespread constituent of soft connective tissues. The functional importance of the inner microfibril network is likely to be mechanical, biochemical (glycosaminoglycan and glycoprotein entrapment), and to a very minor degree hydraulic resistance.

Synovial capillary distribution in relation to altered pressure and permeability in knees of anaesthetized rabbits.

1. The hydraulic conductance of the synovial lining of the rabbit knee increases greatly at intra-articular pressures (IAP) above 9 cmH2O. A structural cause was sought by fixing synovium in situ at less than or equal to 5 cmH2O IAP (ten animals) or 25 cmH2O IAP (five animals) and examining histological sections morphometrically. 2. The synovial lining was found to be a highly deformable sheet of very vascular connective tissue, with 47 x 10(3)-73 X 10(3) capillaries per cm2 section. 150-260 cm2 endothelial surface per cm3 tissue and a vascular volume of 2.4-5.7%. 3. The thickness of the lining averaged 14-19 microns at low IAP and was reduced at high IAP; in suprapatellar synovium, where changes were most marked, thickness fell by 24-47%. The loose subsynovial space expanded. 4. The average distance separating capillary near-edges from the joint cavity approximately halved from 3.75 and 7.47 microns at low IAP (harmonic and arithmetical means respectively) to 1.82 and 3.35 microns at high IAP. Capillaries remained patent and their number density did not change significantly at high IAP. 5. It is concluded that a reduction in the extravascular path length for fluid exchange contributes to the increase synovial conductance at high IAP, but the path length changes were not sufficient to account fully for the conductance changes.

Ultrastructure of transport pathways in stressed synovium of the knee in anaesthetized rabbits.

1. The hydraulic conductance of the synovial lining of a rabbit knee increases greatly when intra-articular pressure (IAP) is raised above approximately 9 cmH2O (yield point). To investigate the cause, synovium was fixed in situ by perfusion at controlled IAP and prepared for transmission electron microscopy. Micrographs of synovium fixed below yield pressure (atmospheric pressure and 5 cmH2O IAP, ten joints) and above it (25 cmH2O IAP, five joints) were analysed by morphometry. 2. The discontinuous cellular lining consisted of fibroblast-like cells (67%) and macrophage-like cells (33%) separated by interstitium-filled gaps. Interstitium formed 26-36% of the surface below yield pressure. Depending on sample site the surface gaps averaged 1.9 +/- 0.2 to 2.4 +/- 0.2 microns wide below yield pressure (mean +/- S.E.M. throughout). Above yield pressure the mean gap width increased by 42-64% (P less than 0.05, analysis of variance). 3. The qualitative and quantitative composition of the lining varied with distance below the surface. In a plane 5 microns deep, the intercellular distances and interstitial area fraction were almost double those at the surface. Classic periodic collagen fibrils (diameter 50 +/- 3 nm) abounded at 5 microns depth whereas the surface interstitium was richer in Ruthenium Red-staining microfibrils (diameter 9.3 +/- 0.7 nm) associated with 93 nm period fibrous long-spacing bundles. 4. Averaging over all the tissue between the surface and the 5 microns deep plane, the mean interstitial volume fraction was 0.61 +/- 0.05 at 5 cmH2O and 0.67 +/- 0.02 at 25 cmH2O (n.s.). 5. Capillary fenestrae (8.5 +/- 1.1 per fenestrated profile) and intercellular junctions were unaltered at high IAP. The tortuosity of the capillary-to-joint cavity path was 1.50 +/- 0.01 below yield pressure and 1.86 +/- 0.24 at 25 cmH2O (n.s.). 6. Intra-articular tracers (ferrocyanide, ferritin and glycogen) permeated synovial interstitium without evidence of preferential pathways. Ferrocyanide delineated the capillary intercellular junction as a permeable channel. Ferritin and glycogen were phagocytosed by the macrophages. 7. In suprapatellar areolar synovium, the most extensive and most altered tissue, the ratio of interstitial area to path length increased maximally 4.1 times between 5 and 25 cmH2O IAP. This represents a substantial contribution to the physiologically estimated rise in interstitial conductance (14 x) but does not wholly explain it.

Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy.

Using low-temperature postembedding techniques for immunoelectron microscopy, we succeeded in demonstrating the precise localization of bullous pemphigoid antigen (BP-Ag) in normal human skin. Small pieces (less than 1 mm3) of normal adult skin were rapidly frozen in liquid propane at -190 degrees C and subjected to freeze substitution with 100% methanol at -80 degrees C. Specimens were embedded in Lowicryl K11M at -60 degrees C which was polymerized under ultraviolet radiation at -60 degrees C. Ultrathin sections were incubated with BP sera followed by rabbit anti-human IgG and colloidal-gold conjugated anti-rabbit IgG. Epidermal ultrastructure was generally well preserved: the basal cell plasma membrane and intra- and extracellular components of hemidesmosomes could be resolved. Gold particles were mainly distributed on and around the hemidesmosomes in both intra- and extracellular sites, with most of the labelling being inside the basal keratinocytes and within about 300 nm of the basal plasma membrane. No specific labelling was observed beneath melanocytes or when normal human serum was used as a control instead of BP serum. Our observations indicate that BP-Ag is localized in and around hemidesmosomes in normal human skin and that the antigen has both intracellular and extracellular domains with the major component occurring inside the cells.

Morphology of surface synoviocytes in situ at normal and raised joint pressure, studied by scanning electron microscopy.

The synovial surface in rabbit knees was examined by scanning electron microscopy (SEM) to define normal surface contour, cell shape, and interstitial exposure. Comparison was made between specimens excised before immersion fixation (I), fixed in situ by vascular perfusion (V) before excision, or fixed in situ under an effusion pressure of 5-25 cmH2O (E). The deeply convoluted appearance of rabbit areolar-muscular synovium fixed after excision (I) was found to be an artefact; areolar-muscular synovium fixed in situ (V) was much smoother. The well documented cobblestone contour of immersion fixed adipose synovium was present after fixation in situ, but may be exaggerated by the SEM preparative process. At higher magnification the synoviocytes showed evidence of considerable surface activity (smooth granules, larger cauliflower-like excrescences, thin lamelliform filopodia). Cell shape was variable but many synoviocytes extended long cytoplasmic processes along the surface, producing fibroblastoid and even stellate outlines. At an intra-articular pressure of 25 cmH2O (E) the cytoplasmic processes were elongated and branched, creating a highly dendritic outline. Also, the exposure of interstitium increased markedly at the higher pressure. It is concluded that extension of lengthy cytoplasmic processes is a feature of normal healthy synoviocytes, and that an increase in interstitial area with joint pressure contributes to the increased hydraulic permeability of synovium at raised pressure.

Synovial fluid--its mass, macromolecular content and pressure in major limb joints of the rabbit.

Synovial fluid from rabbit elbow, shoulder, hip and knee was analysed to establish the normal levels of parameters relevant to fluid exchange viz. fluid mass per joint, hydraulic pressure, temperature, colloid osmotic pressure (COP), protein, albumin and glycosaminoglycan (GAG) concentrations. Fluid mass was greatest in the least congruous joint, the shoulder (43 +/- 4 mg), cf. 6 +/- 2 mg in the highly congruous hip. In the knee (24 +/- 4 mg) the mean thickness of the fluid layer was calculated to be 30 microns. Fluid pressure was subatmospheric in all joints (mean -2.8 +/- 0.4 cmH2O, elbow, to -5.7 +/- 0.3 cmH2O, knee), as in many other interstitial spaces. Colloid osmotic pressure was substantial (mean 11.4 +/- 0.9 cmH2O, shoulder, to 13.1 +/- 1.0 cmH2O, elbow), being 40-46% of serum level. Comparison of synovial fluid results with COP versus concentration curves in vitro indicated that the fluid's COP was primarily generated by its protein content (22-30 g l-1, 64% albumin) rather than GAG (4.0-5.8 g l-1). The GAG was 95% hyaluronate and 5% sulphated GAG. Algebraic summation of the hydraulic and colloid osmotic pressures of synovial fluid and serum indicated a net filtration gradient from blood to joint cavity. When serum COP was reduced by intravenous saline infusion, synovial fluid mass increased, in accordance with the ultrafiltration hypothesis of synovial fluid formation. The fluid's colloid concentration declined as volume increased. The relation was not a simple dilution curve, but indicated that the newly formed synovial fluid contained greater than or equal to 8 g protein l-1 (14.5% serum concentration); and that hyaluronate was entering the synovial cavity at a rate of greater than or equal to 6.5 micrograms h-1 per joint.

Relation between trans-synovial flow and plasma osmotic pressure, with an estimation of the albumin reflection coefficient in the rabbit knee.

The volume of synovial fluid in a joint correlates inversely with plasma colloid osmotic pressure (COP). The inferred influence of plasma osmotic forces on trans-synovial flow was investigated directly here, in isolated perfused hindquarters of sixteen rabbits. Flow of intra-articularly infused Krebs solution across the synovial lining of the cannulated knee was recorded at controlled intra-articular pressure (18 cmH2O). Colloid osmotic pressure in the synovial microcirculation was varied by perfusion with oxygenated red cells resuspended in albumin solution or plasma from an extra-corporeal system at constant perfusion pressure. Studies in vitro showed that the COP versus concentration curve for commercial bovine albumin samples was variable and not reliably described by a widely used polynomial. The rate of trans-synovial absorption Qs was a positive linear function of intravascular COP pi p (r = 0.936, P less than 0.001, n = 83). The average slope dQs/d pi p was 0.20 microliter min-1 cmH2O-1 (S.E. +/- 0.01 microliter min-1 cmH2O-1), the slope depending on hydraulic conductance and osmotic reflection coefficient. Trans-synovial flow was a negative linear function of synovial capillary pressure (Pc). Absolute slope dQs/d pi p was on average only 78% of dQs/dPc in the same joint. The osmotic reflection coefficient of the blood-joint barrier to serum albumin was estimated from these slopes as 0.78-0.81 (S.E.M. +/- 0.06). Vascular perfusion with a hyperosmolar solution of glucose, sucrose or NaCl generated a transient, rapidly decaying osmotic absorption from the joint cavity, with a half-life of 17-60 s. A reversed osmotic transient occurred on reperfusion with isotonic fluid. It was concluded that the blood-joint barrier, which comprises fenestrated endothelium and synovial intima, approximates to an imperfect semipermeable membrane for albumin solutions, justifying the application of Starling's hypothesis to trans-synovial flow. For small solutes the tissues form a highly permeable but nevertheless slightly osmotically reflective membrane.