Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells.

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells.

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing.

A microbiopsy system for fast excision and transfer of biological specimens from donor to high-pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm x 1.2 mm x 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self-made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high-pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well-instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high-pressure frozen in the new specimen platelet.

Gross anatomy in the surgical curriculum in Switzerland: improved cadaver preservation, anatomical models, and course development.

The invention of new techniques for surgery and interventional radiology demand improved training for ongoing specialists. The Anatomical Institutes in Switzerland support these requirements by establishing hands-on practical training courses by using new procedures for cadaver embalming and model construction. Improvements allow courses to provide students with more realistic simulations of both established and experimental surgical methods. Through these changes, the value of in-depth gross anatomy is enhanced as a topic of fundamental importance for the postgraduate medical and surgical curriculum. The web site http://www.unifr.ch/sgahe/snga.html contains information on courses using the Thiel embalming solution. Details about training courses in Switzerland using anatomical models are available at http://www.heartlab.org, http://www.vascular-international.org, and http://www.elastrat.com.

A new approach for cryofixation by high-pressure freezing.

A newly designed high-pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.

Embryonic central nervous system angiogenesis does not involve blood-borne endothelial progenitors.

We asked, whether, in the blood of avian embryos, endothelial precursor cells circulate that actually contribute to the growing vascular system in and around the central nervous system (CNS). We compared the morphology and distribution of QH1-positive cells after transplantation of quail paraxial mesoderm, after blood transfusion, in quail-chick parabiosis, or after quail bone-marrow transplantation. After head mesoderm transplantation from quail to chick, we observed sprouting endothelial cells (ECs), capillary tube formation, and chimeric endothelial lining of large arteries in the host brain. These QH1-positive quail cells showed EC morphologies that demonstrated three different aspects of CNS angiogenesis: invasion by means of filopodia, clonal proliferation and tube formation, and integration into preexisting EC layers. After blood transfusion or in chick-quail parabiosis, blood-borne QH1+ cells were found in the lumen of but not integrated into the wall of the host vascular system. Neither were QH1+ cells observed in the capillary walls of parabiotic chick chorioallantoic membranes. In both cases, the quail cells showed typical macrophage morphology. In chicks that had received quail bone marrow transplants onto their chorioallantoic membranes, QH1+ cells with macrophage, but not EC shape were occasionally seen near the inoculation site. We conclude that (1) blood-borne cells do not become ECs or directly contribute to angiogenesis inside, or in vascular plexuses around the CNS during embryonic development; (2) blood-borne cells do not contribute to the intraneural macrophage population of the embryonic CNS.

Morphology of the stromal surface and endothelium using two different microkeratomes.

PURPOSE: To compare stromal surface and endothelial morphology after keratectomies and after laser in situ keratomileusis (LASIK), using two different microkeratomes. METHODS: Keratectomies (160-microm and 400-microm) were performed on 82 enucleated porcine eyes using the Chiron Automated Corneal Shaper (52 eyes) and the Microtech Turbokeratome (30 eyes). LASIK procedures of -9.00 D, -27.00 D, and -36.00 D were performed with a Schwind excimer laser. The corneas were immediately fixed in glutaraldehyde or stained with alizarin red and trypan. Scanning electron microscopy was then performed. RESULTS: All keratectomies performed with the Chiron microkeratome displayed a relatively smooth surface. The quality of the keratectomies with the manually advanced Microtech microkeratome was variable, with a high incidence (4 of 9) of incomplete cuts and irregular surfaces. In the eyes in which the stromal laser ablation was performed, a thin layer of condensed stroma (pseudomembrane formation) was seen. Vital staining did not indicate endothelial damage. CONCLUSIONS: The surface morphology was unacceptable for one of the microkeratomes tested. Keratectomies of 160 to 400 microm and LASIK up to -36.00 D did not acutely alter endothelial morphology in porcine eyes.

Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria.

The soluble Ca2+-binding protein parvalbumin (PV) is expressed at high levels in fast-twitch muscles of mice. Deficiency of PV in knockout mice (PV -/-) slows down the speed of twitch relaxation, while maximum force generated during tetanic contraction is unaltered. We observed that PV-deficient fast-twitch muscles were significantly more resistant to fatigue than were the wild type. Thus components involved in Ca2+ homeostasis during the contraction-relaxation cycle were analyzed. No upregulation of another cytosolic Ca2+-binding protein was found. Mitochondria are thought to play a physiological role during muscle relaxation and were thus analyzed. The fractional volume of mitochondria in the fast-twitch muscle extensor digitorum longus (EDL) was almost doubled in PV -/- mice, and this was reflected in an increase of cytochrome c oxidase. A faster removal of intracellular Ca2+ concentration ([Ca2+]i) 200-700 ms after fast-twitch muscle stimulation observed in PV -/- muscles supports the role for mitochondria in late [Ca2+]i removal. The present results also show a significant increase of the density of capillaries in EDL muscles of PV -/- mice. Thus alterations in the dynamics of Ca2+ transients detected in fast-twitch muscles of PV -/- mice might be linked to the increase in mitochondria volume and capillary density, which contribute to the greater fatigue resistance of these muscles.

Tenascin-R associates extracellularly with parvalbumin immunoreactive neurones but is synthesised by another neuronal population in the adult rat cerebral cortex.

The molecular components surrounding a neurone serve as recognition cues for the nerve terminals and glial processes that contact them and the constellations formed by these inputs will therefore be determined by the blend of adhesive and repulsive components therein. Using immunohistochemical methods, we observed that the large extracellular matrix-protein, tenascin-R (Restrictin, J1-160-180, Janusin), associates preferentially with the parvalbumin-positive subpopulation of interneurones within the cerebral cortex. In situ-hybridization indicated that tenascin-R-mRNA was expressed in a subpopulation of nerve cells distinct from that containing parvalbumin, suggesting that this protein's association with the latter is receptor mediated. These nerve cells thus modulate at a distance the composition of the extracellular matrix around parvalbuminneurons.

Effects of the myosin inhibitor 2,3-butanedione monoxime (BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes.

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC(50)=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC(50)=8.4 to 10 m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC(50)=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.

Vascular remnants in the rabbit vitreous body. II. Enzyme digestion and immunohistochemical studies.

The purpose of this study was to evaluate the composition of ghost vessels and the newly identified intravitreal structures type 1 and 2 (IVS-1 and 2) observed in the rabbit vitreous body. Rabbit eyes (n = 10, 0.5- approximately 36 months of age) were fixed and embedded in plastic. Post-embedding immuno transmission electron microscopy and enzyme digestion methods specifically directed at vascular extracellular matrix components (collagen IV, elastin and hyaluronan) were used in order to confirm the postulated vascular origin of IVS-1 and 2. In addition, markers of vitreous extracellular matrix components (collagen II, hyaluronan) were used. The postulated vascular nature of ghost vessels and IVS-1 was confirmed by a positive labelling with anti-collagen IV, whereas the demonstration of elastin (by anti-elastin antibodies and elastase digestion) in IVS-1 and 2 confirms their arterial origin. These vascular remnants were also labelled with a hyaluronan marker and with anti-collagen II. The presence of remnants of the hyaloid artery system throughout the vitreous matrix is in conflict with a strict spatial separation between the primary and secondary vitreous during embryonic development as proposed in the literature. It strongly supports an alternative theory which suggests an interactive remodelling of this matrix. The presence of hyaluronan in remnants of the hyaloid system is inconclusive, since hyaluronan is a component both of the adult vitreous matrix and of the vascular extracellular matrix. The presence of collagen II in vascular structures is highly interesting, since it supports another challenging theory, which suggests that lamellae develop alongside tracts formerly occupied by the larger hyaloid vessels.

Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ.

Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments, where also differences between cell types were noted. We asked whether similar differences between species and between cell types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to 7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in vivo.

Morphological and functional analysis of immortalized human corneal endothelial cells after transplantation.

Approximately 50% of donor corneas are unsuitable for keratoplasty due to an unacceptably low endothelial cell count. One way of overcoming this problem and minimizing wastage of donor corneas may be to transplant cultured human corneal endothelial cells onto these. In this study, we examined the morphological characteristics and functional attributes of endothelial layers formed after the transplantation of immortalized cells in vitro. Cultured human corneal endothelial cells, immortalized by transfection with a plasmid encoding SV40 T-antigen, were seeded onto human corneas denuded of their own endothelium. Seven days after transplantation the newly established monolayers were examined by light, confocal and scanning electron microscopy. Endothelial pump function was gauged by monitoring changes in corneal thickness during perfusion of the endothelial face. The endothelia formed from transplanted immortalized cells had a cobblestone-like appearance, being composed of polygonal units joined by junctional complexes. The stromal hydration state of corneas bearing such endothelial layers could be controlled during perfusion. This was an active process achieved via the Na(+)/K(+)-ATPase-dependent endothelial pump, as demonstrated by inhibiting the enzyme with ouabain. Transplantation of immortalized human corneal endothelial cells onto recipient corneas led to the establishment of new monolayers which had the morphology of the native ones in organ-cultured corneas. This model provides us with a means of studying the formation and function of corneal endothelial layers in vitro.

Hypothalamic accessory nuclei and their relation to the angiotensinergic and vasopressinergic systems.

The existence and colocalization of angiotensin II- and vasopressin-like immunoreactivity in individual magnocellular cell groups of the hypothalamus has been demonstrated by using immunocytochemical methods. These neurosecretory magnocellular groups consist of the paraventricular nucleus and the supraoptic nucleus, as well as different accessory cell groups. The fibers from the neurons of the accessory nuclei project directly to adjacent blood vessels and do not comigrate with the hypothalamo-neurohypophysial fiber pathway. On the basis of these findings it can be concluded that in the hypothalamus two different angiotensinergic and vasopressinergic neurosecretory systems exist: (1) an intrinsic hypothalamic and (2) a hypothalamo-neurohypophysial system. The distribution of the accessory cell groups in the hypothalamus is shown in a 3D reconstruction which includes the connection of these magnocellular nuclei with the vascular system in this area.

Redistribution of surface-bound con A is quantitatively related to the movement of cells developing polarity.

Capping in cells developing polarity has been reinterpreted on the basis of a quantitative analysis of Concanavalin A (Con A) redistribution and cell movement in Walker carcinosarcoma cells. Several new features emerged. Based on the developing asymmetry in the distribution of surface-bound Con A, the direction of cell movement and the prospective front-tail polarity can already be predicted when the cell is spherical. Development of polarity by an initially spherical cell is associated with formation of two parts. The concentrically contracting part (prospective uropod) characterized by surface-associated Con A decreases in size, while the other part is cleared from Con A and grows into formerly unoccupied space. Surface-bound Con A shows isotropic centripetal movement towards the initial position of the centroid of the spherical cell rather than rearward movement. Therefore, the centroid of fluorescence intensity remains either stationary or moves marginally forward with respect to the initial position of the spherical cell. The amount and direction of cell movement measured correlates closely with values predicted by a theoretical model that assumes a unidirectional transfer of volume from a stationary contracting compartment into a protruding compartment. The results suggest that isotropic (cortical) contraction of the initially spherical cells and one-sided relaxation rather than unidirectional retrograde movement of ligand-receptor complexes produces movement in cells developing polarity. Reversible accumulation of surface-bound Con A at the uropod occurring to a similar extent in untreated and colchicine-treated cells is partly due to membrane folding and partly to movement in the plane of the membrane.

Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.

PURPOSE: To evaluate the morphology of endothelial monolayers, which have been regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM). METHODS: Material derived from each of the experimental groups described in part I of this investigation was evaluated in the current study. Recipient corneas, denuded of their native endothelium by mechanical, chemical, or physical debridement, were examined to assess the effectiveness of each technique in killing and removing cells. Porcine or human donor corneal endothelial cells maintained in monolayer culture for up to 10 passages then were seeded onto the denuded Descemet's membranes of recipients in the absence or presence of fibroblast growth factor (FGF). The monolayers thereby established were examined in the SEM, and the morphologic status of individual cells compared with that manifested in normal human donor corneas maintained for 4 weeks in organ culture (reference control). Isolated and cultured human keratocytes regrafted onto the denuded Descemet's membranes of recipient corneas served as nonendothelial control specimens. Tissue was processed for examination in the SEM according to standard techniques. RESULTS: Each of the three methods used to strip recipient corneas of their native endothelium was effective and elicited no gross structural damage to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers. The morphologic status of regrafted cells corresponded to that manifested in monolayer cultures before seeding, porcine ones always being more differentiated than their human counterparts. Poorly differentiated human endothelial cells had a slender, elongated, fibroblast-like appearance, whereas more highly differentiated ones manifested broad, flat, polygonal profiles. Monolayers covered the entire corneal surface and impinged to a variable degree onto the trabecular meshwork, at which juncture cells always assumed a less well-differentiated morphology. FGF consistently effected an increase in differentiation status, and as this became augmented, the capacity of monolayers to violate the corneal-trabecular meshwork border was correspondingly repressed. Seeded keratocytes formed dense, multilayered sheaths, resembling retrocorneal membranes, across the entire corneal surface, trabecular meshwork, and iris root. The surface characteristics of the constituent cells were quite distinct from those manifested by endothelial cells, even the least well-differentiated ones. CONCLUSION: Regrafting of human corneal endothelial cells onto the denuded Descemet's membranes of recipients resulted in the formation of stable monolayers. Because the morphologic status of seeded cells closely mimicked that manifested in monolayer cultures before transplantation, it may be anticipated that efforts to refine and optimize culturing conditions would yield improvements in this parameter after regrafting. If these expectations can be realized, then the possibility of successfully establishing a "new" and functional endothelium on recipient corneas destined for clinical grafting may well be brought to fruition in the not-too-distant future.

Protrusive activity, cytoplasmic compartmentalization, and restriction rings in locomoting blebbing Walker carcinosarcoma cells are related to detachment of cortical actin from the plasma membrane.

The dynamic events at the front of locomoting blebbing Walker carcinosarcoma cells [Keller and Bebie, Cell Motil. Cytoskeleton 33:241-251, 1996] are interpreted on the basis of an analysis of the actin cytoskeleton and its relationship to the plasma membrane in fixed cells using a novel double-staining procedure. The data show that blebs are formed where cortical actin is locally depolymerized and/or by detachment of the plasma membrane from more or less intact cortical actin layers. Dissociation between the cortical actin layer and the plasma membrane, which is stimulated by microtubule disassembly, is achieved by forward movement of the plasma membrane, rather than by retraction of the actin layer. Therefore, the detached actin layers form a boundary between the newly forming protrusions and the rest of the cell. They can be associated with "constriction rings," which we have termed "restriction rings." Detached actin layers can impede entry of organelles and the nucleus into the protrusions and thereby compartmentalize the cytoplasm. Later, detached cortical actin layers depolymerize, allowing for relaxation of the restriction rings and for forward movement of cytoplasmic organelles and the nucleus. Actin may repolymerize along the detached plasma membrane allowing for a new cycle to occur. Estimates indicate that the actin polymerization/depolymerization cycles may be largely confined to the front of blebbing cells. The findings suggest that the dynamic events at the front of blebbing metazoan cells are similar to those previously found in Amoeba proteus [Grebecki, Protoplasma, 154:98-111, 1990] but different from those found in lamellipodia.

Actin accumulation in pseudopods or in the tail of polarized walker carcinosarcoma cells quantitatively correlates with local folding of the cell surface membrane.

We determined the actin distribution and the relationship between actin and the cell surface membrane in polarized Walker carcinosarcoma cells showing lamellipodia or blebs at the front in order to get a better insight into actin's role in shape changes and cell locomotion. Using two different techniques, we found that actin is mainly present as a submembraneous layer. The actin concentration detectable in the cytoplasm was about 16X lower. F-actin staining was increased mainly at the contracted tail and to a lesser extent in lamellipodia. However, there is also accumulation of the cell surface membrane at these sites. The quantitative analysis of electron micrographs showed that the apparent accumulation of F-actin at the tail and in the leading lamellipodia was, on the average, fully explained by increased membrane folding. The cell membrane as well as the cortical actin may fold and unfold during shape changes and polarized cells have reserves of plasma membrane as well as of cortical actin at the tail. In addition, the cells may show spots where the surface membrane was dissociated from the cortical actin layer. Polarized cells showed no increase in actin within the blebs or at the basis of lamellipodia. In this respect, the distribution of polymerized actin was different from other currently studied locomoting metazoan cells. So far, the data are difficult to reconcile with models, postulating that polymerized actin within the protrusions is the direct force driving the membrane forward.

Chondrocyte biosynthesis correlates with local tissue strain in statically compressed adult articular cartilage.

In this study, we investigated the depth-dependent metabolic and structural responses of adult articular cartilage to large-strain, static, unconfined compression. Changes in cell biosynthetic activity and several morphometry-based structural parameters (cell density, cell volume fraction, cell surface-area density, mean cell surface area, and mean cell volume) were measured at eight sites representing different depth-zones between the articular surface and the cartilage/bone border. In addition, local axial strain in the superficial, transitional, upper radial, and lower radial zones was estimated on the basis of the change in cell density values. Static compression of articular cartilage revealed a highly heterogeneous deformation profile through the depth of the sample as well as zone-specific changes in biosynthetic activity, as reflected by incorporation of [3H]proline. The axial strains in the top layers were greater than the applied surface-to-surface strain, whereas axial strains adjacent to the cartilage/bone border were significantly less than the applied strain. Zonal changes in cell density and axial strain that occurred during static compression correlated well with alterations in metabolic activity. These coordinated changes between cell biosynthesis and cartilage structure suggest that zone-specific variations in mechanical stimuli could be responsible for spatially varied patterns of cartilage metabolic activity under load.

Mesoderm-derived cells proliferate in the embryonic central nervous system: confocal microscopy and three-dimensional visualization.

In the chick and quail embryo, two cell populations migrate into the neural tube from the surrounding mesodermal tissues during the fourth day of incubation: individual cells which represent macrophages, and endothelial cells which remain continuous with the extraneural vessels. We report here on the proliferative capacity of these mesoderm-derived cells. A double-immunofluorescence protocol for two monoclonal antibodies of subtype IgG1, the endothelial cell/macrophage marker QH1, and the S-phase marker bromodeoxyuridine, was developed. With confocal laser scanning microscopy of thick microtome sections, labeling indices of intraneural individual QH1-positive cells (12%) and of endothelial cells (10%) were determined. In contrast, the labeling index of extraneural endothelial cells was 25%. With three-dimensional visualization of confocal data, the variable morphology of macrophages was shown. Our results indicate that: (1) proliferative activity of intraneural capillary endothelial cells is less than expected and that it is absent from sprouts; (2) both spheroidal and ramified macrophages proliferate inside the neural tissues; and (3) ramified macrophages frequently make contact with capillary endothelial cells. We conclude that most embryonic microglia may be derived from the early invasive QH1+ macrophages.