In vivo dynamic compression has less detrimental effect than static compression on newly formed bone of a rat caudal vertebra.

Fusionless devices are currently designed to treat spinal deformities such as scoliosis by the application of a controlled mechanical loading. Growth modulation by dynamic compression was shown to preserve soft tissues. The objective of this in vivo study was to characterize the effect of static vs. dynamic loading on the bone formed during growth modulation. Controlled compression was applied during 15 days on the 7(th) caudal vertebra (Cd7) of rats during growth spurt. The load was sustained in the "static" group and sinusoidally oscillating in the "dynamic" group. The effect of surgery and of the device was investigated using control and sham (operated on but no load applied) groups. A high resolution CT-scan of Cd7 was acquired at days 2, 8 and 15 of compression. Growth rates, histomorphometric parameters and mineral density of the newly formed bone were quantified and compared. Static and dynamic loadings significantly reduced the growth rate by 20% compared to the sham group. Dynamic loading preserved newly formed bone histomorphometry and mineral density whereas static loading induced thicker (+31%) and more mineralized (+12%) trabeculae. A significant sham effect was observed. Growth modulation by dynamic compression constitutes a promising way to develop new treatment for skeletal deformities.

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches.

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.

Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A.

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.

Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells.

In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A-gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.

Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells.

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A-gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde-fixed, osmiumpostfixed, and Epon-embedded kidneys. Cationic BSA-DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium.

Transmission of microfilariae and infective larvae of Dipetalonema viteae (Filarioidea) among vector ticks, Ornithodoros tartakowskyi (Argasidae), and loss of microfilariae in coxal fluid.

During studies on the acquisition and transmission of infection with the filaria Dipetalonema viteae by Ornithodoros tartakowskyi, it was found that young nymphs and starved medium-sized ticks feed on recently engorged larger ticks. In this manner young ticks acquired the infection with microfilariae, and the microfilariae thus taken developed normally and after 30 days of development were transmitted to a jird. Ticks harboring infective larvae were able to transfer them to other engorged ticks when attempting to feed on them. Although it is not known whether this occurs in nature, it could be a supplementary mechanism in the natural maintenance of this species of filaria. Ticks eliminated microfilariae in the coxal fluid in small numbers during the 1st hr after the infective meal and in increasing numbers with time, reaching a peak between 3 and 5 hr. This mechanism may prevent some ticks from becoming hyperinfected.

Distribution and movement of infective-stage larvae of Dipetalonema viteae (Filarioidea) in the vector tick, Ornithodoros tartakowskyi (Argasidae).

Histological sections and dissection of infected ticks, Ornithodoros tartakowskyi, showed that in the resting tick the 3rd-stage larvae of Dipetalonema viteae were distributed in clumps throughout the hemocoel. In the biting tick, larvae moved anteriorly and congregated especially in the capitulum; and the forward migration occurred even though no blood was ingested. This suggests that the act of biting and not the ingestion of blood is the critical factor in migration. The larvae may reach the buccal cavity through 4 possible avenues: 1) the junction of the pharynx with the buccal cavity; 2) the esophagus; 3) the salivary ducts; and 4) the roof of the hypostome. Developing forms produce direct injury to muscle fibers, and the migrating larvae further disorganize the muscles, affecting to some extent the normal activities of the ticks.

Behavior of Dipetalonema viteae (Filarioidea) during escape from the vector tick, Ornithodoros tartakowskyi (Argasidae).

To determine the behavior of Dipetalonema viteae in its tick vector, Ornithodoros tartakowskyi, the ticks were fed on jirds at successive intervals of 30 to 35 days after a single infective blood meal, and the number of larvae passing from the tick during each bite was determined by recovery of: 1) adult worms from the jirds' tissues; 2) larvae from skin snips taken at the feeding site immediately after the bite; and 3) larvae from serum and tissue after artificial feeding through a skin-membrane. All methods gave similar results. Ticks harboring few larvae released most of them (82%) during the first bite, and required only 2 bites to transmit all. Ticks with moderate or heavy infections required 3 or 4 bites to transfer all larvae. Factors which may explain this are: 1) relatively short duration of the bite of heavily infected ticks due to irritation and damage to the muscles of mouthparts and pharynx by the larvae; 2) resistance of the anterior alimentary tract to penetration by the larvae; and 3) retarding effects of crowding on development and migration of larvae. Aging of infection in the tick apparently did not influence the rate of transfer of larvae. Infection adversely affected the feeding and retarded the molting of young nymphs, but with the loss of larvae in successive bites the ability to suck blood was regained.

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication.

The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats.

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

Glomerular handling of native albumin in the presence of circulating modified albumins by the normal rat kidney.

Persistent hyperglycemia, as occurring in diabetes, induces changes in circulating as well as in structural proteins. These changes involve substitution of lysine residues by glucose adducts resulting in early Amadori products that evolve into toxic and active substances, the advanced glycation end adducts. In previous studies, we demonstrated that early glycated (Amadori) albumin infused into the circulation of normal animals induces transitory alterations of glomerular filtration. Attempting to elucidate the mechanisms underlying these changes, various molecular modifications were introduced in vitro to serum albumin. Glycation, acetylation, carboxymethylation, methylation, and succinylation, involving either a few or a significant number of amino acid residues, produced heavier and more anionic albumin molecules compared with the native one. Native and each of the modified albumin molecules were injected intravenously into normal rats, followed, 30 min later, by hapten-tagged native BSA. Changes in glomerular filtration were evaluated by morphometrical analysis of gold immunolabelings. Compared with native albumin, all the modified forms of albumin induced a deeper penetration of the tracer through the glomerular basement membrane revealing alterations in glomerular permselectivity. This was more evident for severely modified albumin molecules which displayed high labelings in the urinary space and endocytic compartments of proximal tubule epithelial cells. These results indicate that modifications of serum albumin, even minimal, as those occurring in early diabetes, could immediately affect the permselectivity properties of the glomerular wall leading, with time, to severe glomerulopathies.

Endothelial cell protrusions in the rat aortic wall. Immunocytochemical evidence for an alternative transendothelial passage of plasma proteins.

Focal morphological changes in the endothelial lining were observed in the aortic wall of control rats. They consisted of endothelial cytoplasmic projections and vacuolar structures protruding towards the luminal space and containing electron-dense material. Some of these structures were observed to open into the subendothelial space. Endogenous albumin was detected in these compartments by applying protein A-gold immunocytochemistry to thin tissue sections of glutaraldehyde-fixed, Lowicryl-embedded aortic segments. The labelling was mainly distributed along the plasma membrane of the projections as well as over the dense content of the endothelial protrusions. The presence of endogenous albumin in these endothelial structures, together with their opening into the subendothelial space, suggests a role for these structures in an alternative transendothelial transport of albumin.

Reabsorption of native and glycated albumin by renal proximal tubular epithelial cells.

The proximal tubule epithelial handling of native and glycated albumin was evaluated by quantitative immunocytochemistry. Native bovine serum albumin (BSA) and its glycated form (gBSA), tagged to different haptens, were simultaneously injected in anesthetized mice and maintained in circulation for 10 or 60 min. Both albumins were localized within the capillary lumen, in glomerular and peritubular basement membranes, the urinary space, and cellular compartments of the proximal tubular epithelial cells. In these cells, both forms of albumin were concomitantly found within the same endocytic-lysosomal system. Morphometric evaluations have indicated higher proportions of gBSA in the urinary space, reflecting probably a significant glomerular filtration of this form of albumin combined to a lesser reabsorptive clearance. Indeed, higher proportions of native BSA were found in the endocytic compartment of the tubular epithelial cells, suggesting its preferential reabsorption. The present study thus supports a preferential glomerular filtration of gBSA with a facilitated filtration of native BSA in the presence of the glycated one. It also demonstrates the tubular reabsorption of BSA and gBSA through a common endocytic pathway, in which the native BSA is preferentially reabsorbed with respect to its glycated form.

Temporary effects of circulating Amadori products on glomerular filtration properties in the normal mouse.

Previous studies have established a preferential glomerular filtration of glycated BSA (gBSA), as well as a facilitated filtration of BSA in the presence of gBSA. We intend to determine whether these modifications are permanent or transitory. gBSA was intravenously injected into anesthetized normal mice and maintained in circulation for 30 min, 1, 2, 24, and 48 h. Five minutes before death, FITC-BSA was injected. On immunocytochemical evaluations, increased glomerular filtration of FITC-BSA was found at all circulating time points. Changes at 24 and 48 h were less pronounced. Glomerular basement membrane (GBM)-to-lumen gBSA labeling ratios were similar at all time points suggesting no accumulation of gBSA in the GBM. Seventy percent of the gBSA was cleared from the circulation and the GBM after 24 h, and 95% after 48 h. This was confirmed in experiments with radiolabeled tracers. These results suggest that the alteration in GBM permeability to BSA in the normal mouse are due to the presence of gBSA and are gradually overcome along with its clearance from circulation. In early diabetes, increasing concentrations of circulating glycated proteins could be responsible for changes in glomerular permselectivity and probably for the alteration in glomerular filtration properties leading to diabetic nephropathy.

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells.

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.

Distribution of endogenous albumin across the rat aortic wall as revealed by quantitative immunocytochemistry.

Endogenous albumin was revealed over thin sections of rat aortic wall, with high resolution and specificity, by applying the protein A-gold immunocytochemical technique. Gold particles, revealing albumin antigenic sites, were observed over plasmalemmal vesicles in endothelial cells and over the interstitial space throughout the thickness of the aortic wall. The distribution of the labeling in the interstitial space varied from region to region and was associated with the collagen fibers, following the orientation of the bundles. The morphometric evaluation of this labeling demonstrated a first peak in labeling intensity in the intima followed by a steep decrease with low levels in the media, and an increasing gradient towards the adventitia. In the subendothelium, a moderate labeling was observed at the base of the endothelial cells of both aortic and capillary endothelia, followed by a decreasing gradient. Ratios between the labeling density in the intima as well as in the adventitia and that in the capillary lumen (plasma albumin) revealed different concentrations of albumin in these compartments. Endogenous albumin, under steady-state conditions, is thus unevenly distributed over the interstitial spaces across the rat aortic wall, and appears associated along the collagen fibers.

Visualization of glycosaminoglycans in rat incisor extracellular matrix using a hyaluronidase-gold complex.

The enzyme-gold technique was used on dental tissues. Hyaluronidase was complexed with gold, and ultrathin sections of rat incisors were incubated with the hyaluronidase-gold complex to localize chondroitin-sulphate and hyaluronic acid at the ultrastructural level. The hyaluronidase-gold complex was found in predentine and dentine, especially at the mineralization front, in interglobular spaces and around dentinal canaliculi. The very young enamel was labelled, but not the later stages of formation. This method allowed a very precise localization of hyaluronic acid and/or chondroitin sulphate in rat incisors extracellular matrices. These findings support the important role of glycosaminoglycans in dentine mineralization.

Glomerular handling of circulating glycated albumin in the normal mouse kidney.

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation. Endogenous albumin, as well as dinitrophenylated native BSA (DNP-BSA) and glycated albumins (DNP-gBSA), were localized within the capillary lumen, glomerular and peritubular basement membranes, and the mesangial matrix. Morphometric evaluation of the labeling over the glomerular basement membrane (GBM) revealed a peak of labeling in the endothelial side for either endogenous albumin or DNP-BSA. In contrast, the labeling distribution for DNP-gBSA showed a shift toward the epithelial side, suggesting a further penetration of the glycated probe into the GBM. When coinjected with gBSA, DNP-BSA was found to display a labeling distribution similar to that displayed by DNP-gBSA. These results indicate that the glycated tracer penetrates the normal glomerular wall deeper than the nonglycated one. Moreover, glycated albumin increases the infiltration of the nonglycated tracer through the normal glomerular wall. Circulating glycated serum proteins thus appear to play an important role in the onset of the glomerular dysfunction and proteinuria, which take place in long-term hyperglycemic states.