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PubMed2002

Fiber diameter distributions in the chinchilla's ampullary nerves.

A morphometric study of the chinchilla's ampullary nerves was conducted to produce an unbiased accounting of the diameter distribution of their constituent fibers. Diameter analyses were determined from 1 microm plastic-embedded nerve sections taken at a plane immediately proximal to the sensory epithelium. We found these nerves to be composed of 2094+/-573 fibers, having diameters that ranged from 0.5 to 8 microm. The distributions of diameters were positively skewed, where approximately 75% of the fibers were found to have diameters less than 3.5 microm. An analysis of the spatial distribution of diameters within the nerve section revealed that the lateralmost areas of the nerve contained larger fractions of fibers within the smallest diameter quintiles, and the central area harbored greater proportions of the larger diameter quintiles. However, significant fractions of all quintiles were found in all areas. These data were integrated with available data of Fernandez et al. (1998) to produce diameter estimates of calyx, dimorphic, and bouton morphology subpopulations. In view of a general relationship between diameter, innervation locus, and an afferent's physiologic characteristics, these data provide the basis for developing a perspective for the in situ distribution of afferent response dynamics.

Animals↗
PubMed2000

Histopathological differences between temporary and permanent threshold shift.

The structural changes associated with noise-induced temporary threshold shift (TTS) were compared to the damage associated with permanent threshold shift (PTS). A within-animal paradigm involving survival-fixation was used to minimize problems with data interpretation from interanimal variability in response to noise. Auditory brainstem response thresholds for clicks and tone pips were determined pre- and 1-2 h post-exposure in 11 chinchillas. The animals were exposed for 24 h to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 86 dB. Three animals (0/0-day) had both cochleas terminal-fixed 2-3 h post-exposure. Two animals (27/27-day) had threshold shifts determined every other day for 1 week, every week thereafter, and underwent terminal-fixation of both cochleas 27 days after exposure. Six animals (0/n-day) had threshold shifts determined in both ears upon removal from the noise; their left cochlea was then survival-fixed 2-3 h post-exposure. Threshold shifts were determined in their right ear every 2-3 days until their hearing either returned to pre-exposure values or stabilized at a reduced level at which time their right cochlea was terminal-fixed (4-13 days post-exposure). All cochleas were prepared as plastic-embedded flat preparations. Missing hair cells were counted and supporting cells and nerve fibers were evaluated throughout the organ of Corti using phase-contrast microscopy. Post-exposure, all animals had moderate TTSs in their left and right ears which averaged 43 dB for 4-12 kHz. In the 0/0-day animals, the only abnormality which correlated with TTS was a buckling of the pillar bodies. In the 0/n-day animals, their left cochlea (survival-fixed 2-3 h post-exposure) had outer hair cell (OHC) stereocilia which were not embedded in the tectorial membrane in the region of the TTS whereas OHC stereocilia were embedded in the tectorial membrane throughout the cochleas of non-noise-exposed, survival-fixed controls. Three of six right cochleas (terminal-fixed 4-13 days post-exposure) from the 0/n-day animals developed a PTS and two of these cochleas had focal losses of inner and outer hair cells and afferent nerve fibers at the corresponding frequency location. The other cochlea with PTS had buckled pillars in the corresponding frequency region. These results suggest that with moderate levels of noise exposure, buckling of the supporting cells results in an uncoupling of the OHC stereocilia from the tectorial membrane which results in a TTS. The mechanisms resulting in TTS appear to be distinct from those that produce permanent hair cell damage and a PTS.

Acoustic Stimulation↗
PubMed2001

Bone histomorphometry in three females with Rett syndrome.

The frequent occurrence of osteoporosis in Rett syndrome raises questions about the etiology of this finding. It is unknown whether there is any relationship between low bone mass and the underlying genetic disorder. We recently had an opportunity to study the status of bone remodeling by quantitative bone histomorphometry in three girls ages 9.75, 13.5, and 14 years, with typical Rett syndrome who required scoliosis surgery.Anterior iliac crest bone biopsies were performed 1-2 days after double labeling of the bone surfaces with tetracycline. Samples were processed for plastic embedding, sectioned, stained, and histomorphometry performed in the cancellous bone. The same observer performed all measurements. Bone volume was reduced, surface parameters of formation (osteoid surface) were normal while parameters of resorption (osteoclast surface and number) were decreased. The rate of bone formation was reduced in the first two girls but could not be measured in the third girl due to lack of double labeling. It may be that the slow rate of bone formation seen in each age group impedes the development and accumulation of peak bone mass and contributes to the decreased bone volume associated with Rett syndrome, although the data is limited. This is the first report to document decreased bone volume determined by quantitative bone histomorphometry in patients with Rett syndrome. With the recent identification of MECP2 mutations in Rett syndrome it is quite likely that genetic factors not only play a major role in brain development but may also influence other organ growth including bone formation.

Adolescent↗
PubMed1997

Time course of nerve-fiber regeneration in the noise-damaged mammalian cochlea.

The time course of events which are essential for nerve-fiber regeneration in the mammalian cochlea was determined using a group of chinchillas that had been exposed for 3.5 hr to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. The animals recovered from 40 min (0 days) to 100 days at which times their inner ears were fixed and the organs of Corti prepared for phase-contrast and bright-field microscopy as plastic-embedded flat preparations. Selected areas identified in the flat preparations were semi-thick and thin sectioned at radial or tangential angles for examination by bright-field and transmission electron microscopy. The following time-ordered events appeared critical for nerve-fiber regeneration: (1) The area of the basilar membrane in which regeneration had a possibility of occurring showed signs of severe injury. Outer hair cells degenerated first followed by outer pillars, inner pillars, inner hair cells and other supporting cells; (2) Myelinated nerve fibers in the osseous spiral lamina became fragmented, starting at the distal ends of the fibers. This degeneration gradually extended back to Rosenthal's canal; (3) Fibrous processes, originating from Schwann-like cells in the osseous spiral lamina, extended laterally on the basilar membrane; (4) Schwann cells lined up medial to the habenulae perforata in the areas of severest damage, apparently ready to migrate through the habenulae onto the basilar membrane; (5) Schwann-cell nuclei appeared on the basilar membrane beneath the developing layer of squamous epithelium which was in the process of replacing the degenerated portion of the organ of Corti; (6) Regenerated nerve fibers with thin myelin sheaths or a simple investment of Schwann cell cytoplasm appeared in areas of total loss of the organ of Corti; and (7) The myelin sheaths on the regenerated nerve fibers gradually became thicker.

Animals↗
PubMed1998

Neutrophils infiltrating bronchial epithelium in chronic obstructive pulmonary disease.

In order to characterize neutrophil and eosinophil presence in the airways of patients with chronic obstructive pulmonary disease (COPD), bronchoscopy with bronchial washings and bronchial biopsies was performed in 12 smoking stable COPD subjects and 18 normal non-smoking control subjects. Bronchial biopsies were examined by light microscopy using plastic embedding and histochemical techniques to identify different cell types. Bronchial washing fluid of COPD patients was characterized by a predominance of neutrophils (P = 0.001), and a slight, but significant (P = 0.03), increase of eosinophil fraction. Subjects with COPD had higher number of neutrophils in the epithelium (P = 0.01), and eosinophils in the lamina propria (P = 0.01) than did control subjects. The thickness of reticular basement membrane was increased for COPD patients in comparison to control subjects (P = 0.01). The present study provides evidence of neutrophil infiltration both in bronchial washing and bronchial epithelium of patients with COPD, suggesting that the source of neutrophils in airway lumen may be the bronchial mucosa. Although less common than in asthma, airways of COPD subjects reveal eosinophil presence and airway remodelling.

Aged↗
PubMed2002

Electron tomography: towards visualizing the molecular organization of the cytoplasm.

Electron tomography of thin sections of plastic-embedded biological material provides valuable insights into the organellar organization of cells. With the advent of automated data-acquisition procedures, it has become possible to study radiation-sensitive specimens, such as frozen-hydrated organelles and whole cells. This opens up exciting perspectives for visualizing the molecular organization of the cytoplasm.

Animals↗
PubMed1997

Adenovirus mediated gene transfer into rat lung grafts at the time of harvest.

OBJECTIVE: New methods to introduce genetic material into cells in vivo may revolutionize current treatment modalities. Expression of functional genes in lung allografts could be used as a prophylactic strategy for reperfusion injury and rejection. We studied the feasibility of ex vivo adenovirus mediated transfection of rat lung allografts. METHODS: In group I (n = 3) donor rat lungs (Fisher) were flushed with Low Potassium Dextran Glucose (LPDG) solution (20 ml, 20 degrees C). 4 x 10(11) viral particles of adenovirus 5 containing the E. coli lacZ reporter gene coding for beta-galactosidase (AdCMV-beta-Gal) were added to the last milliliter of the flush solution. Lung grafts were stored for 3.5 h at room temperature followed by syngenic orthotopic transplantation (Fisher to Fisher) using a microsurgical cuff technique. On postoperative day 5 the heart lung block was extracted and flushed with x-Gal (beta-Gal substrate) and kept in x-Gal for 3 h at 37 degrees C. Color development was observed macroscopically and plastic embedded sections were used for histologic examination. Group II grafts (n = 3) served as controls and were flushed without adenovirus. RESULTS: X-Gal stained the transfected lung grafts blue, indicating high reporter gene expression. Control lungs did not stain with x-Gal. In group I histological examination demonstrated transfection predominantly in type II pneumocytes. Surprisingly endothelial cells showed no beta-Gal activity. CONCLUSION: This study demonstrates that ex vivo transfection of lung grafts at the time of harvest is a feasible method of gene transfer and results in gene expression after transplantation.

Adenoviridae↗
PubMed1996

Types and incidence of human periapical lesions obtained with extracted teeth.

OBJECTIVES: To determine (1) the frequency of the incidence of abscess, granuloma, and radicular cyst among human periapical lesions obtained with extracted teeth; and (2) whether periapical cysts occur in two categories when histologically analyzed in relation to the root canals. STUDY DESIGN: A total of 256 lesions were analyzed. The specimens were decalcified and embedded in plastic. Serial sections or step-serial sections were prepared, and the sections were evaluated on the basis of predefined histopathologic criteria. RESULTS: The 256 specimens consisted of 35% periapical abscess, 50% granuloma, and 15% cysts. The latter occurred in two categories, the apical true cysts and the apical pocket cysts. CONCLUSIONS: These results show (1) the low incidence of radicular cysts among periapical lesions as against the widely held view that almost half of all periapical lesions are cysts; and (2) the occurrence of two classes of radicular cysts. We are of opinion that the pocket cysts may heal after root canal therapy but the true cysts are less likely to be resolved by conventional root canal treatment.

Decalcification Technique↗
PubMed2003

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Aged↗
PubMed2003

Evidence for an extensive collagen type III proximal domain in the rat femur. II. Expansion with exercise.

Exercise in youth may affect bone "quality" as well as quantity. Using the rat model, 1.5-month-old females were divided into four weight-matched groups, exercised short-term (6 weeks, E(s), n = 20) and long-term (14 weeks, E(L), n = 10) by access to monitored running wheels, and corresponding "sedentary" controls (S(S) short-term, n = 20; S(L) long-term, n = 10). Femora were either plastic-embedded or fresh-frozen. Transverse histological slices, 100 microm thick, were cut midshaft, while similar cryosections, 8 microm thick, were prepared from the same site and also coronal to the femoral neck region. An image analyser measured femoral neck and midshaft microarchitecture, while immunostaining localized collagen type III-rich fibres (CIII, an index of Sharpey fibre insertions) and osteopontin-rich osteons (OPN, an index of remodelling). Exercise increased cortical bone (proximal width +18%, midshaft area +7%). It also raised cancellous bone volume (+25%) by trabecular thickening (+30%) with more intraosseous vascularity and new trabecular interconnections (node-terminus ratio, +57%; trabecular pattern factor, -147%; marrow star volume. -48%). In the cortex a prominent discrete subperiosteal domain became wider (+50% midshaft) with exercise and contained more numerous (+15%) CIII-stained fibres. In contrast the encircled inner bone developed more numerous (+14%) OPN-rich osteons. It is concluded that short-term voluntary exercise augments both cortical and cancellous microarchitecture. It also alters protein composition, such that expanding arrays of Sharpey's fibres within a circumferential proximal domain (Part I) interconnect more powerfully with the musculature and interface more robustly with the core bone that in response becomes more vascular and biodynamic, providing further insight into how muscle mass may be skeletally translated.

Animals↗
PubMed1990

Light absorbed by 575-cones trigger rod disc shedding in the frog retina.

Photoreceptors periodically shed light-sensitive membranes; rods at light onset, cones at night. The spectral characteristics of light required to initiate shedding by 502-rods were studied in the frog retina. After 24 h of white light, animals were dark primed for 1 h then presented with 1 h of nearly monochromatic light to induce the shedding response. The light delivered a total photon flux of 2.5 or 0.5 microE/m2s. Following conventional fixation and plastic embedding, 1 micron sections were examined with light microscopy. The number of photoreceptor tips (phagosomes) shed by 100 consecutive rods were counted and plotted as a function of wavelength. Bright light induced at least 15 phagosomes per 100 rods at all wavelengths tested, 420-640 nm, and this shedding was more than doubled with light from 540-600 nm. When the light was dimmed, there was no shedding response except for this 540-600 nm window. This shedding peak closely corresponds to the absorbance curve of the frog's 575-cone photopigment and implies that the 575-cone can drive rod shedding. The broad background effect further indicates that all photoreceptors have an input and suggests that a luminosity cell, such as the internal horizontal cell, may be involved.

Animals↗
PubMed2000

Histochemical evidence for inducible nitric oxide synthase in advanced but non-ruptured human atherosclerotic carotid arteries.

In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.

Aged↗
PubMed2001

Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments.

Our earlier immuno-gold electron microscopic study indicated that the distribution of caldesmon (CaD) on actin filaments is not uniform and is restricted to the vicinity of the myosin filaments (Mabuchi K, Li Y, Tao T, Wang CLA (1996) J Muscle Res Cell Motil 17: 243). This suggested that CaD could effectively inhibit muscle contraction, if those actin filaments in the vicinity of myosin filaments were saturated with CaD. In the present study we further examined the distribution of CaD along isolated, crude and purified native thin filaments (NTF). Individual CaD molecules on purified NTF were visualized with the aid of a chemical crosslinker, 5,5'-dithiobis(2-nitrobenzoic acid), which efficiently crosslinks CaD to actin (Graceffa P, Adam LP, Lehman W (1993) Biochem J294: 63), and of a monoclonal anti-CaD antibody. The results indicated that individual NTF had alternating CaD-rich and CaD-deficient regions. Moreover, we found that the N-termini of all CaD molecules in a given cluster appeared on the same side of an actin filament. Electron microscopic images of crude NTF immunoprecipitated by a polyclonal antibody clearly indicated that the spacing between the CaD clusters is wide enough for myosin heads to interact with actin subunits. Similar clustering of CaD was also observed in plastic embedded tissue sections. These observations raise the possibility that CaD is not acting as a simple on/off switch, but more likely as a modulator, of smooth muscle contraction.

Actin Cytoskeleton↗
PubMed2001

Long-term studies of the juxtaglomerular apparatus in young microalbuminuric type 1 diabetic patients.

AIM: To determine the long-term changes of the juxtaglomerular apparatus in incipient diabetic nephropathy. METHODS: Three renal needle biopsies were performed on 15 young type I diabetic patients with microalbuminuria; at baseline and after an average of 2.4 and 8.2 years. Using light microscopy, 1 microm serial sections of the plastic-embedded biopsies were investigated and volumes of the juxtaglomerular apparatus and glomerulus and areas of the macula densa and lumina of the afferent and efferent arterioles were measured. RESULTS: From baseline to second follow-up there was a significant decrease in JGA relative to glomerular volume. There was an increase in luminal area of the efferent arteriole which was paralleled by (non-significant) changes in the afferent arteriole. CONCLUSION: Over a period of 8.2 years JGA size remained stable, but decreased relative to glomerular size. Also, an increase in luminal area was noted in efferent arterioles. This may be due to increased single nephron blood flow secondary to nephron loss.

Adolescent↗
PubMed1985

Osteoclast enlargement in endstage renal disease.

In normal subjects and patients with endstage renal disease, osteoclast cell volumes were constructed using serial 2-micron thick plastic embedded sections from iliac crest bone biopsy specimens. Four cells randomly selected from each of the subjects were analyzed to give both the cell volume from the cumulative areas and thickness of each cell slice and also the cell axes taking the vector along the bone face as width or Y, thickness from the bone surface as breadth or X and vertical dimension length or Z. The mean cell volume was 6,230 microns3 in the control subjects and was significantly larger being 11,730 microns3 and 13,680 microns3 in the two patient groups. The cells showed polarity with the largest axes, Y and Z, being those in apposition to the bone surface. Howship's lacunae were enlarged in the patients and the cross-sectional area of an individual lacuna corresponded to the area of the contiguous osteoclast, r = 0.62, P less than 0.001. All patients had secondary hyperparathyroidism and osteoclast numbers were increased. There was no correlation between osteoclast size and duration of renal failure, previous vitamin D intake, or aluminum exposure. It is suggested that in ESRD, osteoclasts undergo both an increase in number and size and that these cells, being larger, remove more bone than the smaller cells in normal subjects.

Adult↗
PubMed1994

Light- and electronmicroscopic distribution of taurine, an organic osmolyte, in rat renal tubule cells.

Several lines of evidence suggest that taurine acts as an organic osmolyte in the kidney. We investigated the cellular and subcellular distribution of this amino acid in rat renal tubule cells. Semi- and ultrathin sections of plastic-embedded rat kidney were incubated with an antiserum against conjugated taurine, using peroxidase-antiperoxidase and immunogold procedures, respectively. Extensive control tests confirmed the selectivity of the antiserum. Our immunocytochemical preparations revealed a highly differentiated labeling pattern. Strong labeling (judged visually or by computer-aided calculation of gold particle densities) was found in collecting duct cells throughout cortex and medulla, in proximal straight tubule cells, and in cells of the descending thin limbs of Henle's loop. Intermediate gold particle densities occurred in proximal convoluted tubule cells and intercalated cells of the collecting ducts (the gold particle in the latter being 30% of that in the collecting duct cells). The distal convoluted tubules, and thick and thin ascending limbs were almost immunonegative. It cannot be excluded that the proportion of free taurine that is retained by the fixative varies somewhat among the different cell types. Yet the highly differentiated labeling pattern that was obtained suggests that taurine is heterogeneously distributed among different populations of tubule cells, and that its level varies substantially even among cells that are exposed to the same osmotic stress.

Animals↗
PubMed1999

Inflammatory aetiology of primary oesophageal achalasia: an immunohistochemical and ultrastructural study of Auerbach's plexus.

AIM: Achalasia is a disease of the oesophagus characterized by increased lower oesophageal sphincter (LOS) tone, absence of LOS relaxation with swallowing and aperistalsis of the body of the oesophagus. The aetiology and pathogenesis of idiopathic achalasia is still controversial. METHODS AND RESULTS: We examined 16 oesophageal biopsies and one low oesophagectomy specimen from patients with achalasia. The control group was composed of five autopsy cases with no history of oesophageal disorders, three cases of diffuse oesophageal spasm, one of gastro-oesophageal reflux disease and one patient with oesophageal carcinoma. Sections were immunostained for neurofilaments NF70 and NF200, S100 protein and neurone-specific enolase. Biopsies with inflammatory infiltrates, were in addition immunostained with antibodies against leucocyte common antigen as well as for CD20, CD43, CD68 and CD45RO. All biopsies were examined after plastic embedding, and electron microscopy (EM) was performed on samples containing autonomic plexus. An inflammatory infiltrate of varying intensity was present along the nerve fascicles and around ganglion cells in 90% of the cases of achalasia. T-lymphocytes predominated in all these cases. The autonomic nerves showed loss of fibres and degenerative changes which were discernible only by EM. Although there was no convincing neuronal loss or signs of active neuronal degeneration in biopsied cases, the oesophagectomy specimen revealed total absence of neurones and significant loss of nerve fibres. The control group showed normal plexuses and no inflammation. CONCLUSION: Degeneration and significant loss of nerve fibres associated with predominant T-cell lymphocytic inflammatory infiltrate around the myenteric plexus support the concept for the inflammatory, probably autoimmune, aetiology of autonomic nervous system injury in primary achalasia.

Adult↗
PubMed2003

The use of filter membranes for high-pressure freezing of cell monolayers.

Rapid freezing of cells and tissues, followed by freeze-substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High-pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK1 and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.

Animals↗