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PubMed1994

A procedure for the visualization of bone with hydroxyl-apatite coated implants.

A procedure for the histological visualization of the interface between implants coated with hydroxyl-apatite (HA) and bone, and the reaction of bones to the implants, has been developed. Thick sections of bones with HA-coated stainless-steel implants, were investigated with the implant still in situ with routine and (confocal) fluorescence microscopy. The prosthesis-HA-bone interface, bone cells, necrotic and vascularised bone, bone-ingrowth and bone remodelling could be visualised. After removal of the implant from the sections alternative sections were further processed for routine light microscopy, microradiography and detailed fluorescence studies. It appeared that the HA coating was still intact and the integrity of the HA-bone bond was very well preserved. The HA coating layer had the same appearance as after the first inspection with surface microscopy. However, more details were visible. Prosthesis-related and time-related changes in the appearance of the HA layer were detected. The advantage of the procedure is that observations can be made of bone reactions with the prosthesis still in situ, without previous embedding in plastics, and laborous sectioning and/or grinding procedures. Thereafter, sections can be processed for different histological techniques, e.g. immunocytochemistry, routine histology, microradiography, fluorescence microscopy, apparently without imparing the HA coating layer.

Animals↗
PubMed1989

Zygomatic and mandibular augmentation with proplast and porous hydroxyapatite in rhesus monkeys.

Using an extraoral approach, subperiosteal pockets were created bilaterally over the zygomatic and mandibular regions in six Rhesus monkeys. One side of each animal received a Proplast I (Vitek Inc, Houston) implant and the contralateral side received an equivalent sized block of porous hydroxyapatite (HA). The animals were followed clinically and radiographically. Two animals were killed postoperatively at 3, 6, and 12 months, respectively. The implants were retrieved en bloc and halved. Half of each specimen was decalcified, embedded in paraffin, and stained. The other half was embedded in plastic, and sections were stained or carbon-coated for histometry scanning under electron microscopy. Clinical evaluation revealed that porous HA implants were more stable than Proplast implants. The Proplast implants showed complete encapsulation by infiltration with fibrovascular connective tissue, and progressive fragmentation with giant cell reactions. The porous HA implants were united to the underlying cortex by bony ingrowth. The volume of implants sampled within 2.5 mm of the underlying cortex contained 42.5% HA matrix and 23.8% bony ingrowth, and the surface area of the HA matrix (9.7 mm2/mm3) was 47.8% covered by bony ingrowth. No giant cell response, fragmentation, or biodegradation was observed or measured in the porous HA implants. The data from this primate model further substantiate previous canine studies and permit more reliable estimation of clinical performance. These results provide comparative data that can contribute to the decision-making process in selecting clinical implants.

Alveolar Ridge Augmentation↗
PubMed1989

Tissue response to facial contour augmentation with dense and porous hydroxylapatite in rhesus monkeys.

Using extraoral incisions, subperiosteal pockets were created bilaterally over the zygomatic and mandibular areas in six rhesus monkeys. One side of each anatomic site received a dense block of hydroxylapatite (HA) implant, and the contralateral side received an equivalent-sized block of porous HA implant. Monkeys were killed at 3, 6, and 12 months, and the implants were manually tested for mobility. The implants were then retrieved in block specimens. Half of each specimen was decalcified, embedded in paraffin, and stained. The other half was embedded in plastic, and sections were stained or carbon coated for scanning electron microscope histometry. The dense HA implants showed complete fibrous encapsulation and they popped out when cut in half. The porous HA implants were attached to the underlying cortex by bone ingrowth. The porous implant volume sampled within 2.5 mm of underlying cortex contained 33.0% HA matrix and 35.7% bone. The surface area of the porous HA matrix (4.8 mm2/mm3) was 54.3% covered by bone ingrowth. It is hypothesized that micromotion may have accounted for the lack of osseointegration of the dense HA specimens. In contrast, the early ingrowth of fibrous tissue into the porous HA block might be responsible for reducing micromotion to levels acceptable for ingrowth of bone. The contrasting biologic response of implant sites to two variants of the same implant material supports the value of comparative studies to permit informed surgical selection decisions.

Animals↗
PubMed1992

Diagnosis of medial temporomandibular joint disc displacement with dual space anteroposterior arthrotomography: correlation with cryosectional morphology.

This study was undertaken to determine if it is possible to identify medial disc displacement with dual space anteroposterior (AP) arthrotomography. Contrast medium was injected into the upper and lower joint spaces of 11 fresh temporomandibular joint (TMJ) autopsy specimens and arthrotomograms were obtained with an AP projection. The arthrotomograms of five joints showed an enlargement of the medial recess of the upper joint compartment with folding of the disc inferiorly. This was interpreted as an indication of medial disc displacement. Cryosections confirmed medial displacement of the disc in these five joints and showed a slight medial displacement of a disc in one additional joint. Retrospective evaluation of the arthrotomogram of this joint showed that the disc was slightly medially displaced, but without folding. The results of this study suggest that AP dual space arthrotomography can be useful for the diagnosis of medial disc displacement in cadaver material.

Aged↗
PubMed1995

Bone formation and implant degradation of coralline porous ceramics placed in bone and ectopic sites.

PURPOSE: This study was undertaken to determine the resorption rate of porous ceramic implants. The hypothesis was that implants placed in soft tissues would degrade more rapidly than implants placed in bone. MATERIALS AND METHODS: To test this hypothesis, implants were manufactured by applying a thin coating of hydroxylapatite onto an interconnected, porous calcium carbonate substrate. Control implants were made entirely of hydroxylapatite with identical microstructure. Two adult dogs received a total of 56 implants placed in the femur, skeletal muscle, and subcutaneous tissues. After killing the animals at 4 months, the specimens were removed, embedded in plastic, sectioned, and either stained for light microscopic examination or subjected to quantitative image analysis using a scanning electron microscope. RESULTS: Contrary to the hypothesis, the rate of degradation was faster for implants placed in bone than in soft tissue. Within the 4 months, degradation was 24% to 63% in bone, depending on the composition. However, it was not statistically significant in either intramuscular or subcutaneous tissue. A surprising observation was that bone ingrowth occurred in 67% of the implants placed in soft tissues. On average, it was 4.3% in intramuscular sites and 6.6% in subcutaneous sites. This bone was histologically normal in 71% of the implants containing bone. CONCLUSION: This study demonstrates that porous ceramic implants composed of hydroxylapatite on calcium carbonate will degrade more rapidly in bone defects than in soft tissue sites. In addition, implants with interconnected porosity and surfaces of hydroxylapatite will become ingrown with bone even after placement in soft tissues. The exact mechanisms for both of these phenomena are not understood.

Analysis of Variance↗
PubMed1984

Autoradiographic evidence of 3-methylindole covalent binding to pulmonary epithelial cells in the goat.

3-Methylindole (3MI), the main ruminal fermentation product of L-tryptophan, causes acute pulmonary edema and interstitial emphysema in ruminants. Intravenous infusion of 3MI in goats causes necrosis and sloughing of pneumocytes and bronchial epithelial cells. Previous studies indicate that a reactive metabolite or metabolites of 3MI bind covalently to tissue macromolecules in the lung and this binding is associated with the pneumotoxicity of 3MI. We undertook this autoradiographic study of 3MI covalent binding to test the hypothesis that reactive 3MI metabolite(s) bind to the lung cells susceptible to 3MI-induced injury. We infused goats with [3H]3MI and killed them either 0.5, 2 or 6 h after start of the infusion. Sections of fixed lung were extensively washed, alcohol dehydrated and embedded in plastic. Only covalently bound radioactivity remained. Silver grains were quantitated per area in the developed autoradiographs. There was a 2:1 ratio of binding to the small airway epithelium compared to the interalveolar septa in all the goats. Both ciliated and non-ciliated bronchiolar cells were labeled, as were both types I and II pneumocytes. Normal goat lung slices incubated in vitro with [3H]3MI were labeled in the same pattern. Inclusion of either of the inhibitors of cytochrome P-450, SKF-525-A or piperonyl butoxide significantly reduced this binding to both the pneumocytes and the bronchiolar cells. We consider these results supportive of our hypothesis that 3MI is metabolized to reactive intermediates by the epithelial cells of the lung, where they bind to macromolecules, which may cause cellular damage.

Animals↗
PubMed1986

A versatile test-object for the calibration of ultrasonic Doppler flow instruments.

Versatility in test-objects for ultrasonic Doppler units is desirable. The test-object described comprises a thin-walled plastic tube embedded in tissue-mimicking material. Blood is simulated using a mixture of water and glycerol with sephadex particles to act as scatterers. The test-object is suitable for use with a wide range of Doppler units which employ pulsed and continuous ultrasound in the frequency range 1 to 10 MHz. Calibration of several types of Doppler and imaging duplex systems has been performed for flow velocities up to 100 cm/s through a 10 mm tube. Above this velocity, for this tube dimension, turbulence occurs prevents the accurate measurement of flow velocity. The versatility of the test-object is further enhanced by the feasibility of manufacturing vessels of different shape and size. Slow flow through the tissue equivalent material may also be detected.

Blood Flow Velocity↗
PubMed1992

Formation of muscle spindles in the absence of motor innervation.

Whether muscle spindles can form in muscles innervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius (MG) muscle in newborn rats, and administering nerve growth factor for 10 days afterwards. The nerve-crushed MG muscles reinnervated by afferents in the absence of motor innervation were examined at postnatal (P) days 7, 9 and 30 for the presence of spindles by light and electron microscope. Reinnervated MG muscles contained spindle-like encapsulations of 1-4 fibers at 7, 9 and 30 days after the nerve crush. The number of spindles exceeded that of normal MG muscles, suggestive of de novo formation of spindles. All nerve-muscle contacts in the spindles had features of sensory endings, and intrafusal fibers expressed the spindle-specific slow-tonic myosin heavy chain (MHC) isoform at P30. No motor endplates were visible on any muscle fibers and extrafusal fibers were atrophied, as would be predicted in the absence of motor innervation. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats.

Animals↗
PubMed1992

Photoconverted carbocyanine DiI allows direct visualization of transplanted glial cells at the ultrastructural level.

We have used the carbocyanine fluorochrome, DiI, to trace living glial cells (astrocytes, oligodendrocytes and immortalized Schwann cells) after their transplantation into the newborn shiverer and normal mouse brain. DiI fluorescence first detected on vibratome sections, was photoconverted into a stable, non-diffusible and electron-dense diaminobenzidine product. Both fluorescence and precipitate were found in the same cells and were detectable until 60 days after transplantation. At the ultrastructural level, DiI precipitate was contained within cytoplasmic vesicles scattered in the transplanted cell bodies and processes. Photoconversion did not interfere with the cell fine structure or predicted post-transplantation behavior. DiI is thus a suitable marker to trace, at the ultrastructural level, living cells after their transplantation.

Animals↗
PubMed1984

Synaptic characteristics of identified pyramidal and multipolar non-pyramidal neurons in the visual cortex of young and adult rabbits. A quantitative Golgi-electron microscope study.

The visual cortex of 20 day old rats and rabbits has been considered as mature on the basis of the observations that the dendritic arborization and the overall synaptic population have almost reached their adult stage in these animals. In the present study we have investigated the visual cortex of 20 day and 7 month old (adult) rabbits in order to determine whether this apparent adult appearance also holds for the synaptic organization of individual neurons. Neurons mainly located in layers III and IV of the primary visual cortex (area 17) were Golgi-impregnated, gold toned and deimpregnated and were then, after embedding in plastic, sectioned serially. The number and length of synaptic profiles, and the length of the neuronal boundaries were analysed in every tenth section. From these counts and measurements the size distribution of the synaptic discs, the number of synapses per 100 micron2 neuronal surface and the receptive surface expressed as the percentage of the total neuronal surface covered with synaptic contacts were estimated using stereological methods. At both ages studied, the density of synapses was significantly higher for the non-pyramidal neurons than for the pyramidal neurons. Differences in the amount of receptive surface were parallel to the differences observed for the number of synapses per 100 micron2. At day 20 the receptive surface of the non-pyramidal neurons was significantly larger than that of the pyramidal neurons. The receptive surface of the non-pyramidal neurons in the adult stage was not only larger than that of the pyramidal neurons in the adults, but also larger than that of the day 20 non-pyramidal neurons. From our results the following conclusions can be drawn: (1) The synaptic input received by the pyramidal neurons is mainly established at day 20 of postnatal life, i.e. prior to the establishment of adult visual behaviour. (2) The non-pyramidal neurons complete their maturation in a later stage than the pyramidal neurons. (3) Medium to large sized synaptic contacts are newly formed after day 20 and are mainly added to the synaptic population on dendrites of non-pyramidal neurons. (4) The specific increase in the number of synapses on non-pyramidal neurons is discussed in relation to intracortical inhibition which is thought to be important for the fine regulation of visual function during development.

Animals↗
PubMed1996

Glutamate immunoreactivity of insular cortex afferents to the nucleus tractus solitarius in the rat: a quantitative electron microscopic study.

Corticosolitary axons and their terminals were labeled by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, after injections into the rat insular cortex. The ultrastructure of these cortical afferents was analysed in the medial and commissural subnuclei of the nucleus tractus solitarius. Cortical terminals had a mean area of 0.36 microns 2, and were among the smallest terminals in the nucleus. They made single, asymmetric synaptic contacts with thin dendritic stems or with spines. The average diameter of the dendrites postsynaptic to cortical axons was 0.59 microns, and significantly smaller (P < 0.01, Kolmogorov-Smirnov test) than the mean (0.87 microns) of the population of dendrites in the same region of the nucleus tractus solitarius. Cortical boutons contained closely packed round and clear synaptic vesicles of diameter ca. 28 nm, a few mitochondria, and no dense core vesicles. Postembedding immunogold analysis showed that the anterogradely labeled cortical axon terminals were immunoreactive to glutamate, but not to GABA. Cortical afferents had on average four times the glutamate immunoreactivity (assessed by gold particle density) than local dendrites or terminals making symmetric synaptic contacts. Similarly, most of the unlabeled axon terminals participating in asymmetric synaptic contacts were highly enriched in glutamate immunoreactivity, suggesting that glutamate may be a most prevalent transmitter in the nucleus tractus solitarius. Terminals immunoreactive to GABA always made symmetric synapses, mostly with dendritic shafts and perikarya. We concluded that insular cortex axons made single, asymmetric synaptic contacts with thin, probably distal dendrites in the nucleus tractus solitarius. Cortical terminals are immunoreactive to glutamate, and morphologically different from primary afferents and from terminals immunoreactive to GABA.

Afferent Pathways↗
PubMed1985

Differential susceptibility of avian hair cells to acoustic trauma.

Five groups of 10-day-old chicks were continuously exposed to either 500 or 1500 Hz pure tone at 125 dB for 4 or 12 h and killed 10 days later. The basilar papillae were fixed, embedded in plastic, sectioned, and hair cells were counted according to type: tall or short. Short hair cells were found to be more susceptible to acoustic overstimulation than tall hair cells. Further, the position of maximum short hair cell loss varied along the length of the basilar papilla as a function of the exposure frequency while the position of tall hair cell loss did not. Similarities between morphological response of short hair cells in avians after acoustic trauma and outer hair cells in mammals are discussed.

Animals↗
PubMed1988

Ganglion cell and hair cell loss in Coturnix quail associated with aging.

Hair cells and ganglion cells were examined in young adult (3 month old) and senescent (3 to 6 year old) quail (Coturnix coturnix). Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 micron intervals from basal to apical tip. Hair cells and ganglion cells were counted from three micron thick sections at each interval. Hair cell number remained constant between age groups (less than 10% loss even in the oldest group). Ganglion cell number, on the other hand, was considerably reduced in the senescent birds (20-60% loss). These results are similar to quantitative results in senescent mammals and suggest that ganglion cell loss may be generalized response to aging.

Aging↗
PubMed1989

Ganglion cell loss continues during hair cell regeneration.

Hair cells and ganglion cells were counted in young adult quail (Coturnix coturnix) after acoustic trauma at 10, 30, 60 and 90 day survival times. Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 mu intervals from basal to apical tip. Hair cells and ganglion cells were counted from 3 mu thick sections at each interval. Hair cells were designated as tall or short within the area 30-70% of length from basal tip of the papilla. Both tall and short hair cells were significantly reduced in number 10 days following trauma. Tall hair cells recovered to within 96% of normal after 60-90 days. Short hair cells recovered but to a lesser extent. Ganglion cell loss did not begin until 30 days after trauma and continued without recovery 90 days after trauma. A good correlation was found for position of both types of hair cell loss and position of ganglion cell loss. These results suggest that the initial loss of hair cells, both tall and short, results in retrograde degeneration of neural fibers and ganglion cells.

Animals↗
PubMed1991

Unusual morphology of the stria vascularis in pigmented strain 2/NCR guinea pigs.

This paper reports an abnormality in the morphology of the apical stria vascularis of inbred 2/NCR guinea pigs as compared to outbred animals. Cochleas were embedded in plastic, sectioned, and examined in the light and electron microscopes. In the 2/NCR animals, the apical stria vascularis consisted of a cuboidal epithelium composed of a monolayer of poorly differentiated cells. Few or no capillaries were associated with this epithelium. No melanin pigment was present in the abnormal region of the stria in these animals, although pigmentation appeared normal in lower turns of the cochlea. Measurements of compound action potential thresholds between 2 and 40 kHz revealed no differences in auditory function between the two strains.

Action Potentials↗
PubMed1993

Glutathione S-transferases in the organ of Corti of the rat: enzymatic activity, subunit composition and immunohistochemical localization.

Glutathione S-transferases (GSTs), a family of ubiquitous cytosolic isozymes, catalyze the detoxification of electrophilic substrates with reduced glutathione and participate in intracellular binding and transport of lipophilic substances. This study measured GST activity biochemically in the inner ear of the rat; determined the isozyme profile by Western blotting; and identified, immunohistochemically, the distribution of the mu and pi class GSTs in the organ of Corti. GST enzymatic activity in inner ear tissues ranged from 117 to 348 nmoles glutathione converted/min/mg protein, values somewhat higher than those found in brain (130) and much lower than in liver (1011). Of the GST isoforms, the pi class (identified by antibodies against the Yp subunit) was most prominent, the mu class (Yb1 subunit) clearly evident while the alpha class (Y(a) subunit) was barely detectable on Western blots. Immunocytochemical analysis showed differential distribution of the Yb1 and Yp subunits. The Yb1 subunit was present in the sensory cells, while supporting cells were not specifically stained. At the subcellular level, the isozyme was localized in the apical zones of inner (IHCs) and outer hair cells (OHCs) close to the cuticular plate. The extent of staining, however, varied between OHCs and IHCs. In the OHCs, staining appeared in discrete spots in the apical areas only, whereas in IHCs staining extended further towards the center of the cells. The Yp subunit was mainly localized to Deiters cell processes and pillar cells. Both Yb1 and Yp colocalized with tubulin-specific antibody. The functional significance of GST in the cochlear receptor cells is speculative. However, a role analogous to that in other tissues (detoxification, prostaglandin synthesis) can be assumed. In addition, an association of GST with the microtubule system is possible based on immunohistochemical colocalization with tubulin.

Animals↗
PubMed1994

Morphological gradients in sensory hair cells of the amphibian papilla of the frog, Rana pipiens pipiens.

The sensory hair cells of the amphibian papilla (AP) of the northern leopard frog were examined in a light-microscopic analysis. Hair cell length and cross-sectional area were found to vary systematically along the rostro-caudal axis of the endorgan. The AP was readily divided into three morphological regions. Rostrally-located hair cells are tall, cylindrically-shaped cells with large cross-sectional areas and long stereocilia; caudally-located hair cells are short, goblet-shaped cells with small cross-sectional areas and short stereocilia. In the middle region, hair cells exhibit features intermediate to those of hair cells located at the AP extremes. The detailed pattern of changes in hair cell morphology along the endorgan correlates well with its observed tonotopy and may have implications for the intrinsic tuning of the AP.

Animals↗
PubMed1995

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures.

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CA1 pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CA1 field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CA1 and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the nonphosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and postlesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampal pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.

Animals↗