Expression of ß-catenin and γ-catenin in maxillary bone regeneration.

ß- and γ-catenin are components of catenin family involved in cadherin adhesion function. Recently it has been shown that this family is involved in other functions such as signaling and activation of transcription factors. The final goal of this study was to evaluate the role of ß- and γ-catenin in bone cell physiology and bone regeneration. Formalin-fixed-paraffin embedded specimens of 15 human bone specimens after sinus lift were collected and examined by immunohistochemstry using primary antibodies against ß- and γ-catenin. Staining intensity and cellular localization were evaluated. ß and γ-catenin showed a very high level of expression in human bone tissues. In particular catenins were expressed in cells with morphological findings of osteoblasts in the areas of new bone formation at the junction between mineralized and unmineralized tissue, between osteoid matrix and bone. Osteoclasts showed also positivity for catenins. Osteocytes, cells located in lacunae of mature mineralized bone with function of bone vitality maintenance, showed no expression for catenins. Specimens characterized by high amount of catenins in osteoblasts at 1° month showed high grade of bone maturation at 3° month. Data demonstrated an overall involvement of catenins in human bone tissues and in particular during bone regeneration process. The presence of staining for ß- and γ-catenin particularly in osteoblasts demonstrates a significant role of catenins in functions, other than in cadherin interaction, such as signaling and activation of transcription factors during differentiation of bone tissues.

Age-dependent spontaneous EEG bursts in rats: effects of brain phosphatidylserine.

During aging, male Sprague-Dawley rats display increasing frequency of bursts of seizure-like EEG patterns. They also have a decreased retention of passive avoidance response and a loss of spontaneous alternation in a Y maze. A study was made on the effects of chronic administration of phosphatidylserine in aged rats. It was found that BC-PS reduced by 65% the number of seizures, and by 70% their duration. It also facilitated retention of passive avoidance and of spontaneous alteration behavior. These results suggest that phosphatidylserine can affect electrophysiological and behavioral parameters in aged rats probably by counteracting age-related biochemical changes.

Time course, localization and pharmacological modulation of immediate early inducible genes, brain-derived neurotrophic factor and trkB messenger RNAs in the rat brain following photochemical stroke.

A focal, unilateral thrombotic stroke was produced in the rat sensorimotor cortex. The time course of expression and localization of the immediate early inducible genes: c-fos, c-jun, zif268; nerve growth factor, brain-derived neurotrophic factor and the related tyrosine kinase high-affinity receptor (trkB) messenger RNAs were studied by in situ hybridization. The levels of messenger RNAs for c-fos, zif268, brain-derived neurotrophic factor (but not nerve growth factor) and trkB were consistently increased in cortex ipsilaterally to the lesion, while c-jun messenger RNA content was only slightly increased. The brain-derived neurotrophic factor messenger RNA was increased from 2 to 18 h following the stroke, mainly in cells having a normal morphological appearance. The trkB messenger RNA displayed temporal and spatial increases similar to brain-derived neurotrophic factor messenger RNA. The time course and pattern of expression of immediate early inducible gene and trophic factor messenger RNAs did not clearly support a causal relationship between these two families of factors. The observed messenger RNA increases were greatly attenuated by the non-competitive N-methyl-D-aspartate-sensitive glutamate receptor antagonist (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine , but substantially unaffected by the non-N-methyl-D-aspartate receptor antagonist 2,3-dihydroxy-6-nitrosulphanoylbenzoquinoxaline. The results suggest a major contribution of N-methyl-D-aspartate-sensitive glutamate receptor activation to the transcriptionally directed events subsequent to stroke. Future studies should clarify the contribution of these processes to either the progression of neuronal degeneration or the establishment of protective compensatory responses.

An animal model based on the Sprague Dawley rat for the evaluation of ototoxicity.

Various methodological approaches that can be used to detect ototoxic effects caused by the administration of various substances are presented, using the Sprague-Dawley rat as an animal model. Electrophysiological data are also presented to show how the model behaves with potentially ototoxic (hyaluronic acid) and initially inert (hydroxy-propyl-methyl-cellulose) substances.

Brainstem auditory evoked potentials in rats with streptozotocin-induced diabetes.

Brainstem acoustic evoked potentials (BAEPs) were studied in streptozotocin (STZ)-diabetic rats and age-matched controls at 3 and 5 months from induction of the pathology. The diabetic status of the animals was kept uncontrolled throughout the study. Body weight and glycosylated hemoglobin were markedly altered in the diabetic animals (-42%, and +120% of control values, respectively). Neurophysiological results showed an increase in the latency of the major components of BAEPs; this increase was clearly time-dependent for the peripheral component (peak I). The central component (peak IV) was also significantly delayed. However, no significant impairment of the central conduction time was demonstrated by examining the interpeak I-IV latency. In conclusion, BAEPs prove to be a useful non-invasive neurophysiological technique that may help unravel both the relative involvement of the peripheral and central nervous systems in the course of diabetes mellitus, and the evolution of diabetic neuropathy.

Distribution of phospholipid-malondialdehyde-adduct in the human lens.

The distribution of phospholipid-malondialdehyde adduct was measured in human lenses pooled by age over a range of 13 to 68 years. Lipid extracts from sections of the central core and from the equatorial region of the lens were probed for lipid adduct using laser induced fluorescence. For an excitation of 351.1/363.8 nm the fluorescence maximum occurred at 473 nm for all ages and regions of the lens. The adduct concentration was always highest in the anterior nuclear section of the central core and decreased toward the anterior and posterior surfaces. The adduct concentration was lowest in the equatorial section for all samples. The regional distribution observed suggests that the formation of lipid-malondialdehyde adduct in the lens could be influenced by the cumulative amount of UV radiation absorbed. No changes with age were observed in the concentration of the adduct in the whole lens nor in the cortical and nuclear regions suggesting the possibility of an equilibrium between the formation and degradation of the adduct.

Comparative ototoxic potential of hyaluronic acid and methylcellulose.

In experimental animal studies, exogenous hyaluronan (HA) has been shown to exert beneficial effects on the healing of tympanic membrane perforation. As any other exogenous substance, HA may prove potentially toxic, by filling the middle ear cavity, to the sensory cells of the organ of Corti. Electrophysiological (ABR) and morphological studies were carried out in the rat to examine the auditory function and the structure of the sensory epithelium. Rats received either HA or hydroxy-propyl-methyl-cellulose by trans-tympanic injection (middle ear cavity was completely filled up) and were compared to untreated, age- and weight-matched rats. In both treated groups ABR revealed transitory, mild conduction hearing loss, in particular for high frequencies, until day 7 postinjection. This loss recovered completely within the 15th day. Morphologically, no significant degenerative/necrotic lesions were observed in the organ of Corti, from both treated groups.

Crown fractures: effectiveness of current enamel-dentin adhesives in reattachment of fractured fragments.

OBJECTIVE: The introduction of the current enamel-dentin adhesives has simplified the reattachment of fractured dentinal fragments. The objective of this study was to determine the strength needed to detach coronal fragments reattached with the most recent adhesives. METHOD AND MATERIALS: A complete coronal fracture was caused on the incisal one third of 40 noncarious maxillary and mandibular lateral and central incisors. Each fractured coronal fragment was reattached to its tooth with the enamel-dentin adhesive Scotchbond MP, All-Bond 2, Dentastic, or One-Step (10 specimens each). RESULTS: There was no statistically significant difference between 2 similar fourth-generation adhesives that use orthophosphoric acid as etchant (Dentastic and All-Bond 2) and a fourth-generation adhesive that uses maleic acid as etchant (Scotchbond MP). The values obtained with the fifth-generation adhesive (One-Step) were significantly inferior to those obtained with the other 3 enamel-dentin adhesives. CONCLUSION: In the reattachment of fractured tooth fragments, fourth-generation adhesives can guarantee a bonding force stronger than fifth-generation adhesives.

Effectiveness of the current enamel-dentinal adhesives: a new methodology for its evaluation.

Two Class II boxes of standardized dimensions were prepared in each of 10 extracted third molar teeth. The dentinal adhesives Syntac, Gluma 2000, Scotchbond MP, and All-bond 2 were applied to five boxes each. All the boxes were restored with the same photopolymerizing resin composite. The microleakage of each restoration was measured in the permeability cell. The physiologic solution able to seep between the walls of the cavity and resin composite was measured and the flux of the liquid over time was calculated. All-Bond 2 was the only adhesive found, in some specimens, to make the dentin completely impermeable and to form an extremely precise seal between the resin complete material and the underlying dentinal surface. The flux values for all four adhesives were significantly different; Syntac allowed the greatest amount of microleakage. However, these in vitro results should not be considered absolute values.

[Spontaneous reparation of post-traumatic tympanic perforation: an experimental study in rats]. / Riparazione spontanea della perforazione timpanica post-traumatica: studio sperimentale nel ratto.

In most patients tympanic membrane perforation spontaneously repairs itself. Nonetheless, in several cases the perforation persists due either to traumatic or phlogistic damage. Although the membrane perforation healing process has been under study for over a century there are still two contrasting theories. One of these theories asserts that repair takes place through the growth of granulation tissue while the other believes that epithelial migration is at the basis of healing. In the present study an experimental animal model (rat) was used in order to assess the staging of the natural evolution of post-traumatic membrane perforation and to characterize, from a morphological and sub-microscopic point of view, the cellular population and microstructural aspects of the extracellular matrix. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to evaluate the progress in tympanic membrane healing and to make a comparison with the most recent theories. Eighteen Sprague-Dawley rats weighing 230-260 gr of the same age (10 weeks) were used in the study. The animals were anesthetized by intraperitoneal injections of Na-pentobarbital (NembutalR) (45 mg/Kg) and, under operating microscope, bilateral perforation of the upper rear quadrant of the pars tensa was performed with a myringotomy lancet. The animals were subjected to periodic follow-ups over next 30 days. Three animals were sacrificed during each control and the tympanic membrane was removed for TEM and SEM study. On the basis of the present study the following conclusions can be drawn: a) primary healing of the lesion is through granulation tissue; b) the healed T.M. is composed of three normal layers as is the normal T.M.; c) the presence of fibroblasts in the intermediate neoformed fibrous layer leads one to conclude that its extracellular matrix is produced and organized "in situ"; d) the neoformed lamina propria has a disorganized, fibrous structure.

[The ultrastructural aspects of the carious lesion]. / Aspetti ultrastrutturali della lesione cariosa.

The aim of the present study was to analyse under SEM the ultrastructural alterations in tubular content and peri- and intertubular dentin in teeth subject to active carious lesions. The samples used were teeth with grade 2 active carious lesions without signs of abrasion, wear, erosion and conservative reconstruction. The coronal fragments obtained by fracture were fixated, dehydrated and metallized before SEM analysis. The study was performed by analysing samples of amelo-dentinal junctions towards the pulpo-dentinal edge. By correlating histopathological lesions to their topographical distribution and to the physiopathological mechanisms involved, it was possible to subdivide dentin underlying a carious lesion into three main zones: demineralisation zone, sclerosis zone, subsclerotic dentin zone. The individualization of these sectors was based on the identification of basic lesions linked to changes in tubular lumen and peri and intertubular dentin. The demineralisation zone was in fact characterised by: dentinal infection and reversal of normal tissue morphology in the outermost layers; the fibrous appearance of intertubular dentin and disappearance of peritubular dentin; modifications in the tubular content secondary to demineralisation, such as sheaths, hollow rods and intratubular crystals. The sclerosis zone was characterised by: fully occluded tubules, tubules fully occluded by rods, tubules partially occluded by hollow rods and by thickening of peritubular dentin. The third zone was the result of tissue-forming activity on the pulpodentinal edge in response to carious lesions and was represented by regular and irregular tertiary dentin. From an analysis of all data collected it is possible to hypothesize that changes in the demineralisation zone are the results of a passive tissue response exclusively linked to physicochemical mechanisms; whereas those in the sclerosis zone are due to the realisation of a lithogenic environment of multifactorial genesis at a intratubular level. The site in which there is the greatest probability of observing a cellular response is the subsclerotic dentinal zone.

Power spectrum analysis of cardiovascular variability monitored by telemetry in conscious unrestrained rats.

Beat-to-beat variability of arterial pressure and heart period (R-R) was studied in eight conscious freely-moving adult male rats in which telemetric recordings of arterial pressure, ECG and respiratory movements were obtained under unrestrained and unstressed conditions. The beat-to-beat time series of these signals (systolic arterial pressure, diastolic arterial pressure and R-R) were analyzed, in the frequency domain, using autoregressive spectral analysis in order to detect and quantify the rhythmic components. In basal conditions, the systolic arterial pressure variability spectrum was characterized by three major spectral components which had central frequencies respectively of 0.08 +/- 0.03 Hz (very low frequency), 0.43 +/- 0.02 Hz (low frequency) and 1.36 +/- 0.19 Hz (high frequency). Similar rhythmic components were found in R-R signal variability. The very low frequency component included a higher percentage of total power in R-R variability spectrum (75.3%) than in systolic arterial pressure variability spectrum (58.4%). The low frequency component was more pronounced in both systolic and diastolic arterial pressure variability spectra. The high frequency component of R-R, systolic and diastolic arterial pressure was synchronous with respiration. Cross-spectral analysis revealed a high statistical coherence between R-R and arterial pressure variabilities in all the three frequency bands. An alpha-adrenergic blocker (phentolamine) specifically abolished the low frequency components of systolic and diastolic arterial pressure variability spectra, thus suggesting that low frequency is a marker of sympathetic modulation of vasomotor activity. The low frequency component of R-R variability spectrum was also markedly blunted. We suggest that cardiovascular variability signals, (R-R, systolic and diastolic arterial pressure) are composed almost of two main rhythms linked to respiration and vasomotor activity. These rhythms can be quantified in conscious unrestrained rats by using telemetry and spectral analysis. This approach seems to offer a new powerful tool for pharmacological studies in conscious small animals.

Protective effects of a monosialoganglioside derivative following transitory forebrain ischemia in rats.

We evaluated the effects of treatment with the inner ester derivative of the monosialoganglioside GM1 on cortical electroencephalographic activity and hippocampal CA1 morphology after transitory, near-complete cerebral ischemia in rats. Ischemia was induced by the four-vessel occlusion method, and we studied only the 58 rats that showed flattening of the cortical electroencephalogram for the entire 30 minutes of occlusion. The ganglioside (n = 30) or saline (n = 28) was administered intravenously immediately after release of the carotid clips and then intramuscularly for 21 days of observation. Cortical electroencephalographic activity was monitored throughout the experiment. After 21 days of recirculation we assessed hippocampal CA1 damage by light microscopy. The results indicate that treatment with the ganglioside reduces postischemic secondary damage to the cortical circuitry (as indicated by significantly higher cortical electroencephalographic activity late after reperfusion) and limits neuronal loss in the CA1 region. Our results lend support to the possible therapeutic use of ganglioside in human pathologic conditions associated with cerebrovascular insufficiencies.