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.

Intra-articular hyaluronic acid in the treatment of osteoarthritis of the knee: clinical and morphological study.

OBJECTIVE: To evaluate, in a pilot, open clinical trial on 40 patients with knee osteoarthritis, the structural changes in the synovial membrane and cartilage following treatment with intra-articular hyaluronic acid (HA-Hyalgan). METHODS: The structural effects of HA given as 5 weekly injections (20 mg/2 ml once a week for 5 weeks), were evaluated by microarthroscopy and morphological analysis of biopsy samples taken at baseline and after 6 months, under blind conditions. Clinical efficacy was also evaluated using visual analogue scales for pain and functional parameters. RESULTS: At 6 months, the microarthroscopic evaluation indicated that the majority of the patients (60%) showed no changes compared to baseline, while 32.5% of the patients showed improvement in the grading and/or extension of cartilage lesions and 7.5% showed a worsened condition. These changes were accompanied by a statistically significant reduction in the synovial inflammation (p = 0.001). The results were confirmed by morphological examination of the cartilage and synovial membrane. At 6 months compared to baseline, a statistically significant reconstitution of the superficial amorphous layer of the cartilage (p = 0.0039), an improvement in the chondrocyte density (p = 0.0023) and vitality (p = 0.05), and a statistically significant reduction in synovial inflammation (p = 0.0001) accompanied by a significant increase in the synovial repair process (p = 0.0001) were observed. Significant and long lasting improvement in pain and joint mobility were also seen after HA treatment. Joint effusion, when present, was reduced. The treatment was well tolerated. CONCLUSION: Hyalgan represents a useful therapy for knee OA, with long-lasting symptomatic efficacy and potential positive effects on joint tissues. Other studies, in particular placebo-controlled studies, are warranted to confirm these promising results observed on joint tissues.

Aging of the human synovium: an in vivo and ex vivo morphological study.

Age-associated changes of the human synovium have been investigated by microarthroscopy, optical and electron microscopy, immunohistochemistry, and cytochemistry. The knee joints of nineteen 15- to 56-year-old subjects, classified as normal by inspection, were carefully examined by microarthroscopy; small synovial tissue biopsy specimens from both the suprapatellar pouch and the medial tibiofemoral gutter were taken. Microarthroscopy showed that the villi were more numerous and the vascular network and cell distribution and profiles less regular in aged individuals. These data were confirmed by scanning electron microscopy, which also showed large areas of the synovial surface devoid of cells and collagen bundles in contact with the joint cavity in aged subjects. Light and transmission electron microscopy confirmed these data and allowed evaluation of the number, distribution, shape, and internal organization of cells as well as the distribution of vessels and the organization of the extracellular matrix in the full thickness of the synovium (down to 2 mm). Particular attention was paid to synovial lining cells, among which three main phenotypes could be recognized: synthetic type (present at all ages and hypertrophied in aged subjects), macrophagelike (increasing with age), and fibroblastlike. Collagen increased with age. Further studies are needed for comprehensive understanding of age-associated changes in the human synovium.

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.

[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.

Ependymoma of the foramen of Monro: ultrastructural characterization.

The characteristics of clear cells of an ependymoma of the foramen of Monro have been studied by electron microscopy to precisely define its organellar composition and to establish the tumor histogenesis. Our data confirm that the once-thought oligodendroglial is, in fact, an ependymal tumor. Both the scarce number of organelles, owing to the low degree of differentiation, and the abundance of hyaloplasmic lipid vacuoles can account for the clear appearance of these tumor cells.

Elastofibroma: an in vivo model of abnormal neoelastogenesis.

Thirteen cases of elastofibroma have been studied by conventional light and electron microscopy, as well as by histochemistry and immunohistochemistry. By light microscopy elastinophilic material appeared as huge fibers crossing collagen bundles. Immunohistochemistry demonstrated a strong positivity for elastin in numerous and circumscribed areas of the extracellular matrix. By electron microscopy, collagen consisted of 40-50-nm wide fibrils, and elastin was made of large aggregates of moderately electron-dense material surrounding a very thin, apparently normal, elastin core. At high magnification these aggregates consisted of short tubules, often in regular arrays, surrounded by microfibrils and microfilaments. These data, associated with selective digestions on thin sections with elastase, purified collagenase, hyaluronidase, and chondroitinase ABC, revealed that elastic fibers in elastofibroma seem to be made of true elastin surrounded by an enormous amount of hydrophilic material, in which some elastin, chondroitin sulfates, and collagenase type-VII sensitive material are aggregated forming a rather ordered array of short tubules.

MELAS syndrome: characteristic migrainous and epileptic features and maternal transmission.

Severe prolonged migrainous symptoms and prolonged partial status epilepticus are characteristic features of the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Maternal transmission previously found in myoclonus epilepsy and ragged-red fibers (MERRF), another mitochondrial disease, is suggested in this disorder as well.

Ultrastructural and immunohistochemical contribution to the histogenesis of human cardiac myxoma.

The ultrastructural features of 8 human cardiac myxomas were analyzed and correlated with immunohistochemical data, with the aim to clarify the characteristics of the cell lines involved in the tumor genesis. Immunohistochemical studies were performed to detect the presence and the distribution of intracytoplasmic filaments (vimentin, desmin, actin, myosin) as well as myoglobin and factor VIII-related antigen, albumin, and lysozyme. Eighty percent of myxoma cells were simultaneously positive for vimentin, desmin, and actin, whereas 30% of them stained with antifactor VIII and antivimentin antibodies. The submicroscopic analysis revealed two main cell populations: (1) one composed of stellate-shaped cells with scanty organelles and sparse hyaloplasmic filaments scattered throughout the myxoid stroma and forming a loose network with their projections; (2) another one included cells with more cytoplasmic organelles, intermediate filaments, and myofilaments arranged either singly or in both solid and hollow cord-like structures. Our results support the hypothesis that cardiac myxoma may originate from a reserve multipotent mesenchymal cell able to differentiate more or less completely along two major evolutional lines: myoid and endothelial. The tumor tissue thus seems to be involved in vessel formation, suggesting a growth pattern akin to that manifested in other forms of endocardial pathological reactivity in which reserve mesenchymal cells are engaged.

Human cytomegalovirus morphogenesis: an ultrastructural study of the late cytoplasmic phases.

The late cytoplasmic phases of human Cytomegalovirus (HCMV) morphogenesis in cultured fibroblasts have been studied by transmission electron microscopy focusing attention on the relationship between the viral particles and host cell organelles. The results obtained largely reflect changes in cells subjected to sublethal injurious stimuli induced by many viruses as well as different noxious agents. A great increase in the number of Golgi apparatuses and lysosomes was observed, both of them interacting with the viral progeny. HCMV seems to acquire its final envelope from Golgi-derived structures and, less frequently, from the plasma membrane.

Congenital muscular dystrophy with central nervous system involvement: case report.

A case of a mediterranean boy with congenital muscular dystrophy (CMD) and central nervous system (CNS) involvement with mild intellectual impairment and seizures is reported. Muscular dystrophy involved both skeletal and mimic muscles, and histological findings were consistent with a congenital dystrophy. EEG recordings showed generalized and localized paroxysmal activities. CT scan demonstrated low-density periventricular areas. Ophthalmoplegia was also observed. A literature review disclosed that in some western cases of CMD plus CNS involvement, cranial muscles other than mimic muscles may be involved.

Mitochondrial paracrystalline inclusions in the peroneus brevis muscle of patients with peripheral neuropathy.

In a series of 50 consecutive biopsies of peroneus brevis muscle (PBm) taken from patients with ascertained or suspected polyneuropathy in the course of sural nerve biopsy, we found a high incidence (26%) of intramitochondrial paracrystalline inclusions (MPI). Five out of these 13 patients were also submitted to an additional biopsy of a proximal muscle, which in no case confirmed the finding of MPI. Six out of the 13 patients with MPI were affected by diseases with a presumably important ischemic component. The mean age of patients with MPI was quite elevated (60.7), and the difference in distribution of age between patients with and without MPI was statistically significant. No significant difference in histochemical changes was found between the group of MPI patients and a control group of age-matched patients without MPI, thus excluding that MPI in the PBm are specifically associated with other neurogenic or myopathic aspects. We conclude that aging and, probably, ischemia are largely responsible for the frequent presence of MPI in the PBm. In addition, factors intrinsic to the muscle itself, possibly related to morphological, physiological, or biochemical peculiarities, may also influence the development of MPI.

A 48-hour preservation of an isolated human uterus: endometrial responses to sex steroids.

Human uteri were perfused with Krebs-Ringer bicarbonate-glucose buffer with and without estrogens and progesterone for a period of up to 48 hours to preserve a viable organ, which was responsive to hormones. Flow rates of 12 to 35 ml/minute per artery were fully distributed into the organ, with pressure values ranging from 80 to 120 mm Hg. Arteriovenous gradients of oxygen and carbon dioxide tensions as well as the levels of lactate, lactic dehydrogenase, and creatine kinase released in the perfusate, indicators of tissue ischemia or cell necrosis, showed a good preservation of the organ for up to 48 hours. The light- and electron-microscopic examinations of endometrial and myometrial tissues taken before and during perfusion confirmed this result. The extracorporeal perfusion of uteri with buffer containing estrogens plus progesterone exhibited secretive modifications of the proliferative endometrium, thus suggesting the viability of the organ and its responsiveness to sex steroids.

Abnormal muscle mitochondria in ischemic claudication.

The gastrocnemius and quadriceps muscles findings in 18 patients with chronic arterial insufficiency were reviewed with regard to mitochondrial changes. Prominent mitochondrial alterations were present in eight out of 18 patients. The comparison of clinical data between patients with and without mitochondrial changes revealed that in this latter group all patients were at stage IV, whereas the degree of ischemic disease was milder in the other group: the difference in distribution of patients at stage IV between the two groups was statistically significant. This supports the view that mitochondrial changes are expression of adaptive modification rather than damage.

Cell ultrastructure in disease.

The doctor of today must adopt the 'cellular way of thinking' in the evaluation of diseases. This ultrastructural outlook provides him with much indispensable information that also serves a practical purpose. A diseased cell organelle is at the basis of every clinical sign and any attempt of therapy must be aimed at that specific point of lesion. We intend, in the light of a long experience, to propose to clinicians a new way of thinking in which a precise correlation between symptoms and submicroscopic changes of the cell is considered. Many different examples amply justify this proposal. Electron microscopy can contribute by enabling identification of structural subcellular modifications suitable for the finest differential diagnosis, more and more complete understanding of pathogenic pathways of various diseases, the establishment of guidelines for precise pharmacological interventions at the molecular level.