Complex histopathologic response in rat kidney to oral ß-myrcene: an unusual dose-related nephrosis and low-dose alpha2u-globulin nephropathy.

Oral gavage studies with ß-myrcene in male F344 rats showed a complex renal pathology comprising both alpha2u-globulin (α2u-g) nephropathy, an unusual nephrosis involving the outer stripe of outer medulla (OSOM), and an increased incidence of renal tubule tumors by 2 years. In the 90-day and 2-year studies, respectively, α2u-g nephropathy and linear papillary mineralization were observed in males at the two lower doses but were absent from the high dose. Nephrosis was characterized by dilation of the S3 tubules, nuclear enlargement (including karyomegaly), and luminal pyknotic cells, all in the outermost OSOM. Nephrosis was minimal at the higher doses in the 90-day study, but progressed to a severe grade in males dosed with 1,000 mg/kg for 2 years. Renal tubule tumors developed in treated groups with incidences up to 30% in the 250 and 500 mg/kg male dose groups. Tumors at the lower doses in males may have been associated with α2u-g nephropathy, while those at higher doses in both sexes may have been due to the nephrosis. Because ß-myrcene induced a complex spectrum of renal pathology, the α2u-g nephropathy mechanism cannot be the sole mechanism of carcinogenesis in these rats.

Nanomechanical mapping of the osteochondral interface with contact resonance force microscopy and nanoindentation.

The bone-cartilage, or osteochondral, interface resists remarkably high shear stresses and rarely fails, yet its mechanical characteristics are largely unknown. A complete understanding of this hierarchical system requires mechanical-property information at the length scales of both the interface and the connecting tissues. Here, we combined nanoindentation and atomic force microscopy (AFM) methods to investigate the multiscale mechanical properties across the osteochondral region. The nanoindentation modulus M ranged from that of the subchondral bone (M=22.8±1.8GPa) to that of hyaline articular cartilage embedded in PMMA (M=5.7±1.0GPa) across a narrow transition region <5µm wide. Contact resonance force microscopy (CR-FM), which measures the frequency and quality factor of the AFM cantilever's vibrational resonance in contact mode, was used to determine the relative storage modulus and loss tangent of the osteochondral interface. With better spatial resolution than nanoindentation, CR-FM measurements indicated an even narrower interface width of 2.3±1.2µm. Furthermore, CR-FM revealed a 24% increase in the viscoelastic loss tangent from the articular calcified cartilage into the PMMA-embedded hyaline articular cartilage. Quantitative backscattered electron imaging provided complementary measurement of mineral content. Our results provide insight into the multiscale functionality of the osteochondral interface that will advance understanding of disease states such as osteoarthritis and aid in the development of biomimetic interfaces.

Effect of laser phototherapy on the hyalinization following orthodontic tooth movement in rats.

OBJECTIVE: We aimed to assess histologic changes after the use of laser phototherapy (LPT) during induced tooth movement with 40 g/F on young adult male rats. BACKGROUND DATA: Hyalinization is a sterile necrosis at the pressure zone of the periodontal ligament observed during the initial stages of the orthodontic movement, and extensive hyaline areas might cause an important delay in the tooth movement. The use of LPT is considered an enhancement factor for bone repair, as it stimulates microcirculation as well as the cellular metabolism. MATERIALS AND METHODS: Thirty animals were divided into two groups (n=15), named according to the time of animal death (7, 13, and 19 days). Half of the animals in each group were subjected to irradiation with infrared (IR) laser (λ790 nm, round shaped beam, 40 mW, continuous wave (CW), diameter=2 mm (0.0314 cm(2)), 1.273 W/cm(2), time=2×112 sec+1×275 sec (total time 499 sec), 2×142.6/4.48 J+1×350/11 J, 635.2 J/cm(2)/20 J/ session), during orthodontic movement, the other half were used as nonirradiated controls. After animal death, specimens were sectioned, processed, and stained with hematoxylin and eosin (HE) and Sirius Red, and were used for semi-quantitative histologic analysis by light microscopy. Data were statistically analyzed. RESULTS: We demonstrated that LPT positively affected an important aspect of dental movement; the hyalinization. In the present study, we found a significant reduced expression of hyalinization after 19 days. On irradiated subjects, hyalinization was increased at day 7 with significant reduction at day 13. CONCLUSIONS: It is possible to conclude that the use of laser light caused histologic alterations during the orthodontic movement characterized by increased formation of areas of hyalinization at early stages, and late reduction when compared to nonirradiated animals.

Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats.

AIM OF THE STUDY: Rhubarb is well used to treat chronic renal failure (CRF) in China and Japan, but recent studies reported that the anthraquinone derivatives contained in rhubarb had nephrotoxicity. In this investigation an attempt was made to assess the value and toxic potential of rhubarb to treat CRF. MATERIALS AND METHODS: Histopathologic and biochemical tests combined with toxicokinetic analysis were performed to investigate the nephrotoxic potential and protective effect of rhubarb extract. RESULTS: In normal rat groups, no death was observed and no renal lesion was found after repetitive administration of rhubarb for 3 weeks. The survival rate, pathologic conditions and biochemical indexes of CRF rats treated with rhubarb at two dosages were all improved and significant amelioration was found in the low dosage group compared to the untreated CRF group. Rhein was the mainly absorbable anthraquinone derivative into systemic circulation after oral administration and the area under curve of rhein in CRF groups was lower than that in normal groups at same dosage. CONCLUSIONS: After 3 weeks of administration of rhubarb extract, there was evidence of protective effect to CRF rats, while incidences of hepatotoxicity with minimal to mild hyaline droplets were also observed in normal rats.

Deleted in Malignant Brain Tumors 1 (DMBT1) is present in hyaline membranes and modulates surface tension of surfactant.

BACKGROUND: Deleted in Malignant Brain Tumors 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein that binds various bacteria and is thought to participate in innate pulmonary host defense. We hypothesized that pulmonary DMBT1 could contribute to respiratory distress syndrome in neonates by modulating surfactant function. METHODS: DMBT1 expression was studied by immunohistochemistry and mRNA in situ hybridization in post-mortem lungs of preterm and full-term neonates with pulmonary hyaline membranes. The effect of human recombinant DMBT1 on the function of bovine and porcine surfactant was measured by a capillary surfactometer. DMBT1-levels in tracheal aspirates of ventilated preterm and term infants were determined by ELISA. RESULTS: Pulmonary DMBT1 was localized in hyaline membranes during respiratory distress syndrome. In vitro addition of human recombinant DMBT1 to the surfactants increased surface tension in a dose-dependent manner. The DMBT1-mediated effect was reverted by the addition of calcium depending on the surfactant preparation. CONCLUSION: Our data showed pulmonary DMBT1 expression in hyaline membranes during respiratory distress syndrome and demonstrated that DMBT1 increases lung surface tension in vitro. This raises the possibility that DMBT1 could antagonize surfactant supplementation in respiratory distress syndrome and could represent a candidate target molecule for therapeutic intervention in neonatal lung disease.

Ligneous conjunctivitis: a clinicopathological, immunohistochemical, and genetic study including the treatment of two sisters with multiorgan involvement.

Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, beta(2)-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results.

P-Nitrobenzoic acid alpha2u nephropathy in 13-week studies is not associated with renal carcinogenesis in 2-year feed studies.

The objective of this study was to characterize the renal toxicity and carcinogenicity of p-nitrobenzoic acid in F344 rats. Dose levels in 13-week and 2-year studies ranged from 630-10,000 ppm and 1,250-5,000 ppm, respectively. At 13 weeks, renal lesions included minimal to mild hyaline droplet accumulation in male rats and karyomegaly in male and female rats. At 2 years, renal lesions included proximal tubule epithelial cell hyperplasia in male rats and oncocytic hyperplasia in high-dose male and female rats, and a decreased severity of nephropathy in males and females. The hvaline droplets in renal tubular epithelial cells of male rats at 13 weeks were morphologically similar to those described in alpha2u-globulin nephropathy. Using immunohistochemical methods, alpha2u-globulin accumulation was associated with the hyaline droplets. In addition, at 13 weeks, cell proliferation as detected by PCNA immunohistochemistry was significantly increased in males exposed to 5,000 and 10,000 ppm when compared to controls. Cytotoxicity associated with alpha2U-globulin nephropathy such as single-cell necrosis of the P2 segment epithelium or accumulation of granular casts in the outer medulla did not occur in the 13-week study. In addition, chronic treatment related nephrotoxic lesions attributed to accumulation of alpha2u-globulin such as linear foci of mineralization within the renal papilla, hyperplasia of the renal pelvis urothelium and kidney tumors were not observed. Although there was histologic evidence of alpha2u-globulin accumulation in male rats at 13 weeks, the minimal severity of nephropathy suggests that the degree of cytotoxicity was below the threshold, which would contribute to the development of renal tumors at 2 years.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Subchronic toxicity (90-day) study with para-nonylphenol in rats.

As a component to the risk assessment process for para-nonylphenol (NP; CASRN 84852-15-3), a 90-day study was conducted in rats following U.S. EPA TSCA guidelines and Good Laboratory Practice regulations. NP was administered to four groups of rats at dietary concentrations of 0, 200, 650, or 2000 ppm which corresponded to approximate dietary intakes of 0, 15, 50, or 150 mg/kg/day, respectively. There were 25 rats/sex/group in the control and high-dose groups and 15 rats/sex/group in the low- and middose groups. Ten of the 25 rats/sex in the control and high-dose groups were designated as recovery animals and were maintained on control diets for 4 weeks after completion of the 90-day exposure period to assess the reversibility of any effects which might be observed. To evaluate for the possible weak estrogen-like activity that has been reported for NP in a number of screening assays, estrous cyclicity was monitored using vaginal cytology during Week 8 of the study, and sperm count, motility, and morphology were evaluated at termination. In-life effects from NP exposure were limited to small decreases in body weight and food consumption in the 2000-ppm dose group. Postmortem measurements at Week 14 indicated a dose-related kidney weight increase in males and a decrease in renal hyaline globules/droplets in males from the high-dose group. The kidney weights showed complete recovery following the 4-week postdosing recovery period. Due to the small magnitude of the changes (i.e., all weights were within or near laboratory historical control values) and the lack of correlating clinical or histopathological changes, the kidney weight alterations were not considered toxicologically significant. The biological significance of reduced hyaline in the kidneys of male rats from the high-dose group is uncertain. Renal tubular hyaline is associated with the rat-specific protein, alpha-2u-globulin, and, therefore, this finding was not considered toxicologically relevant to humans. No other effects attributable to NP were observed. No changes were observed for estrous cycling, sperm evaluations, or effects on endocrine organs. NP, therefore, did not manifest any estrogen-like activity as measured in these parameters at dietary concentrations as high as 2000 ppm, the maximum dose administered in this study. Based on the minor findings for the 2000-ppm dose group, the NOAEL (no-observed-adverse-effect level) for NP in this study is considered to be 650 ppm in the diet, corresponding to an approximate intake of 50 mg/kg/day.

Hepatoprotective effects of emodin from Ventilago leiocarpa.

A major component of ethyl acetate (EtOAc) and chloroform (CHCl3) fractions of Ventilago leiocarpa Bunge (Rhamnaceae), emodin, was isolated and exhibited hepatoprotective effects on carbon tetrachloride (CCl4) as well as D-galactosamine (D-GalN)-induced liver damage. The histopathological examination also clearly showed that emodin reduced lymphocyte cells, Kupffer cells, ballooning degeneration, cell necrosis and hyaline degeneration on CCl4 and D-galactosamine-induced tests.

Migration of hyaline cells into the chick basilar papilla during severe noise damage.

Severe acoustic damage in the chick cochlea causes a destruction of both hair cells and supporting cells in a localized area on the basilar papilla. In this region, the sensory cells are replaced by a layer of flattened epithelial cells. We have employed scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to examine the structure and cytoskeletal changes involved in this process. Immunocytochemical staining for actin indicates that the flattened cells are derived from the hyaline cells normally located along the inferior edge of the basilar papilla. In control cochleae the hyaline cells contain dense bundles of actin filaments that anchor into the basal surface of the cells. The hyaline cells appear to redistribute into the severely damaged region by extending the actin bundles at their basal surfaces. Moreover, the efferent nerves that normally form a network among the hyaline cells move into the severely damaged area along with the hyaline cells. In moderately damaged cochleae, where only hair cells are lost, the hyaline cells do not spread into the damaged region. The functional role of this hyaline cell migration is unknown, but it may be involved in maintenance or repair of the severely damaged cochlea.

Relation of alcoholic liver injury to cirrhosis.

The earliest and most reproduceable lesion associated with chronic alcohol abuse is fatty liver. In some alcoholics this may be superseded by alcoholic hepatitis, which may represent the link between the early lesion and cirrhosis. Alcoholic cirrhosis usually begins as a regular, monolobular variety, but is eventually transformed into an irregular, multilobular type. All stages of alcoholic liver injury have now been produced in the baboon, despite high protein and vitamin supplemented diets. Alcohol may therefore now be regarded as a direct hepatotoxin. Epidemiological studies have indicated that alcoholic liver injury begins with an intake of more than 80 g ethanol a day, and that cirrhosis is generally not seen with an intake of less than 160 g per day. The development of cirrhosis correlates with the total duration and amount of alcohol ingested. Complications of alcoholic cirrhosis include iron overload and primary hepatic carcinoma.