A spontaneously occurring malignant pituicytoma in a male sprague dawley rat.

Pituicytoma is an extremely rare neoplasm derived from pituicytes, which are glial cells in the posterior lobe of the pituitary gland. A malignant pituicytoma was found in the intracranial cavity of a 55-week-old male Sprague-Dawley rat. Macroscopically, the tumor was located on the sphenoid bone and involved the pituitary gland. The tumor was composed of sheets of fusiform cells with spindle- or pleomorphic-shaped nuclei and abundant eosinophilic cytoplasms. The cells were arranged in a whirling or irregular growth pattern. Some tumor cells were bizarre multinucleated giant cells with cytoplasmic eosinophilic hyaline droplets. Many tumor cells were strongly positive for vimentin and glial fibrillary acidic protein, and some cells were positive for ED-1 and S-100. These findings closely resembled those of a giant cell glioblastoma derived from the pituitary gland, suggesting anaplastic pituicytoma. From our review of the literature, we believe this is the first report of a spontaneous malignant pituicytoma in a rodent.

Arachidonic acid supplementation during gestational, lactational and post-weaning periods prevents retinal degeneration induced in a rodent model.

Fatty acids and their derivatives play a role in the response to retinal injury. The effects of dietary arachidonic acid (AA) supplementation on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was investigated in young Lewis rats during the gestational, lactational and post-weaning periods. Dams were fed 0·1, 0·5 or 2·0% AA diets or a basal (< 0·01% AA) diet. On postnatal day 21 (at weaning), male pups received a single intraperitoneal injection of 50 mg MNU/kg or vehicle, and were fed the same diet as their mother for 7 d. Retinal apoptosis was analysed by the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling (TUNEL) assay 24 h after the MNU treatment, and retinal morphology was examined 7 d post-MNU. Histologically, all rats that received MNU and were fed the basal and 0·1% AA diets developed retinal degeneration characterised by the loss of photoreceptor cells (disappearance of the outer nuclear layer and the photoreceptor layer) in the central retina. The 0·5 and 2·0% AA diets rescued rats from retinal damage. Morphometrically, in parallel with the AA dose (0·5 and 2·0% AA), the photoreceptor ratio significantly increased and the retinal damage ratio decreased in the central retina, compared with the corresponding ratios in basal diet-fed rats. In parallel with the increase in serum and retinal AA levels and the AA:DHA ratio, the apoptotic index in the central retina was dose-dependently decreased in rats fed the 0·5 and 2·0% AA diets. In conclusion, an AA-rich diet during the gestation, lactation and post-weaning periods rescued young Lewis rats from MNU-induced retinal degeneration via the inhibition of photoreceptor apoptosis. Therefore, an AA-enriched diet in the prenatal and postnatal periods may be an important strategy to suppress the degree of photoreceptor injury in humans.

N-methyl-N-nitrosourea-induced changes in epithelial rests of Malassez and the development of odontomas in rats.

Morphological changes in the epithelial rests of Malassez (ERM) and the development of odontogenic tumors in the molars of female Lewis rats treated at 4 weeks of age with a single intraperitoneal injection of 50 mg/kg of N-methyl-N-nitrosourea (MNU) were examined at 12, 18 and 30 weeks of age. Following MNU exposure, the total number and average area of ERM in the cervical and furcational regions of the first, second and third molars of the mandible and maxilla were compared with age-matched control animals. The number of ERM at each time point was significantly greater in the MNU-treated group compared to the control group, but there was no time-dependent increase in the number of ERM in either group. The area of ERM was significantly larger in the MNU-treated group compared to the control group at each time point, and it increased in a time-dependent manner in the MNU-treated group. No increases in the number or area of ERM were observed in the control group. At 30 weeks of age, 23% of the MNU-treated rats had developed odontomas (complex type) in the molar region as well as in the incisor region. Immunohistochemically, the expression of tyrosine receptor kinase A (TrkA) and cytokeratin 14 (CK14) decreased, whereas p63 expression remained high during ERM enlargement. In tumors, ameloblast-like cells were positive for amelogenin, TrkA and CK14 but negative for p63, whereas odontoblast-like cells were negative for all antigens examined. In conclusion, a single intraperitoneal injection of MNU caused the development of odontomas in the molar region; these tumors were possibly derived from ERM.

Spontaneously occurring intracranial lipomatous hamartoma in a young BALB/c mouse and a literature review.

An intracranial lipomatous hamartoma was found in the third ventricle of a 7-week-old female BALB/cAnNCrlCrlj mouse. The nodule was composed of mature white adipose cells, which contained one large fat droplet, and there was no evidence of cytological atypia. The brain parenchyma at the retrosplenial granular cortex and the hippocampus in the cerebrum were slightly compressed, and the choroid plexus was dislocated downward. Scattered capillary vessels penetrated the nodule from the surrounding tissue. Based on these findings, the lesion was diagnosed as a lipomatous hamartoma that occurred from the roof of the third ventricle. This extremely rare tumor-like nodule represents an overgrowth of the mature adipocyte population as a malformation rather than a true neoplasm.

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats.

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.

Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells.

Resveratrol (Res; 3,4',5-trihydroxy-trans-stilbene), which is a polyphenol found in grapes, can block cell proliferation and induce growth arrest and/or cell death in several types of cancer cells. However, the precise mechanisms by which Res exerts anticancer effects remain poorly understood. Res blocked both anchorage-dependent and -independent growth of HT-29 and COLO 201 human colon cancer cells in a dose- and time-dependent manner. Annexin V staining and Western blot analysis revealed that Res induced apoptosis accompanied by an increase in Caspase-8 and Caspase-3 cleavage. In HT-29 cells, Res caused autophagy as characterized by the appearance of autophagic vacuoles by electron microscopy and elevation of microtubule-associated protein 1 light chain 3 (LC3)-II by immunoblotting, which was associated with the punctuate pattern of LC3 detected by fluorescein microscopy. Inhibition of Res-induced autophagy by the autophagy inhibitor 3-methyladenine caused a significant decrease in apoptosis accompanied by decreased cleavage of Casapse-8 and Caspase-3, indicating that Res-induced autophagy was cytotoxic. However, inhibition of Res-induced apoptosis by the pan-caspase inhibitor Z-VAD(OMe)-FMK did not decrease autophagy but elevated LC3-II levels. Interestingly, Res increased the intracellular reactive oxygen species (ROS) level, which correlated to the induction of Casapse-8 and Caspase-3 cleavage and the elevation of LC3-II; treatment with ROS scavenger N-acetyl cysteine diminished this effect. Therefore, the effect of Res on the induction of apoptosis via autophagy is mediated through ROS in human colon cancer cells.

Corneal damage induced in adult mice by a single intraperitoneal injection of N-ethyl-N-nitrosourea.

AIM: The morphological response and cell kinetics of the mouse cornea to various doses of N-ethyl-N-nitrosourea (ENU) was examined. MATERIALS AND METHODS: ENU at a dose of 50, 100, 200, 400, or 600 mg/kg was injected intraperitoneally into female BALB/c mice at seven weeks of age. Sequential morphological features and cell kinetics (TUNEL assay as apoptosis marker, PCNA immunostaining as proliferative activity marker, and p63 immunostaining as corneal stem cell marker) of corneal damage caused by 600 mg/kg of ENU were also analyzed 6, 12, 24 and 72 h, and 7 days after exposure. Moreover, older mice (25 to 34 weeks of age) received the same dosage and were sacrificed 7 days later. Both eyes of all mice were analyzed histopathologically and morphometrically, by using the parameters of corneal epithelial thickness. RESULTS: All ENU-treated mice in the 600 mg/kg group developed corneal damage characterized by desquamation and loss of epithelial cells within 7 days. Corneal epithelial thickness was significantly reduced in the 600 mg/kg group as compared to the control group and decreased to approximately half of the normal thickness. Although the number of TUNEL-positive epithelial cells in the ENU-treated mice was similar to that of the control mice, ENU inhibited the proliferative activity of epithelial cells showing PCNA-positivity 72 h after treatment. The p63-positivity of epithelial cells decreased in the central cornea of mice treated with 600 mg/kg of ENU. Older mice did not develop corneal damage from exposure to ENU. CONCLUSION: ENU induced corneal damage in adult mice, and epithelial cell loss was caused by the inhibition of corneal epithelial proliferation. This is the first report to describe ENU-induced corneal injury in adult mice.

Sulforaphane inhibits the growth of KPL-1 human breast cancer cells in vitro and suppresses the growth and metastasis of orthotopically transplanted KPL-1 cells in female athymic mice.

The anticancer effects of sulforaphane (SFN), which is found in cruciferous vegetables, were studied on KPL-1 human breast cancer cells in vitro and in vivo. Cell proliferation in vitro was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and tumor growth and metastasis in vivo were examined in orthotopically (right thoracic mammary fat pad) transplanted KPL-1 cells in female athymic BALB/c mice. The MTT assay showed that SFN directly inhibited KPL-1 cell growth in vitro (IC50 at 48 h, 19.1 µM; IC50 at 72 h, 17.8 µM). Athymic mice received a KPL-1 cell transplant, and SFN treatment (intraperitoneal injection of 25 or 50 mg/kg SFN) was started the next day. Mice received five injections each week during the 26-day experimental period (for a total of 20 injections). Compared with the SFN-untreated controls, SFN suppressed primary tumor growth. At the termination of the experiment, the final tumor volume was 686±94 mm3 for the control group, 516±70 mm3 (75% of control value) for the 25 mg/kg SFN group and 351±55 mm3 (51% of control value) for the 50 mg/kg SFN group. The final tumor weight was 571±69 mg in the control group, 416±63 mg (73% of the control value) in the 25 mg/kg SFN group and 338±56 mg (59% of the control value) in the 50 mg/kg SFN group. SFN caused a dose-dependent decrease in the proliferation ratio and an increase in the apoptotic ratio of the primary tumor cells. SFN treatment tended to reduce regional (axillary) lymph node metastasis. Treatment with 50 mg/kg SFN significantly inhibited KPL-1 cell growth in vivo by suppressing cell proliferation and inducing apoptosis, and it tended to reduce axillary lymph node metastasis of KPL-1 human breast cancer cell xenografts in female athymic mice.

Retinal degeneration induced in adult mice by a single intraperitoneal injection of N-ethyl-N-nitrosourea.

Seven-week-old female BALB/c mice received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (50, 100, 200, 400, or 600 mg/kg), and retinal damage was evaluated after 7 days. Sequential morphological features of the retina and retinal apoptosis, as determined by the TUNEL assay, were analyzed 6, 12, 24, and 72 hr and 7 days after treatment with 600 mg/kg of ENU. Moreover, older mice (25 to 34 weeks of age) received an intraperitoneal injection of 600 mg/kg ENU and were sacrificed 7 days later. All animals were necropsied, and both eyes were examined histopathologically. Two of the 5 mice that received 600 mg/kg ENU died during the experimental period. Histopathologically, all mice that received 600 mg/kg of ENU experienced retinal degeneration characterized by the loss of photoreceptor cells (disappearance of the outer nuclear layer and photoreceptor layer) in both the central and peripheral retina within 7 days. One of 5 mice treated with 400 mg/kg ENU exhibited retinal damage that was restricted to the central retina. Older mice treated with 600 mg/kg ENU exhibited retinal damage that was similar to the retinal damage in younger mice. In the 600 mg/kg ENU-treated mice, TUNEL-positive photoreceptor cells peaked 72 hr after ENU treatment. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after treatment. In conclusion, ENU induces retinal degeneration in adult mice that is characterized by photoreceptor cell apoptosis.

Effects of short-term pregnancy hormone treatment against N-methyl-N-nitrosourea-induced mammary carcinomas in female Lewis rats.

AIM: To study the effects of short-term pregnancy hormone treatment, estradiol and progesterone or prolactin, against pre-existing mammary cancer as well as newly developing mammary cancer by using the N-methyl-N-nitrosourea (MNU)-induced rat mammary cancer model. MATERIALS AND METHODS: Three-week-old female Lewis rats (n=103) received an intraperitoneal injection of 50 mg/kg MNU. Nine weeks after the MNU injection, the rats received 3 weeks of hormone treatments, consisting of a subcutaneously implanted 0.5 mg estradiol- and 32.5 mg progesterone-containing pellet (E/P group, n=35) or four subcutaneous injections per week of 5 mg/kg perphenazine (PPZ group, n=38), a prolactin-releasing agent. The remaining rats did not receive hormones (control group, n=30). At 12 weeks of age (when the hormone treatments were started), 6 rats in each of the three groups had palpable tumors. These rats were sacrificed at 15 weeks of age, and the remaining rats were sacrificed at 19 weeks of age. The entire mammary glands and the palpable mammary tumors were histologically examined, and the development and growth of mammary tumors was compared. RESULTS: Newly developing mammary tumors were suppressed and pre-existing ones regressed in the E/P group, while the development and growth of both newly developing cancers and pre-existing tumors were accelerated in the PPZ group as compared with the control group. CONCLUSION: Short-term, pregnancy levels of estradiol and progesterone (but not prolactin) may be the most effective treatment against MNU-induced mammary tumors in female Lewis rats.

Review: Animal models of N-Methyl-N-nitrosourea-induced mammary cancer and retinal degeneration with special emphasis on therapeutic trials.

N-Methyl-N-nitrosourea (MNU) is a direct-acting alkylating agent that interacts with DNA. Accumulation of mutations may enhance cancer risk in target organs or cause cell death in susceptible tissues or cells when excessive DNA damage is not repaired. MNU targets various organs in a variety of animal species. MNU-induced carcinogenesis can be used as organ-specific animal models for human cancer, and MNU has been most extensively utilized for the induction of mammary cancer in rats. MNU-induced rat mammary tumors possess many similarities to those of human breast cancer, and the model is utilized for screening cancer modulators. MNU-induced cell disruption is also seen in several organs and tissues, especially when MNU is applied before maturity. However, photoreceptor cells in adults are highly sensitive to MNU, which causes cell death due to apoptosis. MNU-induced photoreceptor apoptosis mimics human retinitis pigmentosa and can be used for studies of therapeutic intervention. In this review, the targets of MNU in various animal species are described, and special emphasis is given to therapeutic trials against MNU-induced mammary cancer and retinal degeneration in animal models.

Assessing binaural/bimodal advantages using auditory event-related potentials in subjects with cochlear implants.

OBJECTIVE: To assess the advantage of binaural and bimodal hearing for subjects with cochlear implant (CI) using auditory event-related potentials as well as speech perception tests. SUBJECTS AND METHODS: The subjects comprised four binaural CI users (CI/CI group) and eleven bimodal CI users, who wore a hearing aid (HA) contralaterally (CI/HA group). All subjects had used binaural or bimodal fitting for over 6 months. Their speech perception was examined in a quiet environment using monosyllabic words. Late cortical waves were measured while subjects were engaged in an oddball task of 1kHz frequent and 2kHz rare tone stimuli. The latencies of event-related potential (N1, N2, P3) were compared for monaural, binaural, and bimodal hearing conditions. RESULTS: Significantly (p<0.01, paired t-test) better speech perception for monosyllabic words was found for both binaural and bimodal hearing than monaural hearing. The latency of N1 did not significantly change for either binaural or bimodal hearing. On the other hand, the latency of N2 was significantly (p<0.01, paired t-test) shorter for binaural and bimodal hearing than for monaural hearing. The latency of P3 was shorter for binaural and bimodal hearing than monaural hearing in all subjects, and the difference was statistically significant in both CI/CI and CI/HA groups (p<0.01, paired t-test). CONCLUSIONS: Better speech perception was obtained for binaural and bimodal hearing than for monaural hearing in CI subjects. The results obtained in the comparison of P3 latency agreed with that of speech perception. Thus, using bilateral hearing devices is recommended for CI subjects. We also found that event-related potentials were useful as an objective tool to assess the advantage of binaural and bimodal hearing for CI subjects.

[Implantation of a vibratory mass transducer on the round window: a report of two cases].

The Vibrant Soundbridge (VSB, Med-El, Austria) is a semiimplantable hearing aid usually attached to the long process of the incus to vibrate the ossicular chain in patients with moderate to severe mixed hearing loss. We implanted a VSB vibratory transducer on the round window membrane of the left ear in two cases not treated effectively by tympanoplastic surgeries. Pure tone audiography did not differ significantly in the two cases pre- or postoperatively, indicating that the small mass transducer does not adversely affect middle-ear vibration. Postoperative hearing thresholds with VSBs were similar to those when patient 1 wore an air-conductive hearing aid and patient 2 wore a bone-anchored hearing aid (BAHA). VSB implantation on the round window could thus potentially benefit many patients with mixed hearing loss.

Auditory perception and speech production skills of children with cochlear implant assessed by means of questionnaire batteries.

OBJECTIVE: The aims of this study were to evaluate early auditory perception and speech production skills, and to compare the outcomes between two groups of children wearing the Nucleus cochlear implant (CI) and Clarion CI. METHODS: A total number of subjects were 68 prelingually deaf children who were implanted with a CI at our center, 31 subjects with the Nucleus CI (N-group), and 37 with the Clarion CI (C-group). The early time course of auditory perception and speech production skills were assessed by means of parental interviews using three kinds of questionnaires, the Listening in Progress (LIP) score, the Infant-Toddler Meaningful Auditory Integration Scale (IT-MAIS) and the Meaningful Use of Speech Scale (MUSS). RESULTS: The basement scores for all three test batteries before the use of CI were poorer for the C-group than the N-group. The scores improved rapidly over the initial 6 months and gradually reached a plateauover a 6- to 12-month period. The speech production skills evaluated by MUSS developed much more slowly and took over 3 years to reach a given level. CONCLUSIONS: Our pediatric CI users showed steady development of auditory perception and speech production skills after CI without any significant difference between the two groups.

A naphthoquinone derivative, shikonin, has insulin-like actions by inhibiting both phosphatase and tensin homolog deleted on chromosome 10 and tyrosine phosphatases.

The 1,4-naphthoquinone derivative, shikonin, has been shown to increase glucose uptake by adipocytes and myocytes with minor effects on protein tyrosine phosphorylation in the cells (Biochem Biophys Res Commun 292:642-651, 2002). The present study was performed to examine the mechanism of this action of shikonin. Shikonin inhibited the phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) phosphatase activity of recombinant phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with an IC50 value of 2.7 microM. Shikonin induced marked accumulation of PtdIns-3,4,5-P3 and activation of protein kinase B (PKB) in Chinese hamster ovary cells expressing insulin receptors. In addition to its effect on PTEN, shikonin was found to inhibit several protein phosphatases in cell-free systems. Its effect on tyrosine phosphorylation in intact cells was far weaker than that of pervanadate, a widely used tyrosine phosphatase inhibitor, despite the observation that the effect of shikonin on PKB was more potent than that of pervanadate. These results suggested that the inhibition of PTEN provides a clue to its potent insulin-like actions. We also found that naphthoquinones, including 1,2-naphthoquinone, inhibit PTEN in the cell-free system, which suggested that the effect on PTEN (and thus the effect on phosphatidylinositol 3-kinase signaling) should be taken into account when examining the pharmacological actions of naphthoquinone derivatives.

Factors relating to the vertigo control and hearing changes following intratympanic gentamicin for intractable Ménière's disease.

OBJECTIVE: To look for factors relating to the vertigo control and hearing changes after intratympanic injections of gentamicin (GM). STUDY DESIGN: Prospective. SETTING: Tertiary referral medical center. PATIENTS: Twenty-eight patients with intractable Ménière's disease. INTERVENTIONS: Three intratympanic injections of GM (once per day for three consecutive days). MAIN OUTCOME MEASURES: Although five patients needed further GM injections or vestibular neurectomy because of poor control (Group I), 23 patients had their vertigo controlled for more than two years without further treatment (Group II). The number of vertigo spells per month, pure-tone audiometry, electrocochleography, caloric response, post-head shake nystagmus, and plasma vasopressin as a stress marker were examined. RESULTS: Before GM injections, there was no difference in the number of vertigo spells per month between Groups I and II. However, the hearing thresholds were higher in Group I. Hearing improvement, increase in percentage of canal paresis and induction of post-head shake nystagmus were observed after GM injections only in Group II. Even in the 11 patients who showed an improvement in hearing of more than 10 dB (hearing improvement group), percentage of canal paresis was increased after GM. More, premedication plasma vasopressin levels were lower in the hearing improvement group as compared with the hearing loss/no changes group. Four of eight patients became negative for dominant negative summating potential in electrocochleography after GM injections in the hearing improvement group. CONCLUSION: Our data indicate that the frequency of vertigo is not a key factor in the vertigo control after GM injections, that induction of vestibular damage in the injected ear is essential for the control of vertigo and this effect is mostly pronounced in patients with milder hearing loss, and that hearing improvement is not only a consequence of good vertigo control but also affected by the stress level before treatment.

Activation of insulin receptors by lagerstroemin.

Lagerstroemin, an ellagitannin isolated from the leaves of Lagerstroemia speciosa (L.) Pers. (Lythraceae), was examined for its biological activities. In rat adipocytes, the compound increased the rate of glucose uptake and decreased the isoproterenol-induced glycerol release. In Chinese hamster ovary cells expressing human insulin receptors, it increased the Erk activity. These insulin-like actions were accompanied by the increased tyrosine-phosphorylation of the beta-subunit of the insulin receptors. Tryptic digestion of the extracellular sites of the insulin receptors markedly increased the effective concentrations of insulin without changing those of lagerstroemin. Thus lagerstroemin was considered to cause its insulin-like actions by a mechanism different from that employed by insulin.