Ultrastructure of early mineral deposition during hyaline layer formation in rat molars

There is no consensus on whether the first mineralized layer, the hyaline layer, that is juxtaposed to root dentine is a variety of dentine or cementum or even a tissue of epithelial origin. Some suggest that there is no intermediate tissue between the acellular extrinsic fibre cementum (AEFC) and the root dentine. Here, to study hyaline layer formation and mineralization we examined by transmission electron microscopy the early stages of root development in upper molars from 10 to 13 day old Wistar rats. In addition to conventionally processed material, undemineralized and unstained sections were examined, which showed the deposition of fine mineral crystals in contact with the mineralized surface of root dentine. Early mineralization of the hyaline layer occurred in the region of the inner basement membrane, which persisted between the inner cellular layer of Hertwig's epithelial root sheath and the outer mineralized root dentine. When the root sheath began its fragment, collagen fibrils From the developing periodontal ligament began to insert into the mineralising hyaline layer, which was 0.5-0.8 mum wide. As the fragmentation of the root sheath HERS increased, more collagen fibrils appeared intermingled with the mineralising hyaline layer. In more advanced stages, when the hyaline layer had become fully mineralized and the formation of the AEFC began, the hyaline layer could no longer be identified. Thus, the hyaline layer is clearly discernible at early stages of periodontal development. Subsequently, it is masked by intermingling of cementum and dentine and therefore it is not possible to detect it in the formed roots of rat molars. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Potentiation of panic-like behaviors of the rat by subconvulsive doses of strychnine

The present study was carried out to determine possible panicogenic effects of strychnine administered in subconvulsive doses to rats. Two experiments were conducted to assess two major features of panic in animal models: panic-related flight (through the observation of wild running [WR]) and defensive fights. In the first one, 20 adult male Wistar rats were injected with six different doses of strychnine ranging from 0.5 to 4.0 mg/kg. After 15 min of free observation, the animals were submitted to high-intensity acoustic stimulation and the incidence of WR was recorded. Higher doses of strychnine (above 2.5 mg/kg) easily evoked seizures, but lower doses raised the incidence of WR in a dose-dependent manner. The most effective dose for WR (1.5 mg/kg) was used in the second experiment, in which we investigated the effects of strychnine on sleep-deprivation-induced fights (SDIFs) that have defensive characteristics. For this purpose, 40 subjects were submitted to 5 days of REM-sleep deprivation by the single-platform method and were then assigned into two groups, i.e., strychnine vs. control. After the injections, the animals were observed in social groupings for SDIF recordings over a period of 60 min. The strychnine-treated groups had more SDIF than the control groups (P<.05, Mann-Whitney U test). We conclude that the high level of neural excitability promoted by partial blockade of the glycinergic system can contribute to the manifestation of panic reactions. (C) 2003 Elsevier B.V. All rights reserved.

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na+ and K+ gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CNI 100 (TM) electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity. (c) 2007 Elsevier Ltd. All rights reserved.

Longitudinal study of the spectral composition of behavioral rhythms in the rat

Four. male Wistar rats were housed in pairs of siblings, on LD 12:12h and 22 degrees C + 2 degrees C. Food and water were provided ad libitum. Behavior was videotaped from the 1st to the 3rd month of life. In each age-bracket the spectral composition of rhythmic expressions of the following behavioral categories was analyzed: rest, eat, drink, cage exploration, self-grooming, and social interaction. Rats maintained a stable rank order of time engaged in different behaviors through development, despite modification of time spent in grooming, drinking and social interaction as they got older. Spectral composition of behaviors followed a general ontogenetic pattern: ultradian frequencies of 12-h and 8-h were the strongest in the 1st month and circadian periodicity was predominant in the 3rd month. The increase of circadian power compared with ultradian power components agrees with literature findings. To our knowledge, self-grooming and social interaction have not been investigated before in this context. The similarities between siblings suggest the mutual influence of partners and/ or the existence of genetic factors. Ongoing studies are examining the importance of the social surroundings in which animals develop to the acquisition of adult rhythmic pattern.

Abnormal expression of bcl-2 and bax in rat tongue mucosa during the development of squamous cell carcinoma induced by 4-nitroquinoline 1-oxide

4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the expression of bcl-2 and bax during tongue carcinogenesis induced by 4NQO. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Although no histological changes were induced in the epithelium after 4 weeks of carcinogen exposure, bcl-2 and bax were over-expressed (P < 0.01) in all layers of the 'normal' epithelium. The expression levels were the same in all layers of epithelium for both the antibodies used (bcl-2 or bax). In dysplastic lesions at 12 weeks following carcinogen administration, the levels of bcl-2 and bax expression did not increase when compared to negative control with the immunoreactivity for bcl-2 being restricted to the superficial layer of epithelium. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, bcl-2 was expressed in some cells of tumour islands. on the other hand, immunostaining for bax was widely observed at the tumour nests. The labelling index for bcl-2 and bax showed an increase (P < 0.05) after only 4 weeks of 4NQO administration. In conclusion, our results suggest that abnormalities in the apoptosis pathways are associated with the development of persistent clones of mutated-epithelial cells in the oral mucosa. Bcl-2 and bax expression appears to be associated with a risk factor in the progression of oral cancer.

Myosin heavy chain expression and atrophy in rat skeletal muscle during transition from cardiac hypertrophy to heart failure

The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.

Diuron lacks promoting potential in a rat liver bioassay

The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

Background and Objectives: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis.Methods: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy.Results: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles - images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez.Conclusions: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Placental glutathione S-transferase correlates with cellular proliferation during rat tonguie carcinogenesis induced by 4-nitroquinoline 1-oxide

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure (p < 0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion. (c) 2007 Published by Elsevier GmbH.

Anti-inflammatory effects of low-level laser therapy (LLLT) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema

It has been suggested that low-level laser therapy (LLLT) can modulate inflammatory processes. The aim of this experiment was to investigate what effects red laser irradiation with two different wavelengths (660 nm and 684 nm) on carrageenan-induced rat paw edema and histology. Thirty two male Wistar rats were randomly divided into four groups. One group received a sterile saline injection, while inflammation was induced by a sub-plantar injection of carrageenan (1 mg/paw) in the three other groups. After 1 h, LLLT was administered to the paw in two of the carrageenan-injected groups. Continuous wave 660 nm and 684 nm red lasers respectively with mean optical outputs of 30 mW and doses of 7.5 J/cm(2) were used. The 660 nm and 684 nm laser groups developed significantly (P < 0.01) less edema (0.58 ml [SE +/- 0.17] ml and 0.76 ml [SE +/- 0.10] respectively) than the control group (1.67 ml [SE +/- 0.191) at 4 h after injections. Similarly, both laser groups showed a significantly lower number of inflammatory cells in the muscular and conjunctive sub-plantar tissues than the control group.We conclude that both 660 nm and 684 nm red wavelengths of LLLT are effective in reducing edema formation and inflammatory cell migration when a dose of 7.5 J/cm(2) is used. (c) 2007 Elsevier B.V. All rights reserved.

Ultrastructural and histochemical examination of alveolar bone at the pressure areas of rat molars submitted to continuous orthodontic force

It is usually believed that repair in alveolar bone during orthodontic movement occurs after decreasing of force. However, we have recently observed signs of repair in previously resorbed cementum from human teeth exposed to continuous forces. In order to test the hypothesis that bone resorption and deposition occur concomitantly at the pressure areas, a continuous 15 cN force was applied in a buccal direction to upper first molars from eight 2.5-month-old male Wistar rats for 3 d (n=4) and 7 d (n=4). As a control, two additional rats did not have their molars moved. Maxillae were fixed in 2% glutaraldehyde + 2.5% formaldehyde, under microwave irradiation, decalcified in ethylenediaminetetraacetic acid, and processed for transmission electron microscopy. Specimens from one rat from each group were processed for tartrate-resistant acid phosphatase (TRAP) histochemistry. At both the times studied, the alveolar bone surface at the pressure areas showed numerous TRAP-positive osteoclasts, which were apposed to resorption lacunae. In addition, osteoblasts with numerous synthesis organelles were present in the neighboring areas overlying an organic matrix. Thus, this study provides evidence that the application of continuous forces produces concomitant bone resorption and formation at the pressure areas in rat molars.

Effect of maternal obesity on diabetes development in adult rat offspring

This study aimed to evaluate whether maternal obesity leads to the onset of diabetes in adult Wistar rats offspring. MSG solution neonatally administration induced obesity in rats (F(1)MSG group, n = 30); and saline solution was also administrated to control rats (F1CON group, n = 13). In 3rd month of age, both control and MS G groups were mated for offspring (generation FA named as F2CON, n = 28 and F(2)MSG groups, n = 15; and so both generations were studied until 7th month of life. Lee Index was measured for experimental obesity validation from 5th to 7th month. Glycemia was weekly determined during pregnancy and monthly from 3rd to 7th month. In the end of experimental period all rats were submitted to oral glucose tolerance test (OGTT), with estimation of total area under the curve (AUC); and insulin tolerance test (ITT). Rats were then anesthetized and killed. Data were statistically analyzed with significance level of p < 0.05. Lee Index has confirmed obesity in all MSG rats. Glycemic levels comparisons between generations showed significant maternal interference in control and MSG groups. OGTT analysis showed higher glycemia in obese rats (F(1)MSG) and their offspring (F(2)MSG) as compared to their respective controls; and MSG groups increased AUC from OGTT. As regards ITT, F(2)MSG showed higher glycemia at 30 and 120 min, suggesting a delay of insulin action decreasing. Although glucose intolerance and insulin resistance clinical conditions represent as a factors for type 2 Diabetes mellitus development, this experimental model proposal was not efficient to induce type 2 Diabetes mellitus, but for obesity developing, glucose intolerance and insulin resistance in successive generations of rats. (c) 2007 Elsevier B.V. All rights reserved.

Effect of dipyrone, L-NAME and L-arginine on endotoxin-induced rat paw edema

Paw edema was induced in male Wistar rats (200-250 g) by intraplantar (ipl) administration of 2.5 mu g endotoxin (Etx). Etx, like carrageenin, produced two distinct edema formation phases, an early phase (75 min) followed by a late phase (7 h). We showed that the edema formation in the early phase was antagonized by dipyrone (80 mg/kg, ip) and indomethacin (1 mg/kg, ip) by 52% and 55%, respectively, and that the late phase was resistant to these drugs. These results suggest that in the early phase prostaglandins appear to be involved in the process. However, the activation of the kinin cascade leading to the release of other mediators may be involved in the increase of edema in the late phase. To test this hypothesis, we investigated whether the release of nitric oxide (NO) is involved in the mechanism of endotoxin-induced rat paw edema during the late phase, using N omega-nitro-L-arginine methyl ester (L-NAME) (50 mu g, ipl) as inhibitor of NO synthase and L-arginine (1 mg, ipl) as substrate of NO synthase. The paw edema induced by Etx was inhibited by L-NAME by 56% and increased by L-arginine by 81%. Furthermore, L-arginine given in combination with L-NAME completely reversed the inhibition of Etx-induced edema produced by L-NAME. These results support the hypothesis that in the late phase NO production is associated with the edema evoked by Etx.

Differential vascular adaptive response in spontaneously hypertensive and Wistar rats: Role of nitric oxide, and prehypertensive and hypertensive states

Stress-induced vascular adaptive response in SHR was investigated, focusing on the endothelium. Noradrenaline responses were studied in intact and denuded aortas from 6-week-old (prehypertensive) and 14-week-old (hypertensive) SHR and age-matched Wistar rats submitted or not to acute stress (20-min swimming and I-h immobilization 25 min apart), preceded or not by chronic stress (2 sessions 2 days apart of 1-h day immobilization for 5-consecutive days). Stress did not alter the reactivity of denuded aorta. Moreover, no alteration in the EC50 values was observed after stress exposure. In intact aortas, acute stress-induced hyporeactivity to noradrenaline similar between strains at both age. Chronic stress potentiated this adaptive response in 6- and 14-week-old Wistar but not in 6-week-old SHR, and did not alter the reactivity of 14-week-old SHR. Maximum response (g) in intact aortas [6-week-old: Wistar 3.25 +/- 0.12, Wistar/acute 1.95 +/- 0.12*, Wistar/chronic 1.36 +/- 0.21*(+), SHR 1.75 +/- 0.11, SHR/acute 0.88 +/- 0.08*, SHR/chronic 0.85 +/- 0.05*; 14-week-old: Wistar 3.83 +/- 0.13, Wistar/acute 2.72 +/- 0.13*, Wistar/chronic 1.91 +/- 0.19*', SHR 4.03 +/- 0.17, SHR/acute 2.26 +/- 0.12*, SHR/chronic 4.10 +/- 0.23; inside the same strain: *P < 0.05 relate to non-stressed rat, (+)P < 0.05 related to acute stressed rat; n = 6-18]. Independent of age and strain, L-NAME and endothelium removal abolished the stress-induced aorta hyporeactivity. Conclusion: the vascular adaptive response to stress is impaired in SHR, independently of the hypertensive state. Moreover, this vascular adaptive response is characterized by endothelial nitric oxide-system hyperactivity in both strains. (c) 2006 Elsevier B.V. All rights reserved.

Amifostine protective effect on cisplatin-treated rat testis

Cisplatin is a potent drug used in clinical oncology but causes spermatogenesis damage. Amifostine is a drug used against toxicity caused by ionizing irradiation and chemotherapeutic drugs. Since cisplatin provokes fertility and induces germ cell apoptosis and necrosis, we proposed to evaluate the amifostine cytoprotective action on testes of cisplatin-treated rats. Thirty-day-old prepubertal Wistar rats received a single cisplatin dose of 5 mg/kg and were killed after 3, 6, and 12 hr. The hematoxylin-eosin stained testicular sections were submitted to histological, morphometric, and stereological analysis. The terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) method was used to label apoptotic cells. TUNEL-positive and TUNEL-negative germ cells with abnormal nuclear morphology (ANM) were scored. Significant alterations of greater part of the parameters occurred in the cisplatin-treated group (CE) compared to the group that received amifostine before the cisplatin-treatment (ACE); however, testicular weight and volume did not vary between these groups. Tubular diameter was reduced in CE in comparison to ACE rats, while interstitial tissue and lymphatic space volume and volume density were significantly higher in CE rats; interstitial testicular edema probably occurred in cisplatin-treated rats. CE rats showed important histological alterations, which were more accentuated than in ACE rats. The numerical densities of apoptotic germ cells and TUNEL-negative cells with ANM were lower in ACE than in CE rats. In conclusion, the amifostine previously administered to prepubertal rats reduced the testicular damage caused by cisplatin. We conclude that amifostine partially protected the rat seminiferous epithelium against cisplatin toxicity.