Doxycycline reduces liver and kidney injuries in a rat hemorrhagic shock model.

BACKGROUND: Hemorrhagic shock (HS), which causes insufficient tissue perfusion, can result in multiple organ failure (MOF) and death. This study aimed to evaluate whether doxycycline (DOX) protects cardiovascular, kidney, and liver tissue from damage in a rat model of HS. Immediately before the resuscitation, DOX (10 mg/kg; i.v.) was administered, and its protective effects were assessed 24 h later. Mean arterial pressure, renal blood flow, heart rate, vasoactive drug response, and blood markers such as urea, creatinine, AST, ALT, CPK, CPR, and NOx levels were determined. RESULTS: We showed that DOX has a significant effect on renal blood flow and on urea, creatinine, AST, ALT, CPK, and NOx. Morphologically, DOX reduced the inflammatory process in the liver tissue. CONCLUSIONS: We conclude that DOX protects the liver and kidney against injury and dysfunction in a HS model and could be a strategy to reduce organ damage associated with ischemia-and-reperfusion injury.

MT1-MMP and TIMP-2 are first expressed in the colon glands after a single dose of azoxymethane (AOM).

Tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane-type 1-matrix metalloproteinase (MT1-MMP) are always expressed during the cancer process. The aim was to identify which regions of the colon mucosa MT1-MMP and TIMP-2 begin to express themselves, as well as to establish their expression in relation to cell proliferation and mucin production. After intraperitoneal injection of 15 mg/kg of azoxymethane (AOM) at 4, 12, and 20 weeks, histological sections of the middle segment of the rat colon mucosa were evaluated by immunohistochemistry for cell proliferation and expression of MT1-MMP and TIMP-2 and histochemistry for mucin. As a result, a single dose of AOM initially increased the intensity of MT1-MMP and TIMP-2 expression in the conjunctive cells and glands, concurrently with alterations in the distribution of the mucin produced in the gland of the large intestine mucosa and cell proliferation. As a result, at 4 and 12 weeks, a single dose of AOM initially stimulated the expression of MT1-MMP and TIMP-2 in the conjunctive cells and glands with greater intensity. Changes in the cell proliferation and distribution of the mucin produced in the large intestine mucosa gland were observed. We conclude that MT1-MMP and TIMP-2 were first and strongly expressed in all cells of the colon glands, concurrently with an increase in cell proliferation and a diffuse dispersion of mucin, indicating the onset of the dysplasia process following a single dosage of AOM.

Effects of obesity on the rat incisor enamel and dentine thickness, as well as on the hemimandible shape over generations.

Obesity has several effects on the general body metabolism. However, little is known about the impact of obesity on the growth and shape of mineralized tissues like mandibles and teeth, as well as if it effects are passed down from generation to next. Therefore, in this study, we aimed to evaluate, over nine generations using the consanguineous mating (inbreeding), the effect of the obesity condition produced by the reduction in the number of rats per litter during the lactation period on the hemimandible shape, dentine, and enamel of the rat incisor. Litters were reduced to two males and two females after birth, and were consanguinity mated in adulthood for nine generations. For all evaluations performed in this investigation, only males were used. The control group was formed by a non-consanguineous litter containing eight males. The parameters evaluated were food consumption, body weight, Lee Index, and bone density of the hemimandible bone. Incisor enamel and dentine thickness were also evaluated. The hemimandible shape was evaluated using geometric morphometry. The results show a significant and progressive increase in food intake, Lee Index, body weight, hemimandible weight, and enamel thickness, and a decrease in dentine thickness. The linear measurements of the length of the ramus ascending hemimandibular segment were found to be shorter, while its height was increased. In contrast, the geometric morphometry shows that the general hemimandible shape changed over the consanguineous obesity generations. We conclude that over generations, obesity increases and maintains the parameters evaluated with significant changes in hemimandible shape as well as in the dimensions of enamel and dentine of incisors, suggesting that enamel and dentine could be used as phenotype biomarkers to detect changes in tooth and craniofacial development related to obesity effects.

Effects of a Single Dose of X-Ray Irradiation on MMP-9 Expression and Morphology of the Cerebellum Cortex of Adult Rats.

Although radiation is a strategy widely used to inhibit cancer progression, which includes those of the neck and head, there are still few experimental reports on radiation effects in the cerebellum, particularly on the morphology of its cortex layers and on the Matrix metalloproteinases' (MMPs') expression, which, recently, seems to be involved in the progression of some mental disorders. Therefore, in the present study, we evaluated the morphology of the cerebellum close to the expression of MMP-9 from 4 up to 60 days after a 15-Gy X-ray single dose of X-ray irradiation had been applied to the heads of healthy adult male rats. The cerebellum of the control and irradiated groups was submitted for an analysis of cell Purkinje count, nuclear perimeter, and chromatin density using morphometric estimatives obtained from the Feulgen histochemistry reaction. In addition, immunolocalization and estimative for MMP-9 expression were determined in the cerebellar cortex on days 4, 9, 14, 25, and 60 after the irradiation procedure. Results demonstrated that irradiation produced a significant reduction in the total number of Purkinje cells and a reduction in their nuclear perimeter, along with an increase in chromatin condensation and visible nuclear fragmentation, which was also detected in the granular layer. MMP-9 expression was significantly increased on 4, 9, and 14 days, being detected around the Purkinje cells and in parallel fibres at the molecular layer. We conclude that the effects of a single dose of 15-Gy X-ray irradiation in the cerebellum were an increase in MMP-9 expression in the first 2 weeks after irradiation, especially surrounding the Purkinje cells and in the molecular layers, with morphological changes in the Purkinje cell and granular cell layers, suggesting a continuous cell loss throughout the days evaluated after irradiation.

Evaluation of the inflammatory process, collagen production, and MMP-2 and MMP-9 expressions produced by Luffa aegyptiaca Mill using the subcutaneous rat implanted model.

The subcutaneous rat implanted model is a preclinical approach used in studies to characterize the histocompatibility of materials that could be used as biomaterials. Biomaterials are obtained synthetically or from the environment, and they can be used to treat or replace any tissues or organs that the body has lost. To execute their roles, the biomaterials must present any level of histocompatibility and a lower level of inflammatory reaction. This work aimed to evaluate some aspects of histocompatibility, such as the inflammatory process, collagen production, and MMP-2 and 9 expression as responses to the Luffa aegyptiaca Mill using the subcutaneous rat implanted model. Luffa fragments were implanted into the dorsal subcutaneous region of twelve male Wistar rats, and the number of eosinophils, mast cells, the production of collagen to form the fibrous capsule, and the expression of MMP-2 and MMP-9 were evaluated on the 15th, 45th, and 90th days. Results showed statistical differences (p < 0.05) in the number of eosinophils and mast cells present inside and outside the fibrous capsule among the days evaluated. The permanent presence of macrophages and giant foreign body cells circumjacent to all implants was also observed. A progressive increase in the production of collagen was also detected, along with a significant reduction on day 90 (p < 0.05). The expression of MMP-9 was detected as being specifically expressed in the giant foreign body cells on all days evaluated, while the expression of MMP-2 was detected in fat cells present around the implants, mainly on day 90. Taken together, these results indicate a general reduction level for the inflammatory process during the days evaluated, which allows us to conclude that Luffa, being a natural product that is simple to obtain, could be a potential candidate to become a biomaterial to be tested in further approaches.

Initial damage produced by a single 15-Gy x-ray irradiation to the rat calvaria skin.

BACKGROUND: Calvaria skin has a reduced thickness, and its initial damage produced by irradiation was scarcely reported. We aimed to identify the initial effects of x-ray irradiation in the rat calvaria skin. METHODS: After approval by the Animal Ethical Committee, calvaria skin sections of five Wistar rats per time point were evaluated on days 4, 9, 14, and 25 following a single 15-Gy x-ray irradiation of the head. The control group was composed of five rats and evaluated on day 4. Sections were assessed using hematoxylin-eosin and Masson's trichrome staining for morphology, inflammation, and fibrosis. Fibrosis was also evaluated by the collagen maturation index from Picrosirius red staining and by cell proliferation using the immunohistochemistry, after 5-bromo-2-deoxyuridine intraperitoneal injection. RESULTS: In irradiated rats, we observed a reduction in epithelial cell proliferation (p = 0.004) and in matrix metalloproteinase-9 expression (p < 0.001), an increase in the maturation index, and with a predominance in the type I collagen fibers, on days 9 and 14 (1.19 and 1.17, respectively). A progressive disorganization in the morphology of the collagen fibers at all time points and changes in morphology of the sebaceous gland cells and hair follicle were present until day 14. CONCLUSIONS: The initial damage produced by a single 15-Gy x-ray irradiation to the rat calvaria skin was a change in the normal morphology of collagen fibers to an amorphous aspect, a temporary absence of the sebaceous gland and hair follicles, and without a visible inflammatory process, cell proliferation, or fibrosis process in the dermis.

Expression of the matrix metalloproteinases 2 and 9 in the rat small intestine during intrauterine and postnatal life.

The expressions of matrix metalloproteinases 2 and 9 have been described during the development, as an example in heart and tooth but not in the small intestine yet. In this context, this study aimed to evaluate the expressions of MMP-2 and MMP-9 in the small intestine of Wistar rats during intrauterine (IU) and postnatal (PN) life. Expressions were determined on the 15th and 18th days of IU life and the 3rd, 10th, 17th, 25th, and 32nd days of PN life. Intestinal samples obtained from six animals were submitted to zymography, immunohistochemistry, and staining with Masson's trichrome. The results showed that MMP-2 and MMP-9 were not expressed during IU life; however, after birth, MMP-9 was immunolocalized in the goblet and mast cells. In conclusion, our results showed that MMP-2 and MMP-9 were not expressed in absorptive epithelial cells during the IU period of the small intestine but after birth, MMP-9 was expressed in the goblet cells, and mast cells present in the lamina propria.

Goblet cells and intestinal Alkaline phosphatase expression (IAP) during the development of the rat small intestine.

This study aimed to evaluate the temporal and spacial distribution of the mucins produced by goblet cells and intestinal alkaline phosphatase (IAP) expression during the development of the small intestine of the rat. Intestines were removed from rats on the 15th, 17th and 18th days of intratuterine life (i.u.) and on the 3rd, 10th, 17th and 25th days after birth (a.b.). Intestines were processed for routine histological procedures and sections were submitted to histochemistry using PAS to stain neutral glycoproteins and Alcian blue for acidic glycoproteins, as well as immunohistochemistry to detect IAP. In rats, glycoprotein production was seen to begin in the intestinal epithelium cell at around the 17th day of i.u. life; however, this production was not accompanied by morphological indications of the presence of goblet cells. By the 18th i.u. day, the villus epithelium was undergoing differentiation and the first goblet cells could be identified from this time. At around the 10th day a.b., both compartments of the small intestine were detected; i.e. the villi and the crypts. At this timepoint, goblet cells were present in the villi, and also in the upper regions of the crypts. On the 3rd, 10th 17th and 25th days a.b., the presence of the goblet cells increased and presented regional differences in the sections evaluated. IAP was not detected during i.u. life, but was weakly detected in the cells of the villi from the 3rd day a.b., along the entire extension of the villi. On the 10th day, IAP was detected at the tip of the villi, while on the 25th day, it was detected along the extension of the villi, but with a weaker intensity. In conclusion, a temporal and spacial distribution of goblet cells and IAP activity occurs during the development of the small intestine, suggesting a possible regulatory control in accordance with the suckling and weaning phases of food intake in the rat's life.

Human immature dental pulp stem cells (hIDPSCs), their application to cell therapy and bioengineering: an analysis by systematic revision of the last decade of literature.

During recent years, attention has been given to the potential of therapeutic approaches using stem cells obtained from dental pulp tissue. The aim of this study, therefore, was to give an overview of the papers produced during the last 10 years that have described the use of stem cells obtained from human deciduous teeth in cell therapy or bioengineering. The PubMed database was investigated from January 2002 until July 2011 and the papers published during this period were analyzed according to criteria previously established, using the methodology of systematic review. The measurements were done using "stem cell" as the primary keyword, and "human deciduous teeth dental pulp cell" and "human exfoliated deciduous teeth" as the secondary keywords. Four hundred and seventy-five papers were found. The first screening resulted in 276 papers, from which 84 papers were selected. However, only 11 of them attained the aim proposed in our approach. There were few scientific studies related to direct therapeutic application using stem cells of human deciduous teeth and none of them had been applied to humans. However, the results indicated important and promising applications of the pulp stem-cells in cell therapy and bioengineering as demonstrated by studies in animal models of muscular dystrophy, Parkison's disease, and lupus erythematosus.

MT1-MMP expression in the odontogenic region of rat incisors undergoing interrupted eruption.

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.

Immunolocalization and activity of the MMP-9 and MMP-2 in odontogenic region of the rat incisor tooth after post shortening procedure.

MMP-9 and MMP-2 are metalloproteinases which degrade the denatured collagen fibers. However, there is no report about roles of these MMPs in the odontogenic region of the adult rat incisor tooth under different eruption conditions. Male Wistar rats were divided in a normofunctional group (NF) in which their lower teeth remained in a normal eruption. In a hypofunctional group (HP) rats underwent shortening of their lower left incisor tooth every 2 days during 12 days. The eruption rate as well as the expression and activities of MMP-9 and MMP-2 were evaluated using imunohistochemistry and zymography. Although the shortening increased the eruption rate, no changes in the MMP-9 and MMP-2 were observed. We conclude that in adult rats, in opposite to development of tooth, the MMP-9 and MMP-2 present in the odontogenic region does not seem to play a direct role in the remodeling matrix, even after post-shortening procedures which to lead an acceleration of the eruption process in the incisor.

Influence of different decalcifying agents on EGF and EGFR immunostaining.

This study was designed to verify the influence of three demineralizing agents on EGF and EGFR immunostaining as well as on tissue morphology. We chose submandibular glands that are a source of EGF and its receptor and which could be analyzed using a control in which the decalcification step was not carried out. After sacrifice of adult male Wistar rats by perfusion fixation, the submandibular glands and mandibles were excised and placed together in each of the following solutions: (a) 5% nitric acid in 4% formaldehyde; (b) 4.13% EDTA pH 7.4; (c) 5% trichloroacetic acid. Mandibles served as a parameter for decalcification time in each demineralizing solution. A control group was performed with submandibular glands that were not placed in any demineralizing solution. After mandibles were completely decalcified, glands were processed by embedding in Paraplast® and immunohistochemical staining was made to detect EGF and EGFR. It was observed that decalcification did not produce noticeable differences in terms of EGF and EGFR immunoreactivity, but had an effect on the quality of the morphology and staining. Our results indicate there is no problem performing immunostaining of EGF and EGFR in tissues that require decalcification. 4.13% EDTA (pH 7.4) is the best choice for decalcification in cases that are not urgent.

Increase of MT1-MMP, TIMP-2 and Ki-67 proteins in the odontogenic region of the rat incisor post-shortening procedure.

MT1-MMP and TIMP-2 are well known for their roles in remodelling of extracellular matrix components. However, reports are emerging on the involvement of these molecules in cell kinetics. In the rat incisor tooth, a shortening treatment increases the eruption and cell proliferation rates. However, the role of MT1-MMP and TIMP-2 proteins in these processes is still to be evaluated. Male Wistar rats were divided in two groups. In the normofunctional group (NF) the lower teeth of the rats remained in a normal eruption process. In the hypofunctional group (HP) rats their lower left incisor tooth was shortened every 2 days during 12 days. The eruption rate was estimated during the shortening period and MT1-MMP, TIMP-2 and Ki-67 protein expression from the odontogenic region was measured after the treatment. In HP groups an increase in eruption rate, and in MT1-MMP/TIMP-2 and Ki-67 expression were observed. We conclude that there is a relationship between the increase in eruption rate, and in levels of MT1-MMP, TIMP-2 and Ki-67 in the HP group. This suggests that MT1-MMP and TIMP-2 may have some role in cell proliferation during the eruption of the rat incisor tooth.

Effects of fasting at different stages of lighting regimen on the proliferation of jejunal epithelial cells during rat pup weaning.

The lifespan of intestinal epithelial cells is predetermined by the process of cell proliferation that occurs constantly in the crypt. The control of this process involves some endogenous factors, such as hormones, as well as exogenous factors, like food and natural light variations. These last two exogenous factors seem to be the major modulators of the cell proliferation process. Fasting treatment was conducted to assess the role of food and its effect on the metaphase index (MI) of the intestinal epithelium at different times and periods (light and dark) of the day. The effects of short- (5 hr) and long-term (25 hr) fasting on the MI in the jejunal epithelium of young rats were investigated at 09:00 h, 15:00 hr, 21:00 hr, and 02:00 hr using the arrested metaphases method. The present study demonstrates that 5 hr and 25 hr of fasting treatment decrease the MI at 09:00 hr. It was observed from MI analysis that there is an interaction between the fed/fasted status of the animal and the different times of the day. This result suggests that during the transition from youth to adulthood, the control of MI by the light/dark cycle seems to be more pronounced as compared with control by food intake at some periods of the day, although at other times food had a greater impact on the MI.