Planarians are metazoan freshwater flatworms which are free-living organisms. Their body has pluripotent stem cell promoters of tissue regeneration capacity. The water temperature and the potential of hydrogen (pH) of lentic ecosystems are important factors involved in the distribution and abundance of these animals. Although the pH factor is directly related to the physiology and behavior of planarians, their adaptive and regenerating capacities still remain unknown. The Critical Thermal Maximum (CTM) is a very widespread method used in the evaluation of thermal tolerance. In this study, Girardia tigrina (Girard, 1850) and Girardia sp., a species found in Brazil, which is under study as a new species, had their epidermis assessed by scanning electron microscopy (SEM) to analyze their physiological structures before and after exposure to different stressors. SEM was used as a method to evaluate the planarians' epidermis as a result of the increasing temperature (CTM) and pH alterations, the latter with the use of a new methodology defined as Critical Hydrogen ion concentration Maximum (CHM). In increasing temperatures from 20°C to 37°C, both Girardia tigrina and Girardia sp. proved to be adaptable to thermal stress. Girardia sp. was shown to be more resistant to higher temperatures. However, Girardia tigrina was more resistant to extreme pH conditions (4.0 to 10.0). SEM analysis showed morphological differences among planarian species, such as the arrangement of the structures and cell types of the dorsal epidermis. Moreover, planarians demonstrated the ability to change the surrounding pH of their external environment in order to maintain the function of their physiological mechanisms, suggesting that these animals have a complex survival system, possibly related to protonephridia, flame cells and excretory pores..

The mechanism of regeneration does not start to restore the wound until its corresponding epimorphic phase. A bioestimulation of tissues and cells by laser radiation depends on the wavelength, on the dose, and on the intensity of the light. The goal of this work was to verify the effect of the low power laser at 660 nm on the regenerative process of Girardia tigrina. The specimens were maintained in the laboratory under a temperature ranging from 19° up to 24 °C for 21 days. The planarians were anesthetized by placing them on ice and then cut them with a scalpel. The three treatments were as following: animals individually irradiated with 14 sessions with 1 minute duration (treatment 1), 14 sessions with 3 minutes duration (treatment 2), and without irradiation (control). The planarians were amputated and divided in three study treatments: a control group (without radiation), and two other treatments: irradiated for 1 minute, and irradiated for 3 minutes. The animals were irradiated with diode laser (660 nm) with 3.3 ± 0.3 mW of power, using 0.94 mW.mm -2 power density for each irradiation procedure. During the experiment, 14 irradiation sessions were undertaken. The specimens were fixed in Bouin, and stained with hematoxyline and eosin. From observation and histological analysis, it was possible to assess the effects of interaction between laser and tissue. The head fragment after 1 minute of irradiation presented a better organized tissue scheme, when compared with the other treatments. Aspects of the body fragments submitted to 3 minutes of light treatment were very similar to fragments that had not been injured. It can be concluded that there are changes in the quality of regeneration when treated with low power laser under the conditions mentioned above.Keywords: regeneration, Girardia tigrina, laser, Dugesiidae, histology. Um estudo de laser de baixa potência no processo regenerativo deGirardia tigrina (Girard,1850) (Turbellaria; Tricladida; Dugesiidae) Resumo O mecanismo de regeneração não restaura a ferida até sua fase epimórfica correspondente. A bioestimulação de tecidos e células por radiação laser depende do comprimento de onda, da dose e intensidade de luz. O objetivo deste trabalho foi verificar o efeito do laser de baixa potência (660 nm) no processo regenerativo de Girardia tigrina. Estes vermes têm uma elevada capacidade de regeneração. Os espécimes foram mantidos em laboratório sob uma temperatura de 19° a 24 °C por 21 dias. As planárias foram anestesiadas com gelo e amputadas com bisturi. Os 3 tratamentos foram compostos de: animais individualmente irradiados com 14 sessões de 1 minuto de duração (tratamento 1), com 14 sessões de três minutos de duração (tratamento 2) e não irradiados (controle). Elas foram irradiadas com laser diodo (660 nm) 3,3 ± 0,3 mW potência, usando 0.94 mW.mm -2 de densidade de potência para cada irradiação. Durante o experimento foram realizadas 14 sessões. Os espécimes foram fixados em Bouin e corados com hematoxilina e eosina. Com base nas observações e análi...

Abstract. Tissue regeneration is widely studied due to its importance for understanding the biology of stem cells, aiming at their application in medicine for therapeutic and various other purposes. The establishment of experimental models is necessary, as certain invertebrates and vertebrates have different regeneration abilities depending on their taxon position on the evolutionary scale. Planarians are an efficacious in vivo model for stem cell biology, but the correlation between planarian cellular and molecular neoblast pluripotency mechanisms and those of mammalian stem cells is unknown. The present study had the following objectives: i) Establish Girardia tigrina cell culture, ii) determine the time required for complete cell disintegration and iii) obtain neoblasts by cell subdivision. Twenty-four specimens were deprived of food for seven days. After this time, disintegration was performed by incubation protected at three temperatures for 48 h in an antibiotic, antimycotic and trypsin solution, after which the suspension was homogenized and centrifuged. Histopaque ® 1077 was used for cell separation and interphases were collected and monitored by optical and fluorescence microscopy. Optical microscopy analysis informed the nucleus-to-cytoplasm ratio, cell morphology and cell size. Under fluorescence microscopy, interphase 1 (I1) was subdivided into two groups and neoblasts were marked for characterization; one group was stained with 4',6-diamidino-2-phenylindole and the other was immunolabeled with octamer-binding transcription factor 4 (OCT4) and isolated and observed after 10 days of cultivation. Neoblasts predominated in I1 with a small amount of other cell types. In conclusion, sample disintegration with a trypsin and antibiotic solution was effective at 18˚C and Iscove's modified Dulbecco's medium supplemented with fetal bovine serum was adequate for the establishment of primary cell cultures after 48-h incubation and centrifugation. Antibody anti-OCT4 was used for the characterization of stem cells and was successfully labeled with concentrated neoblasts on interphase 1. IntroductionRegeneration is a complex event that occurs in several vertebrates and invertebrates (1). For regeneration to occur, one of the earliest signaling events following a lesion is the production of cells that are capable of rebuilding lost structures. The way that these events occur and the types of cells involved differ between animal groups (2).The choice of planarians as a regeneration study model is based on their plasticity, regeneration ability, rapid response and adult stem cell reservoir (3). Cell and molecular characterization of planarian stem cells has been achieved with various tools, such as inhibition of the function of genes involved in regeneration via RNA interference, western blotting, in situ hybridization and cell isolation by flow cytometry (4-8). These tools are extremely important alternatives for understanding the mechanisms associated with the maintenance and role of stem cells in vivo (9).Stem cell...

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