Establishment of a rapid method for quality assessment of cardiotonic effect of Shengfupian / 药学学报

Based on the similar structure of adrenaline shared by higenamine (HI), salsolinol (SA) and coryneine (CO), a photochemical colorimetric sensor based on the displacement reaction of o-diphenol hydroxyl group and alizarin red S-phenylboric acid system was constructed to quickly distinguish and identify the cardiac strength of Shengfupian. The results show that the optimal condition of the sensor is: the molar ratio of alizarin red S (ARS) to phenylboric acid (PA) is 1∶3, reaction temperature is 0 ℃; The preparation method of the sample solution is optimized as follows: 2.5 g of Shengfupian powder was taken, 10 times the amount of methanol was added, and 300 W, 40 kHz ultrasound was carried out for 15 min; methodological studies showed that the method had good precision, repeatability and stability. The |△G| value (G is green, |△G| = |G after - G before|) of each sample was obtained by response values determination of 14 batches of Shengfupian. LC-MS/MS was used to determine the contents of three cardiac components in Shengfupian. It was found that the order of the total contents of cardiotonic components was basically consistent with |△G|. Then the correlation was analyzed, and the correlation coefficient R2 was as high as 0.87, which proved the scientificity and accuracy of this method. This study fills the methodological gap of rapid evaluation of the quality of Shengfupian, and provides the key technical support for the high quality and good price of Shengfupian in the market circulation and clinical application.

Selective Separation and Preconcentration of Caffeine from Natural and Pharmaceutical Products using New Polyurethane Foams

Abstract Two polyurethane foam-based sorbents (PUF) were synthesized by imprinting and grafting techniques and examined for selective separation and preconcentration of caffeine (CAF) in some pharmaceutical products and in black tea. Molecularly imprinted PUF was synthesized based on hydrogen-bonding interactions between CAF and alizarin yellow G (AYG) and subsequent polymerization into PUF. The static experiments indicated optimum sorption conditions at pH=6.5 and 5.5 for imprinted PUF (AY-IPUF) and grafted PUF (AY-GPUF), respectively. In the online experiments, the suitable preconcentration time was found to be 40 and 20s for (AY-IPUF) and (AY-GPUF), respectively, at a flow rate of 1.75 mL.min-1. Desorption of CAF has been affected by passing 500 µL of 0.05, 0.01 mol.L−1 HCl eluent onto (AY-IPUF) and (AY-GPUF), respectively. The online methods have provided satisfactory enrichment factors of 8.4 and 10.5 for (AY-IPUF) and (AY-GPUF), respectively. The time consumed for preconcentartion, elution and determination steps was 1.48 and 1.05 min, thus, the throughput was 42 and 57 h-1, for (AY-IPUF) and (AY-GPUF), respectively. The developed sorbents were studied for the determination of CAF in pharmaceutical samples which will be helpful to minimize caffeinism. Finally, in silico bioactivity, ADMET and drug-likeness predictive computational studies of caffeine were also carried out

Effect Of Synthetic Red Dye Orange Red And Natural Red Dye Alizarin On Biochemical And Hematological Parameters In Male Wistar Rats

Nowadays synthetic food dyes are mostly preferred than natural plant derived dyes due to low cost and intense coloration. In this study hematological and biochemical parameters were determined in male wistar rats after 30 days treatment with synthetic red dye orange red and natural plant derived red dye alizarin. 25 male wistar rats were divided into 5 groups with 5 animals per group. Group I rats were taken as control treated with normal rat diet and distilled water. Group II and III rats (experimental) were oral gavaged with 50 mg and 150 mg/kg body weight of alizarin dye. Group IV and V rats (experimental) were gavaged with 50 mg and 150 mg/kg body weight of orange red dye. Treatment of group V rats with 150 mg/kg body weight of orange red dye produce significant changes in RBC, Hb, Hct, MCH, serum aminotransferase enzymes and serum protein fraction. In comparison to this in group IV rats a significant change was observed only in Hb, serum aminotransferase enzymes and serum protein fraction when compared with control (group I) rats. However in group II and III alizarin treated rats no significant change was observed in different biochemical and hematological parameters relative to their respective control. In conclusion synthetic orange red dye proved to be more toxic than natural plant derived red dye alizarin.

Effects of atracurium besylate on corneal endothelium of chickens: in vitro study / Avaliação dos efeitos do besilato de atracúrio no endotélio corneano de galinhas: estudo in vitro

The aim of this study was to investigate the acute effects of atracurium besylate on cellular damage in corneal endothelium of chickens. Twenty healthy chicken eyes were assigned to the following groups: Group 1 (G1), experimental group (n=10); and Group 2 (G2), control (n=10). Excised corneoscleral buttons were immediately placed on glass microscopy slides with endothelial region faced up. Corneal endothelium of eyes in G1 were covered with AB (0.2mL, 10mg/mL) for 3 min and then rinsed with balanced salt solution (BSS), while the corneal endothelium of eyes in G2 were covered with BBS for 3 min. Corneas from both groups were stained with alizarin red/trypan blue and visualized by light microscopy. Ten random photographs were taken from each cornea. The area of cellular damage was measured by software in all samples and cell loss of each group was averaged and compared. Endothelial area of denudation and Descemet's membrane exposure were higher in G1 than G2. In conclusion, atracurium besylate induced an acute damage on corneal endothelium of chickens.(AU)'

Objetivou-se avaliar os efeitos agudos do besilato de atracúrio sobre o endotélio corneano de galinhas. Vinte olhos saudáveis de galinhas foram aleatoriamente separados em dois grupos com 10 olhos cada, sendo G1 o grupo controle e G2 o grupo tratamento. Imediatamente após a excisão dos botões corneoesclerais estes foram colocados em lâminas de microscopia de vidro com o lado endotelial voltado para cima. No Grupo 1, o endotélio corneano foi recoberto com 0,2ml de besilato de atracúrio (10mg/ml) durante 3 minutos e depois lavado com solução salina balanceada. No Grupo 2, o endotélio corneano foi recoberto apenas com solução salina balanceada durante 3 min. As córneas de ambos os grupos foram coradas com vermelho de alizarina e azul de tripano e visualizadas com microscópio óptico. Foram obtidas dez fotografias aleatórias de cada amostra. As imagens foram analisadas e com auxílio de um software as áreas com ausência de células endoteliais calculadas. A perda celular endotelial foi significativamente maior no grupo tratamento comparativamente ao grupo controle. Com base nos resultados apresentados foi possível concluir que o besilato de atracúrio induziu dano agudo nas células do endotélio da córnea de galinhas.(AU)

Alizarin Red-S Protocol for Skeletal Staining during Fetal Period in Rabbit

Background: The main purpose of this study was to give detailed information on the staining protocol of Alizarin Red-S to detect the normal and abnormal skeleton of rabbit fetus. Methods: Eleven (9 females and 2 males) of adult rabbit weighing 3-3.5 kg were used. The female rabbits were left with buck to become pregnant then were classified into a treatment and a control group. The former group received oral doses of 400mg/kg of sodium valproate for 15 days starting from the 6th day after mating until 20th day of pregnancy, while the second received water in the same period. The pregnant rabbit was slaughtered at the 29th day of pregnancy. The live rabbit fetuses were collected. The staining protocol included fixation, dehydration, clearing, staining and preservation. The fetuses were examined under dissecting microscope and photos were taken for documentation. Results: The staining protocol made the rabbit fetuses to be clear enough to see their skeleton through the surrounding tissue. The axial skeleton including skull with mandible, vertebral column, ribs and sternum and the appendicular skeleton including the bones of the fore-and hindlimbs took the stain and became red in color. The macroscopic skeletal disorders of the fetuses of the treatment group were observed. The ossification centres were assessed. Conclusion: This protocol which depended on fixation by 95% ethanol, clearing by 1% potassium hydroxide and staining by 0.001 % Alizarin Red-S was effective in detecting normal and abnormal fetal skeletal morphology

Morphology of endothelial cells from different regions of the cornea of dogs / Morfologia das células endoteliais de diferentes regiões da córnea de cães

ABSTRACT: The aim of this study was to evaluate the morphology of endothelial cells from different areas of the cornea of dogs. Twenty healthy eyes from 10 dogs, females or males, of different ages were studied. Corneal endothelium morphology of superior, inferior, central, nasal and temporal areas was assessed by 0.2% alizarin red staining using an optic microscope. One hundred endothelial cells from each corneal area were analyzed. In all areas of the cornea studied were found endothelial cells with four sides, five sides, six sides and seven sides. There was no significant difference regarding endothelial cell morphology in all corneal regions evaluated. Thus, the morphology of the central cornea area represents the entire endothelial mosaic and may be applied to peripheral areas. Therefore, analysis of the central area is sufficient to estimate the shape of endothelial cells of peripheral areas of healthy dog corneas.

RESUMO: Objetivou-se avaliar a morfologia das células endoteliais de diferentes regiões da córnea de cães. Vinte olhos saudáveis de 10 cães, fêmeas ou machos, de diferentes idades foram estudados. A morfologia do endotélio corneano das regiões superior, inferior, central, nasal e temporal foi avaliada pela coloração vermelho de alizarina 0,2% com microscópio óptico. Foram analisadas 100 células endoteliais de cada região da córnea. Em todas as regiões da córnea estudadas foram encontradas células endoteliais com quatro lados, cinco lados, seis lados e sete lados. Não houve diferença significativa em relação à morfologia de células endoteliais da córnea em todos as regiões estudadas. Assim, a morfologia da região central da córnea representa todo o mosaico endotelial e pode ser aplicada em áreas periféricas. Portanto, a análise da área central é suficiente para estimar a forma das células endoteliais das áreas periféricas de córneas de cães saudáveis.

Alizarin induced the function of beta subunits of BKCa channel relaxed interlobar renal artery and reduced SHR systolic pressure / 实用医学杂志

Objective To investigate the effect of alizarin on BKCa channel beta subunit and electrophysiological characteristics in interlobar renal artery of SHR.Methods Tail artery pressure measurement instrument,pressure myograph system,whole cell patch clamp technique and Western blot were used to measure the change of systolic pressure of SHR,renal interlobar arterial diameter,single vascular smooth muscle cell electrophysiological characteristics and expression of the BKCa channel beta subunit before and after alizarin intervention,respectively.Results (1) SHR systolic pressure was decreased after alizarin intervention (P ＜ 0.01).(2) ibTX inhibited alizarin-mediated renal interlobar arterial relaxation (P ＜ 0.01).(3) Alizarin enhanced BKCa channel-mediated outward currents (P ＜ 0.05).(4) Alizarin up-regulated BKCa channel beta subunits after alizarin intervention (P ＜ 0.01).Conclusion Alizarin reduced SHR systolic pressure and relaxed interlobar renal artery by enhancement BKCa currents via mediation of the function of beta subunits.

Efficacy of Human Platelet Rich Fibrin Exudate vs Fetal Bovine Serum on Proliferation and Differentiation of Dental Pulp Stem Cells

BACKGROUND AND OBJECTIVES: The imperative role of dental pulp stem cells (DPSCs) in regenerative therapy demands an in-vitro expansion which must deal with the safety and ethical problems associated with fetal bovine serum (FBS). The primary aim of this study was to compare the effects of human platelet rich fibrin (hPRF) exudate Vs FBS on proliferation and osteodifferentiation of human dental pulp stem cells (hDPSCs). The secondary one was to determine the optimum concentration of hPRF exudate inducing hDPSCs proliferation and osteodifferentiation. METHODS: The direct method was used to prepare hPRF exudate. hDPSCs were isolated from impacted mandibular third molars of twelve donors by the outgrowth method. For cell viability and proliferation rate testing, 96 well plates were used and the assay was done in duplicate and the trial repeated four times under the same conditions. Six wells were used to contain 10% FBS, serum free media, 1%, 5%, 10% and 20% concentrations of hPRF exudates, respectively. The proliferation assay was carried out by MTS tetrazolium cell proliferation assay kit and Elisa reader. The study design for osteodifferentiation protocol was exactly as the proliferation one and instead the assay was carried out by alizarin red with Elisa reader. RESULTS: Compared to 10% FBS, 10% hPRF exudate was the optimum concentration for hDPSCs proliferation, while 1% hPRF exudate was the optimum concentration for osteodifferentiation of hDPSCs. CONCLUSIONS: Avoiding the risk of zoonosis which may be occurred with FBS, it is recommended to use 10% hPRF exudate for proliferation and 1% for osteodifferentiation.

Morphology of endothelial cells from different regions of the equine cornea / Morfologia das células do endotélio de diferentes regiões da córnea de equinos

ABSTRACT: The objective of this study was to evaluate the morphology of different regions of the equine cornea using optical microscopy. Both healthy eyes of eight horses, male or female, of different ages were evaluated. Corneas were stained with alizarin red vital dye and subsequently examined and photographed using optical microscopy. Corneal endothelial morphology of central, superior, inferior, temporal and nasal areas was assessed. One hundred endothelial cells from each corneal area were analyzed. The shape of the corneal endothelial cells of each corneal region was analyzed. Statistical data analysis was conducted using the Student's t test. Values of P<0.01 were considered significant. Regarding morphological analysis, no statistically significant differences were reported between the equine corneal regions analyzed. The present research suggested that there are no morphological differences between regions of the equine cornea. The values obtained in any region of the equine cornea can be extrapolated to other regions of the cornea and are representative of the cell morphology present in all regions of the cornea.

RESUMO: O objetivo deste estudo foi o de avaliar a morfologia das diferentes regiões da córnea equina usando microscopia óptica. Foram avaliados ambos os olhos saudáveis ​​de oito equinos, machos ou fêmeas, de diferentes idades. As córneas foram coradas com corante vital vermelho de alizarina, examinadas com microscópio óptico e fotografadas. A morfologia endotélio corneano de áreas centrais, superior, inferior, temporal e nasal foi avaliada. Foram analisadas ​​células endoteliais da córnea de cada área. A forma das células endoteliais da córnea de cada região da córnea foi analisada. Análise estatística dos dados foi realizada por meio do teste t. Valores de P<0.01 foram considerados significativos. Em relação à análise morfológica estatisticamente significativa, não foram encontradas diferenças entre as regiões da córnea equina analisadas. O presente trabalho sugere que não houve diferença entre a morfologia das regiões da córnea de equino. Os valores obtidos em qualquer região da córnea equina podem ser extrapolados para outras regiões da córnea e são representativos da morfologia celular em todas as regiões da córnea.

Alizarin activates large-conductance Ca2+-activated K+ channels on renal interlobar arterial vascular smooth muscle cells in Wistar rat / 中国药理学通报

Aim To investage the regulatory effect of alizarin on renal interlobar arterial smooth muscle cells. Method The effect of alizarin on BKCa channels was observed by whole-cell patch clamp recording tech-nique. Results Selective BKCa channel blocker ibTX inhibited alizarin-induced outward current(P<0. 05);single smooth muscle cells were incubated in extracel-lular fluid with no Ca2+ 30 minutes, selective L-Ca2+channel blocker nimodipine, selective calcium ion che-lating agent BAPTA-AM and ryanodine inhibited the a-lizarin-induced BKCa channels current, too ( P <0. 05 ) . Conclusion Alizarin relaxes renales interlobar arterial smooth muscle via activation of L-Ca2+ channel firstly, which lead to Ca2+ flowed into cytoplam, and rising of Ca2+ in cytoplam ryanodine receptor indirect-ly, and BKCa is activated at last.

Efficacy and safety of alizarin combined with standard anti-tuberculosis therapy in treatment of multidrug-resistant pulmonary tuberculosis / 中华临床感染病杂志

Objective To evaluate the clinical efficacy and adverse reactions of alizarin combined with anti-tuberculosis therapy for multidrug resistant pulmonary tuberculosis (MDR-PTB).Methods A total of 200 confirmed MDR-PTB patients admitted in the Affiliated Hospital of Hangzhou Normal University during June 2013 and June 2015 were enrolled in the study.Patients were randomly divided into study group and control group (100 in each).Both groups were given standard anti -tuberculosis treatment for 8 months, and additional alizarin was given to study group .Chi-square test was used to assess the differences in clinical efficacy, sputum negative conversion rate, cavity closure and lesion absorption rate , as well as the incidence of adverse reactions between two groups ( including patients categorized according to TCM syndrome ). Results There were 39 markedly effective cases, 51 improved cases, 10 ineffective cases in study group, and 22 markedly effective cases, 35 improved cases, 43 ineffective cases in the control group.The total effective rate in study group was significantly higher than that in control group (90% vs.57%, χ2 =28.262, P 0.05). There was no significant difference in sputum negative conversion rate between two groups (76% vs.55%,χ2 =2.190, P >0.05).The cavity closure and lesion absorption rate in study group ( 91%) was significantly higher than that in the control group (54%,χ2 =38.294, P <0.01).The adverse reaction rate in study group was 27%, which was significantly lower than that in control group (66%, χ2 =30.570, P <0.01).Conclusion Alizarin in combination with standard anti -tuberculosis therapy can improve the clinical efficacy and reduce adverse reactions in treatment of MDR -PTB.

Exploration of in vitro channel tropism research methods of Chinese materia medica by supramolecular imprinting function of alizarin / 中草药

Objective: Based on supramolecular imprinting template self-recognition theory, to lay the foundation for the in vitro channel tropism research methods of Chinese materia medica (CMM). Using alizarin as imprinting template feature monomer, in vitro affinity chromatography of different pig organs was performed to test the mechanism of alizarin belonging to the liver meridian. Methods: The liver, lungs, tongue, abdominal muscle, leg muscle groups, and other tissues of pigs, as stationary phases, were made into column chromatography. Using PBS as the mobile phase, in vitro affinity chromatography experiments with alizarin belonging to the liver meridian and white sugar belonging to the spleen meridian were conducted, the content of eluent alizarin was measured by HPLC and the content of sugar was measured by UV. By using the analysis of total statistical moment, the various chromatographic parameters and chromatography equations were obtained. Results: The chromatographic parameters for the alizarin in liver, lungs, tongue, abdominal muscles, leg muscles were as following: C0: 0.2509-4.813 mg/mL; AUC: 2.509-48.13 mg; VR: 20.42-30.77 mL; σ2: 282.6-532.7 mL2; H: 14.13-26.63 mL; n: 1.212-1.777; S: 0.759 0-1.000. In the positive control group, the adsorption capacity of liver for alizarin was the strongest, and abdominal, leg, lungs, and tongue were weakened in turn. While in the negative control group, the adsorption capacity of liver for glucose (white sugar) was not the strongest. Conclusion: The adsorption capacity of liver for alizarin is the strongest, and liver has an affinity for alizarin by the super molecular imprinting templates, and it matches the theory of meridian belonging to the liver, which would establish the in vitro channel tropism preliminary research methods of CMM.

Protective effect of Cissus quadrangularisLinn. on diabetes induced delayed fetal skeletal ossification.

Background: Delayed fetal skeletal ossification is one of the known complications of maternal diabetes. Objective: The present study was designed to evaluate the protective role of petroleum ether extract of Cissus quadrangularis (PECQ) on diabetes‑induced delayed fetal skeletal ossification. Materials and Methods: Female Wistar rats were rendered diabetic with streptozotocin (STZ, 40 mg/kg, intraperitonial) before mating. After confirmation of pregnancy, the pregnant rats were divided into three groups: normal control group, diabetic control group, and diabetic + CQ group. The diabetic + CQ group pregnant rats were treated with PECQ (500 mg/kg body weight) throughout their gestation period. Immediately after delivery, pups were collected from all three groups and processed for alizarin red S–alcian blue staining in order to examine the pattern of skeletal ossification. Results: Fewer ossification centers and decreased extent of ossification of forelimb and hindlimb bones were observed in the neonatal pups of diabetic control group as compared to those in the normal control group. PECQ pretreatment significantly restored the ossification centers and improved the extent of ossification of forelimb and hindlimb bones in the neonatal pups of diabetic + CQ group as compared to those in the diabetic control group. Conclusions: The results suggested that PECQ treatment is effective against diabetes‑induced delayed fetal skeletal ossification. However, further studies on the isolation and characterization of active constituents of PECQ, which can cross the placental barrier and are responsible for the bone anabolic activity are warranted.

Histopathologic study of lens epithelial cells in different types of cataracts / 中华实验眼科杂志

Background Cataract was directly associated with the damage to the structure and function of lens epithelial cells (LECs).In those patients who suffer from cataracts,morphologic changes of LECs is the most compelling evidence confirming loss of cellular structure and function of LECs.So,learning about the morphological changes of LECs of the different types of cataracts is very important for study on biological behaviors of LECs in different environments or diseases.Objective This study was to evaluate the morphological and ultrastructural changes of the LECs in different types of cataracts.Methods Anterior capsular member from age-related cataracts,diabetic cataracts and high myopia complicated cataract were obtained during the cataract surgery and 15 pieces for each.Trypan blue and alizarin red (TB-AR) stain,haematoxylin and eosin stain were performed in the samples to assess the viability and morphology of LECs.The ultrastructural change of LECs was observed under the transmission electron microscope.The features of the LECs were compared among the different types of cataract.Results TB-AR stain showed that LECs were polygon in shape with the mosaic arrangement and round cell nucleus,and a few dead cells were seen in the samples age-related cataract.In the diabetic samples,LECs largened from swelling with different sizes.More dead cells were found in the high myopia complicated cataract.Haematoxylin and eosin stain exhibited that the anterior capsular membrane presented a homogeneuous membrane,and monolayer LECs attached firmly anterior capsular membrane in the samples of related cataract.Majority of the cells had the intact structure.However,the interspaces between cells and capsular membrane were found in diabetic cataract.Also,smaller LECs were seen in high myopia complicated cataract with the irregular cell morphology.Under the transmission electron microscope,LECs presented the normal shape,intact intercellular tight junction and well attachment between cells and capsular membrane in the samples of the age-related cataract.In the samples of the diabetic cataract,edema of LECs and large intercellular spaces were seen.In addition,the jogged pump and vacuolar degeneration of cytoplasm were revealed in the high myopia complicated cataract.Conclusions The degeneration,necrosis and apoptosis was a common pathological basis of age-related cataract,diabetic cataract and high myopia complicated cataract.However,the damage of LECs was more serious in diabetic cataract and high myopia complicated cataract than that of agerelated cataract.

Prenatal stress caused by movement restriction induces changes in the development of skull bone in CF-1 mice progeny / El estrés prenatal causado por restricción de movimiento induce cambios en la anatomía ósea de cráneo en la progenie de ratones CF-1

Prenatal stress is associated with changes in body weight and size, and with disorders of the skeletal bone development process. However, there is a lack of documentation on the impact of prenatal stress on skull bone anatomy during the gestation period. Therefore, this research focuses on the short-term effects of prenatal stress on the skull bone anatomy of CF-1 mice on the day of birth. Methodology: Gestating females were divided at random into two groups (control and stressed). The experimental group was subjected to the stress of movement restriction during the final week of gestation. Upon birth the body weight of the progeny was evaluated (control group, n=34; stressed group, n=29). They were then cleaned and stained with alizarin red in order to evaluate the length, width and suture spaces of the skull bone anatomy from superior and inferior views. Results: Gestational stress significantly altered the skull bone anatomy (p<0.05) of the offspring at birth in comparison with the control group. Conclusion: Prenatal stress alters the skull bone anatomy of the CF-1 mouse at birth.

El estrés prenatal se ha asociado con alteraciones en el peso y tamaño corporal, además de trastornos en el proceso de osificación del esqueleto en desarrollo. Sin embargo, existen escasos antecedentes acerca del impacto del estrés prenatal sobre la anatomía ósea craneal durante el periodo de gestación. Por lo tanto, la presente investigación estudió los efectos a corto plazo del estrés prenatal sobre la anatomía ósea craneal del ratón CF-1 en el día de nacimiento. Las hembras gestantes fueron divididas aleatoriamente en dos grupos (control y estresado), el grupo experimental fue sometido a estrés por restricción de movimiento durante la última semana de gestación. Al nacimiento se evaluó el peso corporal de la progenie (grupo control n:34; grupo estresado n:29), para posteriormente diafanizar y teñir con alizarina roja, evaluando dimensiones longitudinales, anchos y espacios suturales de la anatomía de los huesos de cráneo por la vista superior e inferior. El estrés gestacional alteró significativamente la anatomía de los huesos de cabeza ósea (p<0,05) de las crías en el momento del nacimiento con respecto a los controles. El estrés prenatal altera la anatomía de los huesos craneales del ratón CF-1 evaluado al nacer.

Evaluation of marking efficiency of different alizarin red S concentrations on body fish structures in Oreochromis niloticus (Perciformes: Cichlidae) juveniles

The use of alizarin red S (ARS) marked tilapias could provide valuable fisheries management information to evaluate fish stocking events and may facilitate aquaculture management practices. As a new technique in fishes, the aim of this study was to compare and evaluate the chemical marks produced in tilapia juveniles by ARS through two treatments: 1) 12 hours of immersion and 2) immersion after osmotic induction. This was analyzed at three concentrations: 50, 75 and 100mg/l, and in three structures: otoliths, fish scales and caudal fin rays of Oreochromis niloticus juveniles. After three culture months 80% of specimens were analyzed and significant differences (p<0.05) in mark intensity were detected between treatments for otoliths and fin rays, but not for fish scales. Significant differences between concentrations were found for the 12h immersion treatment, while no significant differences were detected with osmotic induction. Our results showed that marks appeared at all concentrations, and none of the concentrations produced weak marks. Osmotic induction had a greater mortality than the 12h immersion procedure. After eight culture months the rest of the specimens were analyzed and the mark permanence was observed in all cases. According to the present results we recommend the marking process of 12h immersion treatment at 100mg/L concentration.

El uso de alizarina roja S (ARS) para marcar tilapias podría proporcionar información valiosa para el manejo de su pesquería. Para evaluar pesquerías acuaculturales manejadas con siembras o repoblamientos de peces se comparó y evaluó la marca producida por la alizarina roja S, empleando dos tratamientos: 1) Inmersión en ARS durante 12h; e 2) Inmersión en ARS después de un choque osmótico. El análisis se realizó a tres concentraciones: 50, 75 y 100mg/l y en tres estructuras: otolitos, escamas y radios de la aleta caudal de Oreochromis niloticus. Ochenta por ciento de los ejemplares fueron cultivados durante tres meses y analizados posteriormente. Los resultados mostraron diferencias entre las concentraciones de la marca para el tratamiento de 12h de inmersión mientras que no hubo diferencias entre las concentraciones para el tratamiento con inducción osmótica. Se encontraron diferencias en la intensidad de la marca entre los tratamientos para otolitos y radios de las aletas pero para las escamas no hubo diferencias significativas. Todas las concentraciones produjeron marcas (desde débiles a intensas), sin embargo la concentración de 100mg/l no produjo marcas débiles. El tratamiento por inducción osmótica presentó mayores niveles de mortalidad. Después de ocho meses de cultivo el resto de los ejemplares fueron analizados y se observó la permanencia de las marcas en todos los casos. En vista de lo anterior, para los propósitos de marcaje se recomienda el uso del tratamiento de inmersión por 12h y una concentración de 100mg/l.

Prenatal stress caused by movement restriction induces changes in the appendicular osseous development of CF-1 mice progeny / El estrés prenatal causado por restricción de movimiento induce cambios en el desarrollo óseo apendicular de la progenie de ratones CF-1

Prenatal stress has been associated with alterations in weight and body size, as well as disturbances in the process of developing skeletal ossification, occurring during childbirth and the early stages of life. However, the effect evidence of prenatal stress on bone grow thand development during the gestation period has been low; therefore, it is unknown whether these alterations are associated with potential for growth disorders Because of this, the study aims to determine the short-term effects of prenatal stress on the CF-1mouse bone structure growth in your date of birth. The female mice were divided randomly in two groups: controlled (n=2) and stressed (n=3). The latter was put under stress by means of movement restriction during the last week of gestation. Second, an evaluation of their gestational development was made, obtaining measurements of their weight. Finally, diaphanization with KOH and staining with Alizarin red was used to measure the length of their appendicular bones and their flat pelvic bones, of 53 P0 mice (25 control; 28 stressed during gestation). The stressed mice's body weight (p=0.02) and the length of their appendicular bones (radii, p=0.0011; ulnae, p= 0.0001; humeri, p=0.0001; femorae, p=0.0006; tibiae, p=0.0015) were affected significantly in contrast with the controlled group. On the other hand,there were nosignificant differences inmaternal bodyweightand length ofthe mice pelvic bones (isquium, ilium; p>0.05). The prenatal stress by means of movement restriction alters the osseous appendicular morphology of the CF-1 mouse evaluated at birth...

El estrés prenatal se ha asociado con alteraciones en el peso y el tamaño corporal, así como trastornos en el proceso osificación en el esqueleto en desarrollo, que se producen durante el parto y las primeras etapas de vida. Sin embargo, la evidencia de los efectos del estrés prenatal sobre el crecimiento de los huesos y el desarrollo durante el período de gestación ha sido baja, desconociéndose si estas alteraciones están relacionadas con trastornos del crecimiento. El objetivo fue determinar los efectos a corto plazo del estrés prenatal en la estructura ósea del ratón CF-1 en el día de nacimiento. Las hembras gestantes fueron divididas aleatoriamente en dos grupos: control (n=2) y estresado (n=3). Este último fue puesto bajo estrés por restricción de movimiento durante la última semana de gestación. Evaluando el peso corporal en la progenie al nacer (grupo control n=25; grupo estresado n=28), para posteriormente diafanizar mediante KOH y teñir con alizarina roja, midiendo la longitud de huesos largos apendiculares y huesos planos de pelvis en el plano coronal. El peso de los ratones estresados (p = 0,02) y la longitud de los huesos apendiculares fueron significativamente menores en comparación con el grupo control (radio, p = 0,0011; ulna, p = 0,0001; húmero, p = 0,0001; fémur, p = 0,0006; tibia, p = 0,0015). Por otro lado, no hubo diferencias significativas en el peso corporal de la madre y la longitud de los huesos pélvicos de las crías (isquion, íleon, p> 0,05). El estrés prenatal por restricción de movimiento altera la morfología ósea apendicular del ratón CF-1, evaluados al nacimiento...

In vitro assay for osteoinductive activity of different demineralized freeze-dried bone allograft

PURPOSE: Various bone graft materials have been used for periodontal tissue regeneration. Demineralized freeze-dried bone allograft (DFDBA) is a widely used bone substitute. The current widespread use of DFDBA is based on its potential osteoinductive ability. Due to the lack of verifiable data, the purpose of this study was to assess the osteoinductive activity of different DFDBAs in vitro. METHODS: Sarcoma osteogenic (SaOS-2) cells (human osteoblast-like cells) were exposed to 8 mg/mL and 16 mg/mL concentrations of three commercial types of DFDBA: Osseo+, AlloOss, and Cenobone. The effect of these materials on cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The osteoinductive ability was evaluated using alizarin red staining, and the results were confirmed by evaluating osteogenic gene expression using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In the SaOS-2 cells, an 8 mg/mL concentration of Osseo+ and Cenobone significantly increased cell proliferation in 48 hours after exposure (P<0.001); however, in these two bone materials, the proliferation of cells was significantly decreased after 48 hours of exposure with a 16 mg/mL concentration (P<0.001). The alizarin red staining results demonstrated that the 16 mg/mL concentration of all three tested DFDBA induced complete morphologic differentiation and mineralized nodule production of the SaOS-2 cells. The RT-PCR results revealed osteopontin gene expression at a 16 mg/mL concentration of all three test groups, but not at an 8 mg/mL concentration. CONCLUSIONS: These commercial types of DFDBA are capable of decreasing proliferation and increasing osteogenic differentiation of the SaOS-2 cell line and have osteoinductive activity in vitro.

Estudo microscópico e macroscópico, com enfoque radiográfico e de alizarina, no desenvolvimento embrionário e fetal de gatos domésticos (Felis catus) em diferentes idades gestacionais / Microscopic and macroscopic study focusing ray and alizarin on embryonic and fetal development in cats (Felis catus) in different gestational ages

The domestic cat was named Felis catus by Carolus Linnaeus in his book Systema Na turae, in 1798. The family Felidae has many morphological similarities with wild felines. The study of the embryology of the domestic cat is of great value considering its importance as an experimental model for the wild cats endangered from extinction, especially in the research related to reproductive biology. The objective of this study is the descriptive embryology of the domestic cat at different stages of pregnancy, through macroscopic description of photographic records, radiographic and alizarin technique, and microscopic description of photographic records by light microscopy. In embryos with an estimated gestational age of 17 days we observed macroscopically an expansion corresponding to the rostral forebrain, the placoid site of lens, cervical flexure, the four pharyngeal arches with grooves dividing the cardiac prominence, a sign of the limb bud, and the presence of somites. In the caudal region of the embryo, we saw the cranio-caudal bend, allowing the same position in format of a "C". In embryos with an estimated age of 22 days, we noticed macroscopically the forebrain, optic vesicle pigmentation of the retina, the optic vesicle, fourth ventricle, liver, fore and hind limbs with a slight distinction between the digits and superficial vascularization. In embryos with an estimated age of 25 days we noticed presence of the forebrain and midbrain, the pronounced cervical curvature of the optic vesicle with strong pigmentation of the retina, the optic vesicle, limbs and chest well developed, distinguishing the digits and pronounced the liver Fetuses with estimated age of 52 days have internal and external structures easily identified in adult animals. With respect to the bone structure we noted that they did not have any radial bone formed, only bone shafts. Microscopically, the embryo of the domestic cat with CR of 0.9cm and estimated age of 19 days revealed the presence of beak, oral cavity with upper and lower nasal cavity, eye and opening of the 4th ventricle of the brain, esophagus, heart with atrium and ventricle, lung, liver, mesonephric ridge, primitive gonad, stomach, forelimb bud, spine and spinal cord in development. This paper is of great importance for study of the internal and external morphology of domestic cats for better understanding of the embryonic development of the species.

O gato doméstico (Felis catus) foi nomeado por Carolus Linnaeus em seu livro Systema Naturae, em 1798. A família Felidea apresenta muita semelhança morfológica com os felinos selvagens. O estudo da embriologia do gato doméstico é de grande valia, uma vez que, é considerado um importante modelo animal quando comparado aos gatos selvagem em extinção, especialmente relacionado às pesquisas sobre biologia reprodutiva. Este trabalho objetivou análisar e comparar as fases embrionárias de quatro embriões e um feto de felinos domésticos. Nos embriões com idade gestacional estimada em 17 dias (0,5cm CR) podemos observar pela análise macroscópica a presença de dilatação rostral correspondente ao prosencéfalo, o local placóide do cristalino, a flexura cervical, os quatro arcos faríngeos com os sulcos que o dividem, a proeminência cardíaca, o indício do brotamento do membro pélvico, além da presença de somitos. Na região caudal do embrião, visualizamos a curvatura cranio-caudal, permitindo ao mesmo uma posição em formato de "C". Nos embriões com idade gestacional estimada em 22 dias (1,2cm CR), na análise macroscópica foi visualizado o prosencéfalo, vesícula óptica com pigmentação da retina, vesícula ótica, quarto ventrículo, fígado, membros torácicos e pélvicos com discreta distinção dos dígitos e vascularização superficial. Nos embriões com idade gestacional estimada em 25 dias (1,5cm CR) notamos a presença do prosencéfalo e mesencéfalo, a curvatura cervical pronunciada, vesícula óptica com forte pigmentação da retina, vesícula ótica, membros pélvicos e torácicos bem desenvolvidos, com distinção dos dígitos e fígado bem pronunciado. Os fetos com idade gestacional estimada em 52 dias (10cm CR) possuem estruturas internas e externas facilmente identificadas em animais adultos. Com relação às estruturas ósseas notamos que as mesmas não apresentam nenhuma epífise óssea formada, sendo visíveis somente as diáfises ósseas. Na análise microscópica, o embrião de idade gestacional de 19 dias (0,9cm CR) revelou a presença do rostro, cavidade oral com lábio superior e inferior, cavidade nasal, olho e a abertura do 4º ventrículo encefálico, esôfago, coração com átrio e ventrículo, pulmão, fígado, crista mesonéfrica, gônada primitiva, estômago, broto do membro torácico, coluna vertebral e a medula espinhal em formação. Esse trabalho é de grande importância para o estudo da morfologia externa e interna de gatos domésticos, principalmente no que diz respeito ao desenvolvimento ósseo e articular, considerando as alterações que podem ou não ser promovidas pelo uso de terapias medicamentosas ou celulares durante o desenvolvimento embrionário e fetal.

Skeletogenesis of the pectoral girdle and forelimbs in embryos of Caiman yacare (Daudin, 1802) (Crocodylia, Alligatoridae)

Embryos of Caiman yacare were collected and subjected to the bone clearing and staining protocol in orderto analyze the ontogenetic patterns of ossification of the pectoral girdle and forelimb skeleton. The osseousstructure of the girdle and forelimbs of C. yacare begins to ossify starting at 30 days of incubation, withthe presence of dye retention in the scapula, coracoids, humerus, radius and ulna bones. During embryonicdevelopment, the autopodio of C. yacare has four bones in the carpus, the radial, ulnar, pisiform and carpaldistal 4+5 bone. Their ossification begins at 39 days of incubation with the radial, followed by the ulnar, and at54 days, the pisiform and the distal carpal 4 + 5. Each mesopodio has 5 metacarpi and are present 15 phalanges,two in digits I and V, three in digits II and IV, and four in digit III (phalangeal formula 2:3:4:3:2). Ossificationof the metacarpi starts at 27 days of incubation, following the sequence MCII=MCIII=MCIV>MCI>MCV.The first phalanges begin the process of ossification on day 36, continuing up to the last day of incubation.The sequence of ossification of the proximal phalanges is PPI=PPII=PPIII>PPIV=PPV, that of the medialphalanges is MPII>MPpIII>MPdIII>MPIV, and that of the distal phalanges is DPI>DPII>DPIII>DPV>DPIV.The ontogenetic pattern of the bones of the forepaw of C. yacare generally differs from that of other reptiles,although there are some similarities.