Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

Efeitos da ingestão de cafeína durante a gestação e a lactação sobre a pele de ratas e de filhotes e sua relação com as concentrações séricas do cortisol materno / Effects of caffeine intake during pregnancy and lactation on the skin of rats and offspring and their relationship to maternal serum concentrations of cortisol

O objetivo deste trabalho foi verificar os efeitos da ingestão materna de diferentes doses de cafeína durante a gestação e a lactação, na pele de ratas-mães e filhotes, bem como sua relação com as concentrações séricas do cortisol materno. Vinte e quatro ratas Wistar adultas foram distribuídas em quatro grupos, representados pelo controle e tratados, com cafeína nas doses de 25, 50 e 100mg/kg. Os grupos tratados receberam cafeína por sonda orogástrica durante toda a gestação e a lactação. O controle recebeu água destilada como placebo. Foram avaliados e quantificados os diferentes tipos de folículos pilosos e a espessura da epiderme. A técnica de imuno-histoquímica, com o uso do anticorpo anti-CDC47, foi utilizada para avaliar a proliferação celular da epiderme e dos folículos pilosos das mães. Na mãe, também foram mensurados os níveis séricos de cortisol pela técnica da quimioluminescência. Os dados foram submetidos à análise de variância com comparação das médias pelos testes Kruskal-Wallis e SNK. Nos grupos tratados com cafeína nas doses de 25 e 50mg/kg, tanto as mães quanto seus filhotes apresentaram hipotricose e/ou alopecia focal. Apesar de a frequência de alterações macroscópicas das mães ter sido superior a dos filhotes, nestes as lesões, quando presentes, foram difusas. A análise histológica demonstrou calcinose de folículos pilosos nas mães e nos filhotes. Mas a morfometria somente revelou diferença significativa no número de folículos pilosos das mães, bem como redução significativa da proliferação celular dos folículos pilosos do grupo tratado com 50mg/kg de cafeína. Os níveis de cortisol materno somente foram significativamente elevados no grupo tratado com 100mg/kg de cafeína. Conclui-se que a cafeína ingerida pelas ratas gestantes e lactantes pode causar lesões cutâneas tanto nas mães quanto nos filhotes, caracterizadas por hipotricose e/ou alopecia, independentemente dos níveis séricos do cortisol materno.

The aim of this study was to investigate the effects of maternal caffeine intake during pregnancy and lactation on the skin of rats and their offspring, as well as their relationship to maternal serum levels of cortisol. 24 adult Wistar rats were equally divided into four groups represented by the control and treated with caffeine at doses of 25, 50 and 100mg/kg. The groups received caffeine by orogastric tube during the entire pregnancy and lactation. The control received distilled water as placebo. Different types of hair follicles and the thickness of the epidermis were assessed and quantified. Immunohystochemistry technique using antibody anti-CDC47 was used to evaluate cellular proliferation of the epidermis and hair follicles of the mothers. Also in the mothers, serum levels of cortisol were measured by the chemiluminescence technique. Data were submitted to analysis of variance comparing mediums by Kruskall Wallis Test and SNK. In groups treated with caffeine 25 and 50mg/kg, both mothers and their puppies had focal alopecia and/or hypotrichosis. Despite the higher frequency of macroscopic changes on the mothers, these lesions were diffuse when present on the puppies. Histological analysis showed calcinosis of hair follicles in the mothers and their puppies. But morphometry revealed significant difference in the number of hair follicles from mothers, as well as a significant reduction of cell proliferation of hair follicles in the group treated with 50mg/kg of caffeine. Maternal cortisol levels were significantly elevated in the group treated with 100mg/kg of caffeine. It is concluded that caffeine intake by pregnant and lactating rats can cause skin lesions in both the mothers and their offspring, characterized by alopecia and/or hypotrichosis, regardless of serum levels of maternal cortisol.

Effects of short-term administration of estradiol on reperfusion arrhythmias in rats of different ages

Little is known about age-related differences in short-term effects of estradiol on ischemia-reperfusion (I/R) insults. The present study was designed to evaluate the effects of short-term treatment with estradiol on reperfusion arrhythmias in isolated hearts of 6-7-week-old and 12-14-month-old female rats. Wistar rats were sham-operated, ovariectomized and treated with vehicle or ovariectomized and treated with 17β-estradiol (E2; 5 µg·100 g-1·day-1) for 4 days. Hearts were perfused by the Langendorff technique. Reperfusion arrhythmias, i.e., ventricular tachycardia and/or ventricular fibrillation, were induced by 15 min of left coronary artery ligation and 30 min of reperfusion. The duration and incidence of I/R arrhythmias were significantly higher in young rats compared to middle-aged rats (arrhythmia severity index: 9.4 ± 1.0 vs 3.0 ± 0.3 arbitrary units, respectively, P < 0.05). In addition, middle-aged rats showed lower heart rate, systolic tension and coronary flow. Four-day E2 treatment caused an increase in uterine weight. Although E2 administration had no significant effect on the duration of I/R arrhythmias in middle-aged rats, it induced a marked reduction in the rhythm disturbances of young rats accompanied by a decrease in heart rate of isolated hearts. Also, this reduction was associated with an increase in QT interval. No significant changes were observed in the QT interval of middle-aged E2-treated rats. These data demonstrate that short-term estradiol treatment protects against I/R arrhythmias in hearts of young female rats. The anti-arrhythmogenic effect of estradiol might be related to a lengthening of the QT interval.

Fatores associados com os tipos de heparina usados no tratamento da angina instável em um hospital brasileiro / Factors Associated with the Types of Heparin used in the Treatment of Unstable Angina at a Brazilian Hospital

Unfractionated heparin (UFH) and low-molecularweight heparins (LMWHs) are widely used in curative and preventive treatments of thromboembolic disorders. The aim of the study was to investigate factors associated with the choice of these types of heparin to treat patients with unstable angina under real conditions of hospital use. A cross-sectional study was performed in a private general hospital in Belo Horizonte, Brazil, from January 1st to December 31th, 2001. Data were collected from the hospital electronic database. Inpatients with angina who received enoxaparin or UFH were included in the survey. Data for 555 patients were recorded, including 401 treated with enoxaparin and 154 with UFH. Univariate analysis showed that male and elderly people predominated in both groups, with no statistical difference in the proportions (p>0.05). Multivariate analysis showed 4 factors associated with the use of enoxaparin: cardiac revascularization surgery (OR=0.434), arrhythmias (OR=9.343), risk factors for coronary artery disease (OR=1.333) and private health insurance (OR=0.297). Thus, clinical and organizational factors were associated with the type of heparin used by patients with unstable angina at this hospital. Further drug utilization studies are necessary to expand and improve the data available on the use of heparins in the hospital setting.

A heparina não-fracionada (HNF) e heparinas de baixo peso molecular (HBPM) são amplamente utilizadas em tratamentos curativos e preventivos de tromboembolismo. O objetivo do estudo foi investigar os fatores associados com a escolha desses tipos de heparinas para tratar pacientes com angina instável sob as condições reais de uso hospitalar. Trata-se de um estudo transversal realizado em hospital geral privado, na cidade de Belo Horizonte,MG Brasil, no período de Janeiro a Dezembro de 2001. Para a coleta de dados, utilizou-se o banco de dados informatizado do referido hospital. Pacientes internados com angina que receberam enoxaparina ou HNF foram incluídos no estudo. Registrou-se dados de 555 pacientes, incluindo 401 tratados com enoxaparina e 154 com HNF. Na análise univariada, observouse que o gênero masculino e pacientes idosos foram predominantes em ambos os grupos, sem diferença estatística entre as proporções (p>0,05). A análise multivariada revelou quatro fatores associados ao uso de enoxaparina: cirurgia de revascularização cardíaca (OR=0,434), arritmias (OR=9,343), fatores de risco para doença coronariana (OR=1,333) e atendimento por plano de saúde (OR=0,297). Assim, fatores clínicos e organizacionais estão associados com o tipo de heparina usado por pacientes com angina instável, neste hospital. A realização de mais estudos de utilização de medicamentos é necessária para aprimorar o conhecimento sobre o uso de heparinas, em hospitais.

Análise da utilização de medicamentos antimicrobianos sistêmicos em crianças e adolescentes em dois hospitais de ensino / Analysis of the use of systemic antimicrobial drugs in children and teenagers in two teaching hospitals

A farmacoterapia pediátrica exige uma variedade de formas farmacêuticas adequadas às diferentes fases do desenvolvimento da criança. O objetivo deste estudo foi identificar os antimicrobianos utilizados em dois hospitais e verificar se realmente havia adequação das formas farmacêuticas aos pacientes pediátricos. Os antimicrobianos foram identificados pelo sistema informatizado das farmácias. Os medicamentos foram analisados, levando-se em consideração a necessidade de transformação da forma farmacêutica, o fracionamento e a porcentagem de desperdício. Verificou-se que 97,2% dos medicamentos analisados foram considerados inadequados para utilização em recém-nascido prematuro; 82,5% foram considerados inadequados para uso em recém-nascido a termo e 68,1% foram considerados inadequados para lactentes. A falta de formulações adequadas para essas faixas etárias acarreta frequentes manipulações, o que pode comprometer o uso adequado dos medicamentos, principalmente dos medicamentos parenterais. Identificou-se, também, a necessidade de transformação dos medicamentos sólidos de uso oral. Essas atividades, sem dúvida, elevam o risco de contaminação e/ou de inexatidão da dose. Os resultados mostram uma falta de formulações farmacêuticas de antimicrobianos adequadas para utilização nas diversas faixas etárias pediátricas, sendo, portanto, necessárias ações das autoridades sanitárias para assegurar a disponibilidade de formulações apropriadas, contribuindo para a promoção do uso racional de medicamentos em pediatria.

Pediatric pharmacotherapy requires a variety of drug dosage forms suited to different stages of child development. The aim of this study was to identify the antimicrobial drugs used in the pediatric units of two hospitals and to examine whether the drug dosage forms used were really suitable for pediatric patients. The antimicrobial drugs were identified through the computerized system of the hospital dispensaries. The medicines were analyzed regarding the need to transform or split the drug dosage form and the amount wasted. A total of 92.7% of the medicines analyzed were inappropriate for use with a preterm neonate, 82.5% were unsuitable for a term neonate and 68.1% were unsuitable for infants. The lack of suitable formulations for these age groups may lead to more frequent compounding of drugs, hampering the correct use of medicines, especially parenteral drugs. A need to transform solid oral drug forms was identified. Such activities certainly increase the risk of drug contamination or inaccuracy of dose. The results showed a lack of pharmaceutical formulations of antimicrobial drugs suitable to be given to various pediatric age groups. Therefore, health authorities should ensure the availability of appropriate drug dosage forms for children, in order to promote the rational use of drugs in pediatrics.

Effect of pinealectomy, adrenalectomy, pinealectomy plus adrenalectomy upon the quantification of spermatogenic cells of adult rats

The objectives of this study were to evaluate the effects of pinealectomy, adrenalectomy and pinealectomy-adrenalectomy upon the quantification of spermatogenic cells of rats. As such, 32 adult Wistar rats with a mean body weight of 331.7± 15.5g were assigned into one of the following treatments: (a) a sham-operated control group, consisting of nine animals; (b) ten pinealectomized animals; (c) seven adrenalectomized animals and (d) six pinealectomized plus adrenalectomized animals. No significant differences were observed between groups for the following parameters: body, testes, prostate and seminal vesicle weights, seminiferous tubular diameter, number of cells per seminiferous tubular cross sections (primary spermatocytes at pachytene, round spermatids, Sertoli cells) and numbers of germ cells per Sertoli cell (primary spermatocytes at pachytene and round spermatids ). Although no increase in testicular weight was observed following pinealectomy, a significant (P<0.05) increase of approximately 11.5 percent in the number of round spermatids per Sertoli cell (Sertoli cell ratio) occurred thus suggesting that short-term pinealectomy abolishes the antigonadal effect of the pineal gland upon adult Wistar rat testes

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33 per cent of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ñ 0.28 mM, angiotensin II, N = 9 vs 6.4 ñ 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.

Alterations in plasma prolactin and glucose levels induced by surgical stress in hyperprolactinemic female rats

The effects of hyperprolactinemia on plasma prolactin (PRL) and glucose were investigated in female rats submitted to surgical stress (laparotomy under ether anesthesia). Wistar rats weighing 250-280g received pituitary grafs under the kidney capsule three weeks before the experiments (N = 15) while a control group underwent sham transplantation (N = 14). The sham-operated rats presented a threefold increase of PRL levels as early as after 5 min of surgical stress (P<0.01); the PRL levels reached a peak at about 15 min and returned to baseline at 40 min. The PRL levels of the grafted rats were increased 3.5-fold compared to the sham-operated controls before stress (20.2 + 5.6 ng/ml vs 5.8 + 0.9 ng/ml, respectively; P<0.05), but did not change significantly during the experimental period. Plasma glucose was already significantly increased at 5 min in sham-operated control and grafted rats (P<0.01) and reached maximal concentrations at about 15 min. The grafted rats presented higher glucose levels than sham-operated controls before stress (122.2 + 3.3 vs 100.5 + 4.2 mg/dl; P<0.01) and at 40 min (182.6 + 13.6 vs 146.7 + 8.4 mg/dl; P<0.05). The hyperprolactinemic rats showed impaired surgical stress-induced PRL release and higher glucose levels both at rest and during the first postoperative hour. These results indicate that chronic hyperprolactinemia inhibited PRL secretion and enhanced the hyperglycemic stress response in the female rat.

Effect of prepuberal chronic morphine administration on the onset of puberty in pituitary-grafted female rats

The effect of morphine on the onset of puberty was studied in female Wistar rats bearing pituitary grafts implanted at 21 days of age, or sham operated (SO). Morphine was given sc, daily, from day 22 until the occurrence of vaginal opening (VO), taken as an index of puberty. Two doses of morphine (2 or 8 mg kg-1 day-1) were used and control animals received saline of the same volume. Morphine (both doses) induced delayed puberty in SO rats, as indicated by age at VO: mean ñ SEM, 36.90 ñ 0.75 and 36.33 ñ 1.08 days vs 33.06 ñ 0.69 days for 2 and 8 mg vs control group. Pituitary graft induced precocious puberty and this effect was reversed by the highest dose of morphine (29.47 ñ 0.84 vs 32.80 ñ 0.59 days for saline vs 8 mg morphine, grafted rats). These data show that chronic administration of morphine during the prepuberal period delayed the onset of puberty and reversed the precocious puberty induced by pituitary graft in the female rat

Evidence for adrenal medulla involvement in cold adaptation of preoptic-lesioned rats

The cold-adaptation process was examined in preoptic-lesioned (PO, 18 rats) and adrenaldemedullated-preoptic-lesioned rats (ADPO, 16 rats). PO rats and ADPO female Wistar rats wee cold acclimatized to 5-C for 2 h a day for 2 weeks, or maintained at 25C, starting one week after the operation. Control animals (20 rats) were sham-operated and then treated exactly like the lesioned rats. Lesions int he PO region caused a decrease in the ability to maintain normal body temperature in a cold environment (5-C for 2h). Theri colonic temperature fell even by the end of the 2nd h of cold exposure (-1.1-C for PO rats and -1.25-C for ADPO rats, P<0.01). However, after 2 weeks of cold acclimattion the PO rats, but not the ADPO rats were able to thermoregulate in the cold and showed a normal increase in interscapular brown adipose tissue weight (79%, P < 0.01). These data suggest that the PO is not an essential link for cold adaptation when a mild col-adaptation methods is used. Furthermore, the data show that increased adrenal medullary activity is required for the cold adaptation process in PO rats

Preferential saline or water intake by pinealectomized, adrenalectomized, and pinealectomized-adrenalectomized male rats

Because the pineal gland appears to be involved in the regulation of body electrolytes, saline or water preference was investigated imn pinealecrtomized, adrenalectomized, pinealectomized-adrenalectomized, and sham-operated (SO) control rats. The pinealectomized, adrenalectomized, and pinealectomized-adrenalectomized rats exhibites increased saline consumption during teh two postoperative weeks when compared with the SO-control animals. Water intake was similar for pinealectomized and SO-control rats, but decreased for pinealectomized-adrenalectomized, and adrenalectomized rats. Although total fluid intake increased for pinealectomized, and pinealectomized-adrenalectomized rats, the proportion of saline ingested in preference to water by pinealectomized-adrenalectomized rats was twice that exhibited by pinealectomized rats. The results showed two different behaviors: increased saline intake without changes in water intake by pinealectomized rats, and increased saline intake with decreased water intake by adrenalectomized rats. However, the higher salin intake induced by pinealectomy was further increased by adrenalectomy, suggesting a direct role for the pineal gland in meidating specific salt appetite