Efecto de tres anestésicos en Haliotis tuberculata coccinea (Gastropoda) y la aplicabilidad del aceite de clavo en el cultivo de abalón

[EN]Three chemical methods for induction of relaxation in H. tuberculata coccinea (50-80mm shell length) were tested: sodium pentobarbital (60mg/l), 2-phenoxyethanol (2ml/l) and clove oil at three concentrations (0.02, 0.05 and 0,5ml/l). The time taken for animals to release from aquaria walls and recover from the anaesthetics was recorded. No mortality was observed after one week from experiment. All anaesthetics made animals release from container walls within 10-30 min, but they were ineffective as muscle relaxants, leaving the foot hard and contracted. The most effective concentration tested for clove oil was 0,5ml/l at 22ºC.

Influence of body temperature on bacterial growth rates in experimental pneumococcal meningitis in rabbits

We examined the role of fever as a host defense in experimental pneumococcal meningitis in rabbits. Twelve hours after intracisternal inoculation of an encapsulated type 3 Streptococcus pneumoniae strain, body temperature was manipulated by using two different anesthetic drugs: pentobarbital, which did not affect temperature, and urethane, which mitigated the febrile response to infection. Growth rates of pneumococci in cerebrospinal fluid were dramatically influenced by modification of the febrile response. Rabbits whose fever was not suppressed had mean bacterial doubling times of 2.76 +/- 1.43 h. Animals with a blunted febrile response had a significantly faster mean bacterial growth rate (doubling time = 1.10 +/- 0.27 h; P less than 0.02). When the antipyretic effect of urethane was counteracted by raising the ambient temperature, animals also showed a marked reduction in pneumococcal growth rates. In vitro, the pneumococci grew well at 37 degrees C in Trypticase soy broth (doubling time = 0.61 +/- 0.05 h) and in pooled rabbit cerebrospinal fluid (doubling time = 0.85 +/- 0.07 h). However, at 41 degrees C neither medium supported growth. Thus, body temperature appears to be a critical determinant of pneumococcal growth rates in experimental meningitis, and fever could be a host defense in this disease.

STUDIES ON ENERGY METABOLISM IN THE HIPPOCAMPUS AFTER ISCHEMIA

The gerbil model of ischemia was used to determine the effect of carotid occlusion on energy metabolites in cellular layers of discrete regions of the hippocampus and dentate gyrus. Levels of glucose, glycogen, ATP and phosphocreatine (PCr) were unchanged after 1 minute of ischemia. However, 3 minutes of ischemia produced a dramatic decrease in net levels of all metabolites. No additional decrease was observed after 15 minutes of ischemia. Re-establishment of the blood flow for 5 minutes after a 15 minute ischemic episode returned all metabolites to pre-ischemia levels. Concentrations of glucose and glycogen were elevated in sham-operated animals as a function of the pentobarbital anesthetic employed. In other studies, elevated GABA levels (produced by inhibiting GABA-transaminase with (gamma)-vinyl-GABA (GVG)) were found to decrease the rate of utilization of the high-energy phosphate metabolites ATP and PCr in the mouse cortex. In addition, glucose and glycogen levels were increased. Thus, tonic inhibition by GABA produced decreased cellular activity. Additional experiments demonstrated the attenuation of ischemia-induced metabolite depletion in cellular layers of regions of the hippocampus, dentate gyrus and cortex after GVG administration. Under ether, 1 minute of bilateral carotid occlusion produced a dramatic decrease in metabolite levels. After GVG treatment, the decrease was blocked completely for glucose, glycogen and ATP, and partially for PCr. Therefore, GABA-transaminase inhibition produced increased levels of GABA which subsequently decreased cellular activity. The protection against ischemia may have been due to (a)decreased metabolic rate; the available energy stores were utilized at a slower rate, and (b)increased levels of energy substrates; additional supplies available to maintain viability. These data suggest that the functional state of neural tissue can determine the response to metabolic stress. ^

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

1 The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. 2 In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+](i), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+](i) transients was 28 muM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. 3 In fura-2-loaded neurons, voltage clamped at -60mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 muM) simultaneously inhibited nAChR-induced increases in [Ca2+](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by - 40% at - 120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. 4 The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+](i) by similar to40%. 5 Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+](i), indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. 6 Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 muM), pentobarbital (50 muM) and ketamine (10 muM). 7 In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

Pharmacologically induced and stimulus evoked rhythmic neuronal oscillatory activity in the primary motor cortex in vitro

Parkinson's disease (PD) is associated with enhanced synchronization of neuronal network activity in the beta (15-30 Hz) frequency band across several nuclei of the basal ganglia (BG). Deep brain stimulation of the subthalamic nucleus (STN) appears to reduce this pathological oscillation, thereby alleviating PD symptoms. However, direct stimulation of primary motor cortex (M1) has recently been shown to be effective in reducing symptoms in PD, suggesting a role for cortex in patterning pathological rhythms. Here, we examine the properties of M1 network oscillations in coronal slices taken from rat brain. Oscillations in the high beta frequency range (layer 5, 27.8 +/- 1.1 Hz, n=6) were elicited by co-application of the glutamate receptor agonist kainic acid (400 nM) and muscarinic receptor agonist carbachol (50 mu M). Dual extracellular recordings, local application of tetrodotoxin and recordings in M1 micro-sections indicate that the activity originates within deep layers V/VI. Beta oscillations were unaffected by specific AMPA receptor blockade, abolished by the GABA type A receptor (GABAAR) antagonist picrotoxin and the gap-junction blocker carbenoxolone, and modulated by pentobarbital and zolpidem indicating dependence on networks of GABAergic interneurons and electrical coupling. High frequency stimulation (HFS) at 125 Hz in superficial layers, designed to mimic transdural/transcranial stimulation, generated gamma oscillations in layers 11 and V (incidence 95%, 69.2 +/- 7.3 Hz, n=17) with very fast oscillatory components (VFO; 100-250 Hz). Stimulation at 4 Hz, however, preferentially promoted theta activity (incidence 62.5%, 5.1 +/- 0.6 Hz, n=15) that effected strong amplitude modulation of ongoing beta activity. Stimulation at 20 Hz evoked mixed theta and gamma responses. These data suggest that within M1, evoked theta, gamma and fast oscillations may coexist with and in some cases modulate pharmacologically induced beta oscillations.

Ação do fator de crescimento de fibroblasto básico na cicatrização da aponeurose abdominal de ratos

Objetivo: Trabalho realizado em ratos com o objetivo de estudar o efeito do Fator de Crescimento de Fibroblastos básico (FCFb) na cicatrização da aponeurose abdominal. Métodos: Foram usados 20 ratos Wistar separados aleatoriamente em 2 grupos iguais. Os animais foram anestesiados com pentobarbital sódico na dose de 20 mg/Kg por via intraperitoneal e submetidos a laparotomia mediana de 4 cm, cuja camada aponeurótica foi suturada com mononylon 5-0. No grupo I foi aplicada a dose de 5mg de FCFb sobre a sutura da aponeurose. No grupo II (controle) foi aplicada solução salina 0,9% sobre a linha se sutura. Após observação por 7 dias os animais foram mortos com superdose de anestésico. A camada aponeurótica com 1,5 cm de largura foi submetida a teste de resistência à tensão empregando a Máquina de Ensaios EMIC MF500. Biópsias das zonas de sutura foram processadas e coradas com HE e o tricômico de Masson. Os achados histopatológicos foram quantificados através de sistema digital (Image pro-plus) de captura e processamento de imagens. Os dados obtidos foram analisados pelo teste T com significância 0,05. Resultados: Nos animais do grupo I (experimental) a zona de sutura da camada aponeurótica suportou a carga de 1.103±103,39gf. A quantificação dos dados histopatológicos desse grupo atingiu a densidade média 226±29,32. No grupo II (controle) a carga suportada pela zona de sutura foi de 791,1±92,77 gf. Quando foram comparadas as médias das resistências à tensão dos dois grupos, observou-se uma diferença significante (p<0,01). O exame histopatológico das lâminas desse grupo relevou densidade média 114,1±17,01, correspondendo a uma diferença significante quando comparadas as médias dos dois grupos (p<0,01). Conclusão: Os dados permitem concluir que o FCFb contribuiu para aumentar a resistência da aponeurose suturada e para melhorar os parâmetros histopatológicos da cicatrização.

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen

Repercussões do tempo operatório em pulmões de ratos idosos

A duração das operações pode representar um fator importante para uma série de complicações pós-operatórias, especialmente para os indivíduos idosos. Objetivo: estudar a repercussão nos pulmões, de operações de diferentes tempos de duração. Métodos: Vinte ratos idosos (18 meses) e 20 jovens (3 meses) foram separados aleatoriamente em grupos A e B respectivamente. Os grupos foram divididos em A1, A2, A3, A4, B1, B2, B3 and B4, com cinco ratos cada. Os animais foram anestesiados com pentobarbital (20mg/Kg) intraperitoneal. No subgrupo A1 e B1 foi feita operação com duração de 30 minutos, nos grupos A2 and B2 60 minutos, em A3 and B3 a operação foi feita em 120 minuto e os animais A4 e B4 (controle) não foram operados. O procedimento consistiu de laparotomia xifopubiana que foi aberta e fechada tantas vezes quanto necessário para atingir os tempos estipulados. Após o quinto dia pós-operatório os animais foram mortos com superdose de anestésico e biópsias de ambos os pulmões foram realizadas. Os achados histopatológicos foram transformados em escores. Resultados: os grupos de ratos jovens atingiram os escores: A1= escore 6, A2=11; A3=28; A4=5. Os ratos idosos tiveram os escores: B1=12; B2=34; B3=51 e B4=6. A análise estatística revelou diferenças significantes entre os escores dos grupos A e B. Conclusões: O tempo prolongado nas operações realizadas em ratos idosos contribuiu para o aparecimento de alterações pulmonares de modo significante. Quanto maior o tempo operatório, mais intensas e mais freqüentes as complicações pulmonares

Repercussões do tempo operatório em pulmões de ratos idosos

A duração das operações pode representar um fator importante para uma série de complicações pós-operatórias, especialmente para os indivíduos idosos. Objetivo: estudar a repercussão nos pulmões, de operações de diferentes tempos de duração. Métodos: Vinte ratos idosos (18 meses) e 20 jovens (3 meses) foram separados aleatoriamente em grupos A e B respectivamente. Os grupos foram divididos em A1, A2, A3, A4, B1, B2, B3 and B4, com cinco ratos cada. Os animais foram anestesiados com pentobarbital (20mg/Kg) intraperitoneal. No subgrupo A1 e B1 foi feita operação com duração de 30 minutos, nos grupos A2 and B2 60 minutos, em A3 and B3 a operação foi feita em 120 minuto e os animais A4 e B4 (controle) não foram operados. O procedimento consistiu de laparotomia xifopubiana que foi aberta e fechada tantas vezes quanto necessário para atingir os tempos estipulados. Após o quinto dia pós-operatório os animais foram mortos com superdose de anestésico e biópsias de ambos os pulmões foram realizadas. Os achados histopatológicos foram transformados em escores. Resultados: os grupos de ratos jovens atingiram os escores: A1= escore 6, A2=11; A3=28; A4=5. Os ratos idosos tiveram os escores: B1=12; B2=34; B3=51 e B4=6. A análise estatística revelou diferenças significantes entre os escores dos grupos A e B. Conclusões: O tempo prolongado nas operações realizadas em ratos idosos contribuiu para o aparecimento de alterações pulmonares de modo significante. Quanto maior o tempo operatório, mais intensas e mais freqüentes as complicações pulmonares

Ação do fator de crescimento de fibroblasto básico na cicatrização da aponeurose abdominal de ratos

Objetivo: Trabalho realizado em ratos com o objetivo de estudar o efeito do Fator de Crescimento de Fibroblastos básico (FCFb) na cicatrização da aponeurose abdominal. Métodos: Foram usados 20 ratos Wistar separados aleatoriamente em 2 grupos iguais. Os animais foram anestesiados com pentobarbital sódico na dose de 20 mg/Kg por via intraperitoneal e submetidos a laparotomia mediana de 4 cm, cuja camada aponeurótica foi suturada com mononylon 5-0. No grupo I foi aplicada a dose de 5mg de FCFb sobre a sutura da aponeurose. No grupo II (controle) foi aplicada solução salina 0,9% sobre a linha se sutura. Após observação por 7 dias os animais foram mortos com superdose de anestésico. A camada aponeurótica com 1,5 cm de largura foi submetida a teste de resistência à tensão empregando a Máquina de Ensaios EMIC MF500. Biópsias das zonas de sutura foram processadas e coradas com HE e o tricômico de Masson. Os achados histopatológicos foram quantificados através de sistema digital (Image pro-plus) de captura e processamento de imagens. Os dados obtidos foram analisados pelo teste T com significância 0,05. Resultados: Nos animais do grupo I (experimental) a zona de sutura da camada aponeurótica suportou a carga de 1.103±103,39gf. A quantificação dos dados histopatológicos desse grupo atingiu a densidade média 226±29,32. No grupo II (controle) a carga suportada pela zona de sutura foi de 791,1±92,77 gf. Quando foram comparadas as médias das resistências à tensão dos dois grupos, observou-se uma diferença significante (p<0,01). O exame histopatológico das lâminas desse grupo relevou densidade média 114,1±17,01, correspondendo a uma diferença significante quando comparadas as médias dos dois grupos (p<0,01). Conclusão: Os dados permitem concluir que o FCFb contribuiu para aumentar a resistência da aponeurose suturada e para melhorar os parâmetros histopatológicos da cicatrização.

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen

Repercussões do tempo operatório em pulmões de ratos idosos

A duração das operações pode representar um fator importante para uma série de complicações pós-operatórias, especialmente para os indivíduos idosos. Objetivo: estudar a repercussão nos pulmões, de operações de diferentes tempos de duração. Métodos: Vinte ratos idosos (18 meses) e 20 jovens (3 meses) foram separados aleatoriamente em grupos A e B respectivamente. Os grupos foram divididos em A1, A2, A3, A4, B1, B2, B3 and B4, com cinco ratos cada. Os animais foram anestesiados com pentobarbital (20mg/Kg) intraperitoneal. No subgrupo A1 e B1 foi feita operação com duração de 30 minutos, nos grupos A2 and B2 60 minutos, em A3 and B3 a operação foi feita em 120 minuto e os animais A4 e B4 (controle) não foram operados. O procedimento consistiu de laparotomia xifopubiana que foi aberta e fechada tantas vezes quanto necessário para atingir os tempos estipulados. Após o quinto dia pós-operatório os animais foram mortos com superdose de anestésico e biópsias de ambos os pulmões foram realizadas. Os achados histopatológicos foram transformados em escores. Resultados: os grupos de ratos jovens atingiram os escores: A1= escore 6, A2=11; A3=28; A4=5. Os ratos idosos tiveram os escores: B1=12; B2=34; B3=51 e B4=6. A análise estatística revelou diferenças significantes entre os escores dos grupos A e B. Conclusões: O tempo prolongado nas operações realizadas em ratos idosos contribuiu para o aparecimento de alterações pulmonares de modo significante. Quanto maior o tempo operatório, mais intensas e mais freqüentes as complicações pulmonares