RESUMEN
BACKGROUND: Thickness measurement of the outermost layer of the skin, the stratum corneum (SC), is essential for in-vivo measurement of the cutaneous bioavailability of topically applied drugs and cosmetics. Our aim was to compare SC thickness calculated from confocal Raman spectroscopy (CRS) data with results of SC thickness based on confocal laser scanning microscopy (CLSM) measurements and with literature data, to validate CRS data with CLSM data and vice versa. METHODS: SC thickness was measured with two non-invasive devices, confocal Raman spectroscopy and confocal laser scanning microscopy, on four different areas of the body: volar forearm, leg, face and palm in 18 healthy adult subjects. RESULTS: Comparable results of SC thickness were obtained with both methods, structure analysis of CLSM images, and computation of Fick's first law on water gradients measured with CRS: 20 µm and 19 µm (volar forearm), 21 µm and 22 µm (lower leg), and 13 µm with both methods (cheek), respectively. DISCUSSION: For the first time it was possible to accurately determine the thickness of SC with CRS and CLSM and to validate both systems against each other and with results of literature data. CONCLUSION: Both methods, CRS and CLSM, were found to be suitable to measure SC thickness correctly. Therefore, when using CRS, for example to obtain detailed information about the molecular composition of the skin, it is additionally possible to accurately measure SC thickness with the same device to have an orientation in which skin layer molecules are found.
Asunto(s)
Dermoscopía/métodos , Células Epidérmicas , Epidermis/fisiología , Microscopía Confocal/métodos , Espectrometría Raman/métodos , Adulto , Humanos , Persona de Mediana Edad , Tamaño de los Órganos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.
Asunto(s)
3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Trastornos del Sistema Nervioso Inducidos por Alcohol/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Receptores Opioides kappa/efectos de los fármacos , Convulsiones/tratamiento farmacológico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/uso terapéutico , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Trastornos del Sistema Nervioso Inducidos por Alcohol/prevención & control , Analgésicos no Narcóticos/farmacología , Analgésicos no Narcóticos/uso terapéutico , Analgésicos Opioides/farmacología , Analgésicos Opioides/uso terapéutico , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Encéfalo/metabolismo , Encéfalo/fisiopatología , Depresores del Sistema Nervioso Central/efectos adversos , Modelos Animales de Enfermedad , Interacciones Farmacológicas/fisiología , Sinergismo Farmacológico , Etanol/efectos adversos , Masculino , Naltrexona/efectos adversos , Naltrexona/análogos & derivados , Receptores Opioides kappa/metabolismo , Convulsiones/inducido químicamente , Convulsiones/fisiopatología , Especificidad de la Especie , Síndrome de Abstinencia a Sustancias/fisiopatología , Síndrome de Abstinencia a Sustancias/prevención & control , Resultado del TratamientoRESUMEN
The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. Earlier work indicates that sensitivity to the anticonvulsant effect of ALLO was enhanced during ethanol (EtOH) withdrawal in rats and in C57BL/6 mice, an inbred strain with mild EtOH withdrawal. In contrast, ALLO sensitivity was reduced during EtOH withdrawal in DBA/2 mice, an inbred strain with severe EtOH withdrawal. Thus, the present studies examined ALLO sensitivity during EtOH withdrawal in another animal model of EtOH withdrawal severity, the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines. Male mice were exposed to EtOH vapor or air for 72 h. During peak withdrawal, animals were injected with ALLO [0, 3.2, 5, 10 or 17 mg/kg, intraperitoneally (i.p.)] and tested for their sensitivity to the anticonvulsant effect. In separate studies, potentiation of GABA-stimulated chloride uptake by ALLO (10 nm to 10 microm) was assessed in microsacs prepared from mouse brain mice during peak withdrawal. Notably, WSP mice were cross-tolerant to the anticonvulsant effect of ALLO during EtOH withdrawal (i.e. significant decrease in the efficacy of ALLO) when compared with values in air-exposed mice. In contrast, sensitivity to the anticonvulsant effect of ALLO was unchanged during EtOH withdrawal in the WSR line. Functional sensitivity of GABA(A) receptors to ALLO was significantly decreased during EtOH withdrawal in WSP mice in a manner consistent with the change in behavioral sensitivity to ALLO. These findings suggest that mice selectively bred for differences in EtOH withdrawal severity are differentially sensitive to ALLO during EtOH withdrawal.
Asunto(s)
Convulsiones por Abstinencia de Alcohol/metabolismo , Anticonvulsivantes/metabolismo , Moduladores del GABA/metabolismo , Pregnanolona/metabolismo , Receptores de GABA-A/metabolismo , Convulsiones por Abstinencia de Alcohol/genética , Animales , Anticonvulsivantes/administración & dosificación , Cloruros/metabolismo , Relación Dosis-Respuesta a Droga , Moduladores del GABA/administración & dosificación , Masculino , Ratones , Ratones Endogámicos , Pregnanolona/administración & dosificación , Prosencéfalo/efectos de los fármacos , Prosencéfalo/metabolismo , Receptores de GABA-A/efectos de los fármacos , Especificidad de la Especie , Estadísticas no Paramétricas , Esteroides/administración & dosificación , Esteroides/metabolismoRESUMEN
The hypothesis that kappa-opioid system activity may in part mediate convulsions exhibited during ethanol withdrawal was tested by exposing Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice to chronic ethanol. Whole brain was harvested for RNA isolation and prodynorphin mRNA steady-state levels in whole brain were examined using Northern blot analysis. The data revealed significantly increased levels of prodynorphin mRNA expression in mice susceptible to ethanol withdrawal convulsions after withdrawal, with no corresponding increase in prodynorphin steady-state levels in mice resistant to ethanol withdrawal convulsions. These findings were not due to basal differences in prodynorphin expression between the WSP and WSR mice. To verify that the differences observed were not due to an ethanol-induced global alteration in gene transcription, mRNA levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were measured. Glyceraldehyde-3-phosphate dehydrogenase expression was unchanged following both chronic exposure to ethanol and chronic exposure followed by withdrawal. These results extend our understanding of prodynorphin's role in generalized seizure activity to include ethanol withdrawal-induced convulsions. Our findings suggest that prodynorphin expression is modulated during ethanol withdrawal convulsions, or alternatively, prodynorphin may mediate the severity of ethanol withdrawal convulsions.