Vitamin C administration attenuated artemether induced hepatic injury in rats / La aAdministración de vitamina C atenúa la lesión hepática inducida por artemeter en ratas

This research was designed to investigate the potential protective effect of vitamin C supplementation against hepatocyte ultrastructural alterations induced by artemether (antimalarial drug) administration. Twenty-four adult male albino rats were used in this study and were divided into four groups (n=6). Group I served as a control and rats in group II administrated artemether (4 mg/kg B.W) orally for three consecutive days. Group III administered artemether plus a low dose of vitamin C (2.86 mg/kg/l water) while group IV received artemether plusa high dose of vitamin C (8.56 mg/kg). At the end of the experimental period (14 days), the harvested liver tissues were examined by transmission electron microscopy (TEM), and blood samples were assayed for biomarkers of liver injury and oxidative stress. Artemether significantly (p<0.05) augmented biomarkers of liver injury such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and oxidative stress such as superoxide dismutase (SOD), Glutathione Peroxidase (GPX), and caused degeneration and damage of the rough endoplasmic reticulum and disrupted mitochondria. The blood sinusoids were also damaged with distortion of their canaliculi. Administration of vitamin C showed improvement of liver biomarkers, and liver parenchyma, especially in a high dose of vitamin C.We concludes that vitamin C is a partial protective agent against artemether-induced liver injury.

Esta investigación fue diseñada para investigar el posible efecto protector de la vitamina C contra las alteraciones ultraestructurales de los hepatocitos, inducidas por la administración de arteméter (medicamento antipalúdico). En el estudio se utilizaron 24 ratas albinas macho adultas y se dividieron en cuatro grupos (n = 6). El grupo I fue designado como control y las ratas en el grupo II se adminstró Arteméter (4 mg / kg de peso corporal) por vía oral durante tres días consecutivos. En el grupo III se administró arteméter, además de una dosis baja de vitamina C (2,86 mg / kg / l de agua) mientras que el grupo IV recibió arteméter más una dosis alta de vitamina C (8,56 mg / kg). Al final del período experimental (14 días), los tejidos hepáticos recolectados se examinaron por microscopía electrónica de transmisión (MET), y las muestras de sangre se analizaron en busca de biomarcadores de daño hepático y estrés oxidativo. El arteméter aumentó significativamente (p <0,05) los biomarcadores de daño hepático como alanina aminotransferasa (ALT), aspartato aminotransferasa (AST) y estrés oxidativo como superóxido dismutasa (SOD), glutatión peroxidasa (GPX) y causó degeneración y daño de la retículo endoplásmico rugoso y mitocondrias alteradas. Los sinusoides sanguíneos también fueron dañados con la distorsión de sus canalículos. La administración de vitamina C mostró una mejoría de los biomarcadores hepáticos y el parénquima hepático, especialmente en una dosis alta de vitamina C. Concluimos que la vitamina C es un agente protector parcial contra la lesión hepática inducida por arteméter.

New observations on the effect of camellia oil on fatty liver disease in rats / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Camellia oil has become an important plant oil in China in recent years, but its effects on non-alcoholic fatty liver disease (NAFLD) have not been documented. In this study, the effects of camellia oil, soybean oil, and olive oil on NAFLD were evaluated by analyzing the fatty acid profiles of the plant oils, the serum lipids and lipoproteins of rats fed different oils, and by cytological and ultrastructural observation of the rats' hepatocytes. Analysis of fatty acid profiles showed that the polyunsaturated fatty acid (PUFA) n-6/n-3 ratio was 33.33 in camellia oil, 12.50 in olive oil, and 7.69 in soybean oil. Analyses of serum lipids and lipoproteins of rats showed that the levels of total cholesterol and low-density lipoprotein cholesterol in a camellia oil-fed group (COFG) were lower than those in an olive oil-fed group (OOFG) and higher than those in a soybean oil-fed group (SOFG). However, only the difference in total cholesterol between the COFG and SOFG was statistically significant. Cytological observation showed that the degree of lipid droplet (LD) accumulation in the hepatocytes in the COFG was lower than that in the OOFG, but higher than that in the SOFG. Ultrastructural analysis revealed that the size and number of the LDs in the hepatocytes of rats fed each of the three types of oil were related to the degree of damage to organelles, including the positions of nuclei and the integrity of mitochondria and endoplasmic reticulum. The results revealed that the effect of camellia oil on NAFLD in rats was greater than that of soybean oil, but less than that of olive oil. Although the overall trend was that among the three oil diets, those with a lower n-6/n-3 ratio were associated with a lower risk of NAFLD, and the effect of camellia oil on NAFLD was not entirely related to the n-6/n-3 ratio and may have involved other factors. This provides new insights into the effect of oil diets on NAFLD.

Vitamin E protects against hepatocyte ultrastructural damage induced by high fat diet in a rat model of pre-diabetes / La vitamina E protege contra el daño ultraestructural de los hepatocitos inducido por la dieta alta en grasas en un modelo de pre-diabetes en ratas

SUMMARY: We sought to investigate the potential protective effect of Vitamin E supplementation against hepatocyte ultrastructural alterations induced by high fat diet (HFD) in a rat model of pre-diabetes. Therefore, rats were either fed with HFD (model group) or a standard laboratory chow (control group) for 12 weeks before being sacrificed. The protective group fed on a HFD and started the treatment with vitamin E (100 mg/kg/day, i.p) from day 1 until being sacrificed at week 12. The harvested liver tissues were examined using transmission electron microscopy (TEM) and blood samples were assayed for biomarkers of liver injury and prediabetes. TEM images showed that HFD induced profound pathological changes to the hepatocyte ultrastructure as demonstrated by degenerated hepatocytes with damaged cytoplasm that have mitochondrial swelling, dilation of endoplasmic reticulum, blebbing of plasma membranes, and cytoplasmic accumulations of lipid droplets and vacuoles, which were substantially but not completely protected with vitamin E. In addition, HFD significantly (p<0.05) augmented biomarkers of liver injury and pre-diabetes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), total cholesterol (TC), triglycerides (TG), and low density lipoprotein cholesterol (LDL-C), which were significantly (p<0.05) reduced with vitamin E except TNF-α and TC. Furthermore, none of these biomarkers were reduced to the control level by vitamin E. We conclude that vitamin E is a partial protective agent against HFD-induced liver injury and pre-diabetes.

RESUMEN: El objetivo de este estudio fue investigar el posible efecto protector de la administración de suplementos de vitamina E contra las alteraciones ultraestructurales de los hepatocitos inducidas por una dieta rica en grasas (DRG) en un modelo de prediabetes en ratas. Antes de ser sacrificadas las ratas fueron alimentadas con DRG (grupo modelo) o un alimento estándar de laboratorio (grupo control) durante 12 semanas. El grupo protector se alimentó con una DRG y comenzó el tratamiento con vitamina E (100 mg/kg/día, i.p) desde el día 1 hasta sacrificarlo en la semana 12. Los tejidos hepáticos recolectados se examinaron mediante microscopía electrónica de transmisión (MET) y se tomaron muestras de sangre y se analizaron los biomarcadores de daño hepático y prediabetes. Las imágenes de MET mostraron que el DRG indujo cambios patológicos profundos en la ultraestructura de los hepatocitos, como lo demuestran los hepatocitos degenerados con citoplasma dañado e hinchazón mitocondrial, dilatación del retículo endoplasmático, formación de ampollas en las membranas plasmáticas y acumulaciones citoplásmicas de gotas de lípidos y vacuolas, los que fueron sustancialmente protegidas con vitamina E. Además, DRG aumentó significativamente (p <0,05) los biomarcadores de daño hepático y prediabetes como alanina aminotransferasa (ALT), aspartato aminotransferasa (AST), factor de necrosis tumoral alfa (TNF-α), malondialdehído (MDA), colesterol total (CT), triglicéridos (TG) y lipoproteína de colesterol de baja densidad (LDL-C), la cual se redujo significativamente (p <0,05) con vitamina E, excepto TNF-α y CT. Ninguno de estos biomarcadores se redujo al nivel de control por la vitamina E. Concluimos que la vitamina E es un agente protector parcial contra la lesión hepática inducida por DRG y la prediabetes.

Differential therapeutic effects of Crataegus aronia and simvastatin on the hepatocyte ultrastructure in hepatic steatosis / Efectos terapéuticos diferenciales de Crataegus aronia y simvastatina sobre la estructura del hepatocito en la esteatosis hepática

Complications of fat accumulation in liver, hepatic steatosis such as liver cirrhosis and liver failure are among the common public health problems. We sought to investigate the damage to the hepatocyte ultrastructure induced by high fat diets (HFD) and compared the therapeutic effects at the cellular level of two antioxidant and lipid lowering agents; Crataegus aronia extracts and simvastatin on hepatic steatosis. Rats were either fed with HFD (model group) or low fat diets (LFD) (control group) for 15 weeks before being sacrificed and therapeutic groups started the treatment with these agents after week 11 until the sacrifice day. Harvested liver tissues were examined using transmission electron microscopy (TEM) and liver homogenates were assayed for markers of anti-oxidative stress that are known to be modulated in liver injury. TEM examinations of the model group showed a profound damage to the hepatocytes compared to the control group as demonstrated by steatosis, damaged mitochondria and vaculated cytoplasm, disrupted rough and smooth endoplasmic reticulum and nuclear membrane, dilated intercellular space between hepatocytes, and alterations in lysosomes. In addition, HFD ameliorated the anti-oxidant glutathione (GSH) and augmented the oxidative stress TBARS biomarkers. Both Crataegus aronia and simvastatin significantly reduced lipids and TBARS, and treated damage to hepatic cells, but hepatocyte structures were differentially responded to these agents. However, only Crataegus aronia induced GSH (p=0.001). We conclude that HFD-induced hepatic steatosis caused a substantial damage to the hepatocyte's ultrastructures, and Crataegus aronia and simvastatin treatments differentially reversed hepatic injuries.

Las complicaciones de la acumulación de grasa en el hígado, la esteatosis hepática como la cirrosis hepática y la insuficiencia hepática se encuentran entre los problemas comunes de salud pública. Se intentó investigar el daño a la ultraestructura de los hepatocitos inducido por la dieta alta en grasas (DAG) y se compararon los efectos terapéuticos a nivel celular de dos antioxidantes y agentes hipolipemiantes; Extracto de Crataegus aronia y simvastatina sobre esteatosis hepática. Las ratas fueron alimentadas con DAG (grupo modelo) o dieta baja en grasa (DBG) (grupo control) durante 15 semanas antes de sacrificarse y los grupos terapéuticos comenzaron el tratamiento con estos agentes después de la semana 11 hasta el día del sacrificio. Se examinaron los tejidos hepáticos usando microscopía electrónica de transmisión (MET) y se analizaron homogeneizados de hígado para marcadores de estrés anti-oxidativo, que se sabe están modulados en la lesión hepática. Los exámenes MET del grupo DAG mostraron un grave daño de los hepatocitos en comparación con el grupo control, demostrado por esteatosis, daño mitocondrial y citoplasma vacío, retículo endoplásmico rugoso y liso y membrana nuclear, el espacio intercelular dilatado entre hepatocitos y alteraciones en los lisosomas. Además, DAG mejoró el anti-oxidante glutatión (GSH) y aumentó el estrés oxidativo TBARS biomarcadores. Tanto Crataegus aronia como simvastatina redujeron significativamente los lípidos y TBARS, trataron el daño a las células hepáticas, pero las estructuras de hepatocitos respondieron diferencialmente a estos agentes. Sin embargo, sólo Crataegus aronia indujo GSH (p = 0,001). Concluimos que la esteatosis hepática inducida por HFD causó un daño sustancial a la ultraestructura del hepatocito y los tratamientos de Crataegus aronia y simvastatina diferenciaron las lesiones hepáticas.

Effect of cigarette smoking on the structure of hepatocytes: TEM study / Efecto del tabaquismo sobre la estructura de los hepatocitos: estudio MET

The liver is one of the major organs that is indirectly affected by cigarette smoke. The aim of this project is to define the histologic and ultrastructural changes in normal liver cells after exposing animals to cigarette smoke. Thirty albino rats were exposed to cigarette smoke for 90 days, followed by morphologic examination of their livers under light microscope and electron microscope. The liver cells of cigarette smoke exposed rats showed mild swelling with increased eosinophilia. Ultrastructural examination of these cells demonstrated cytoplasm with highly proliferated and crowded mitochondria. There were many electron dense mitochondria. These mitochondria were pleomorphic in shape compared to mitochondrias of control rats. Also, loss of mitochondrial cristae and widening of the intermembranous space was noticed. It is concluded that smoking exerts cellular damage and oxidative stress on normal liver cells resulting in ultrastructural changes.

El hígado es uno de los principales órganos indirectamente afectado por el humo del cigarrillo. El objetivo de este trabajo fue definir los cambios histológicos y ultraestructurales de las células normales del hígado después de exponer a los animales al humo del cigarrillo. Treinta ratas albinas fueron expuestas al humo de cigarrillo durante 90 días, seguido de un examen morfológico de los hígados bajo microscopio de luz y microscopio electrónico. Las células hepáticas de las ratas expuestas al humo de cigarrillo mostraron una leve inflamación con un aumento de la eosinofilia. En el examen ultraestructural de estas células se observó el citoplasma mitocondrial altamente proliferado y saturado. Se observó gran cantidad de mitocondrias electrón-densas y éstas presentaban forma pleomórfica en comparación con las mitocondrias del grupo control. Además, se observó pérdida de las crestas mitocondriales y ensanchamiento del espacio intermembranoso. Se concluye que el tabaquismo ejerce daño celular y estrés oxidativo en las células hepáticas normales, lo que resulta en la aparición de cambios ultraestructurales.

Morfometría nuclear de hepatocitos esteatósicos y esteatósicos estimulados con laser infrarrojo / Nuclear morphometric of the steatotic hepatocytes and stimulated steatotisc with infrared laser

Un total de 24 ratas hembras de 4 meses de vida con peso aproximado de 250 g recibieron una solución de alcohol 40 % disuelto en agua lo cual derivó en una esteatosis alcohólica multivesicular. A 12 de estas ratas esteatósicas se le aplicó estimulaciones de láser infrarrojo con dosis de 8 J/cm2 durante 15 días consecutivos. Posteriormente las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado esteatósico como del estimulado para enseguida ser procesadas para microscopía electrónica de transmisión. De hepatocitos esteatósicos y esteatósicos estimulados se obtuvieron microfotografías electrónicas de transmisión con aumentos finales de 9.500 X, las cuales fueron sometidas a estudios morfométricos para determinar tanto el número de poros nucleares como de fracciones volumétricas de los siguientes componentes celulares: Areas celular y nuclear, fracciones volumétricas de núcleos y nucléolos, eu y heterocromatina. De igual manera se determinó la relación nucleo-citoplasmática de ambos tipos celulares. Del análisis de los resultados entre hepatocitos alcohólicos y alcohólicos estimulados se visualiza que existen notables diferencias en todos los componentes celulares cuantificados. Se concluye que los efectos de la estimulación con lásr infrarrojo provoca en los hepatocitos una drástica transformación en su ultraestructura y en su morfología, situación que se traduciría, por ende, en una variación funcional, representando de esta manera el efecto que dicha estimulación provoca en los hepatocitos.

A total of 24 female rats, aged 4 months and weighing approximately 250 g and they given a solution of 40 % alcohol dissolved in water, leading to alcoholic multivesicular steatosis and 12 of rats was given and infrared laser with dose of 8 J/cm2 during 15 d. The rats were then killed and samples of steatosis and stimulated and were taken and processed for examination by transmission electron microscope. Transmission electron microscope microphotographs steatotic hepatocytes and stimulated steatotic were obtained with final magnification of 9,500 X. They were subjected to morphometric studies to determine the number of nuclear pores and volumetric fractions and areas the following components: cellular and nuclear area, volumetric fractions of nucleus, nucleolus, eu and heterochromatin, nucleocytoplamic ratio of each cell type was determined. Analysis of the results between alcoholic hepatocytes and stimulate alcoholic shows that noticeable differences exist in all the cell components quantified. It is concluded that the effects of the stimuli of laser infrared provoke in the hepatocytes, a drastic transformation of their ultrastructure and morphology. This finally leads to functional variations, representing the effects produced by this stimulate in the hepatocytes.

Ultrastructure of fetal mice hepatocytes exposed in utero to diazepam / Ultraestructura de hepatocitos fetales de ratón expuestos in utero a diazepam

Previous research has shown that fetal mice hepatic cells from females treated with diazepam (Valium) during pregnancy depict cytoplasmic and nuclear modifications when observed with photonic microscope. The purpose of this work is to investigate if diazepam administered subcutaneously (SC) to pregnant mice females induces ultraestructural alterations in the cytoplasmic organelles and nucleus to fetal hepatocytes. Transmission electron microscopy observations of fetal hepatocytes from pregnant females treated with a single daily dose of diazepam 2.7 mg/kg/bw/SC administered from 6th to 15th days of gestation revealed that they frequently presented disorganized and dilated rough endoplasmic reticulum cisterns, membranous elements, abundant Golgi complex and glycogen granules, around large vacuoles. The voluminous nucleus shows atypical distribution of chromatin. These alterations could modify the hepatocyte's physiology and probably persist after birth.

Estudios previos muestran que las células hepáticas de fetos de ratón, de hembras tratadas con diazepam (Valium) durante la gestación, presentan modificaciones citoplásmicas y nucleares que se pueden observar con el microscopio fotónico, por lo que el propósito de este trabajo es determinar si el diazepam administrado por vía subcutánea (SC) a hembras gestantes de ratón, induce alteraciones ultraestructurales de los organelos citoplásmicos y del núcleo de los hepatocitos fetales. En los fetos de ratón del grupo experimental de hembras gestantes, tratadas con dosis únicas diarias de 2,7 mg/kg de peso corporal administradas por vía SC del 6° al 15° día de la gestación, se observó con el microscopio electrónico de transmisión que los hepatocitos fetales presentaban con frecuencia retículo endoplásmico rugoso desorganizado, con cisternas dilatadas; había elementos membranosos y complejo de Golgi abundante, al igual que gránulos de glucógeno que rodeaban a grandes vacuolas. Los núcleos eran voluminosos, con la cromatina distribuida atípicamente. Estas alteraciones podrían modificar la fisiología de los hepatocitos y probablemente persistan después del nacimiento.

Microscopía electrónica de nucleos hepáticos estimulados con dosis crecientes de laser infrarrojo / Electron microscopy hepatic nuclei stimulated with increasing doses of infrared laser

Un total de 20 ratas hembras de 4 meses de vida con peso aproximado de 250 gramos fueron divididas en 4 grupos de animales rotulados como A, B, C y D. El grupo A corresponde al control y los demás grupos recibieron respectivamente estimulaciones con laser infrarrojo con dosis crecientes de 4, 8 y 16 Joules por cm2 durante 15 días consecutivos en 5 puntos del hígado. Posteriormente las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado control como de los estimulados con inducciones infrarrojas para enseguida ser procesadas para microscopía electrónica de transmisión. De los hepatocitos se obtuvieron microfotografías electrónicas de transmisión con aumentos finales de 9.500 X, las cuales fueron sometidas a estudios morfométricos para determinar fracciones volumétricas de sus núcleos y estructuras nucleares. De igual manera se cuantificaron las áreas nucleares, celulares y se determinó la relación núcleo-citoplasmática de los tipos celulares estudiados. Analizados los resultados entre los hepatocitos controles e irradiados se visualiza que existen notables diferencias en la totalidad de los parámetros cuantificados concluyéndose que los efectos de las estimulaciónes infrarrojas con dosis crecientes genera transformaciones en su ultraestructura y en su morfología, fundamentalmente en el aumento de los volúmenes nucleares, y celulares, los volúmenes de cromatina y de la relación-núcleo-citoplasmática situación que se traduciría en una variación funcional, representando de esta manera un efecto evidente que estas inducciones infrarrojas generan.

Twenty-four four-month-old female rats weighing approximately 250 grams were divided into four groups labeled A, B, C and D. A corresponds to the normal group and the other groups received stimulation increasing doses with 4, 8 and 16 J/cm2 of infrared laser respectively for 15 consecutive days in five points of the liver. The rats were then sacrificed and samples of normal liver and liver stimulated with infrared inductions were extracted for immediate processing via transmission electron microscopy. From cell types transmission electron microphotographs were obtained at magnifications of 9500 X these were subjected to morphometric studies to determine volumetric fractions of the nuclei and nuclear structures. Likewise, cell and nuclear areas and nuclear-citoplasmatic relation were quantified. Analysis of the results between normal and radiated hepatocytes revealed notable differences in all the cell components quantified. It is concluded that the effects of increasing infrared stimulation doses brings transformation in their ultrastructure and morphology, fundamentally in the considerable increase in nuclear volume, chromatin volume and the nuclear-citoplasmatic relation, which ultimately translates into a functional variation, thus representing an obvious impact produced by these infrared inductions.

Metformin induces ultrastructural alterations in hepatocytes of spontaneously hypertensive rats / La metformina induce alteraciones ultraestructurales en hepatocitos de ratas espontáneamente hipertensas

Metformin, an oral biguanide approved for the treatment of type II diabetes is widely prescribed for other clinical conditions. Currently, metformin is being investigated as potential anti-tumor agent. However, there have been recent concerns about hepatotoxicity associated with the use of metformin. This study, by means of high resolution transmission electron microscopy (TEM) and morphometry, investigated potential ultrastructural changes induced by metformin treatment on the hepatocytes of spontaneously hypertensive rats (SHR). Morphometric analysis was carried out on images of randomly selected cells from sectioned gluteraldehyde-osmium-fixed, Epon embedded liver tissue. One-way analysis of variance (ANOVA) on morphometric data showed statistically significant differences in the mean volume density (MVD) of lipid bodies (F=136.48, P<0.0001)and mean surface density (MSD) of endoplasmic reticulum (ER) (F=12.45, P<0.003) between hepatocytes of control (n=8) and metformin-treated (MT) (n=8) animals. MVD for control group was 5.42% (±0.36 SEM) but decreased significantly in the MT group (1.13%, ±0.04 SEM). Similarly, MSD of ER for control was 24.7 µm2/µm3 (±1.64 SEM) but decreased for MT animals (18.90 µm2/µm3, ±0.28 SEM). These data are most likely consistent with the effects of metformin on lipid metabolism, and may not reflect on hepatotoxicity induced by the drug, in SHRs.

La metformina, una biguanida oral aprobada para el tratamiento de la diabetes tipo II, es también ampliamente prescrita para otros cuadros clínicos. Actualmente, la metformina está siendo investigada como posible agente anti-tumoral. Sin embargo, ha habido recientes preocupaciones acerca de la hepatotoxicidad asociada con el uso de metformina. En este estudio, por medio de la microscopía electrónica de transmisión (MET) de alta resolución y morfometría, se investigaron los posibles cambios ultraestructurales, inducidos por el tratamiento con metformina, en los hepatocitos de ratas espontáneamente hipertensas (REH). El análisis morfométrico se llevó a cabo en imágenes de células seleccionadas al azar a partir de tejido hepático seccionado, fijado con glutaraldehído-osmio e inmerso en Epon. El análisis de la varianza (ANOVA) de los datos morfométricos mostró diferencias significativas en la densidad de volumen medio (DVM) de cuerpos lipídicos (F=136,48, P<0,0001) y la densidad de superficie media (DSM) del retículo endoplasmático (RE) (F=12,45, P<0,003) entre los hepatocitos control (n=8) y los animales tratados con metformina (MT) (n=8). La DVM para el grupo control fue de 5,42% (±0,36 EEM), pero disminuyó significativamente en el grupo MT (1,13%, ±0,04 EEM). Del mismo modo, la DSM del RE para el grupo control fue de 24,7 µm2/µm3 (±1,64 EEM), pero disminuyó para los animales MT (18,90 µm2/µm3, ±0,28 EEM). Estos datos están probablemente más relacionados con los efectos de la metformina sobre el metabolismo de los lípidos, y no se relacionarían con la hepatotoxicidad por inducción de la droga, en REH.

El retículo endoplasmático liso en hepatocitos estimulados con distintas dosis de láser infrarrojo / The smooth endoplasmic reticulum in hepatocytes stimulated with different doses of infrared laser

Veinticuatro ratas hembras Sprague Dawley de 4 meses de vida con peso aproximado de 250 g, fueron divididas en cuatro grupos (A, B, C y D), donde el grupo A (control) no recibió estimulación infrarroja, B se irradió con láser infrarrojo 4 J/cm², C con dosis de 8 J/cm² y D con 16 J/cm². La estimulación infrarroja se realizó diariamente, por 15 días ininterrumpidos. Las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado normal (control) como estimulado con las distintas dosis infrarrojas, las que fueron procesadas para microscopía electrónica de transmisión. De los hepatocitos normales y estimulados, se obtuvieron microfotografías con aumentos finales de hasta 36.500 X, que fueron sometidas a estudios morfométricos para determinar fracciones volumétricas con especial énfasis en el retículo endoplásmico liso (REL) y de los siguientes componentes celulares: retículo endoplasmático rugoso (RER), mitocondrias, glicógeno, eu y heterocromatina. De igual manera se cuantificaron las áreas celulares y nucleares. Del análisis de los resultados entre hepatocitos normales y estimulados con diferentes dosis infrarrojas, se visualiza que existen notables diferencias en todos los componentes celulares cuantificados particularmente el REL. Se concluye que las estimulaciones infrarrojas provocan una drástica transformación en la ultraestructura y morfología de los hepatocitos, lo que provocaría una variación funcional, representando de esta manera el efecto que estas estimulaciones provocan en este tipo celular.

A total of 24 female Sprague-Dawley rats aged 4 months and weighing approximately 250 g, were divided into four groups labeled A, B, C and D. Group A received no infrared stimulation and served as control. Group B was radiated with a dose of 4 J/cm² of infrared laser, Group C with doses of 8 J/cm² and Group D with 16 J/cm². This infrared stimulation was carried out daily for 15 days uninterrupted. The rats were then sacrificed and samples of both normal-control liver and liver stimulated with the different infrared doses were extracted for immediate processing via transmission electron microscopy. Transmission electron microphotographs were obtained at magnifications of 21300X from both normal and stimulated hepatocytes; these were subjected to morphometric studies to determine volumetric fractions with special emphasis on the smooth endoplasmic reticulum (SER) and the following cell components: rough endoplasmic reticulum (RER), mitochondria, glycogen, eu and heterochromatin. Likewise, cell and nuclear areas were quantified. Analysis of the results of normal and stimulated hepatocytes with different infrared doses showed considerable differences in all the quantified cell components and particularly from the SER it is concluded that the effects of these stimulations bring about a drastic transformation in the ultrastructure and morphology of the hepatocytes, which may ultimately translate into a functional variation, thus representing the effect that these stimulations cause in this cell type.

Micro-ultrastructure features of liver in Chinese Taihe black-bone silky fowl (Gallus gallus domesticus Brisson) / Características ultraestructurales de hígado en la gallina silky China Taihe de hueso negro (Gallus gallus domesticus Brisson)

In this work, the morphological features of liver in Chinese Taihe black-bone silky Fowl (BSF) were analyzed by light microscope and transmission electron microscopy. The results showed that two kinds of hepatocytes were present in Taihe BSF liver, i.e., the dark and the light hepatocyte. The dark hepatocyte was electron-dense and lager, with many organelles, mitochondria especially. The light hepatocytes were smaller than the dark. They had electron-lucent cytoplasm with a small number of organelles. Furthermore, there were lipolysosomes in the light hepatocyte. The numerous long and serried finger-like microvilli spread into bile canaliculus lumen. The glycogen granules intensely stained, spread in some hepatocytes. Numerous glycogen granules scattered in cytoplasm especially near bile canaliculi. However, lipid droplets were not observed in any hepatocytes. The natural apoptotic hepatocytes were observed in Taihe BSF liver. The hepatocytes which contain abundant uesicae-like endoplasmic reticulum closed the apoptotic hepatocytes and spread the process to approach the cell residual bodies. Besides, there was a macrophage with several phagosomes. In conclusion, the dark and the light hepatocyte were present in Chinese Taihe BSF liver. They were different from electron-dense and organelles. The hepatocytes of Taihe BSF could undergo natural apoptosis, regeneration and renew ability.

Fueron analizadas las características morfológicas ultraestructurales de hígado en la gallina sedosa china de hueso negro por microscopía óptica y microscopía electrónica de transmisión. Los resultados mostraron que se encontraron hepatocitos claros y oscuros en el hígado de la gallina china Taihe. El hepatocito oscuro era denso y de mayor tamaño. Tenía numerosos organelos, especialmente mitocondrias. Los hepatocitos claros eran más pequeños que los oscuros. El citoplasma presentó un pequeño número de organelos. Además, había lipolisosomas en los hepatocitos claros. Numerosas microvellosidades se extendían hacia los canalículos biliares. En algunos hepatocitos se observó una tinción marcada en los gránulos de glucógeno. Sin embargo, no se observaron gotas de lípidos en los hepatocitos. Se observaron los hepatocitos apoptóticos naturales en el hígado de la gallina Silky Taihe. Aquellos hepatocitos que contenían abundante retículo endoplásmico, cerraban los hepatocitos apoptóticos y extendían el proceso de acercamiento a cuerpos residuales celulares. También hubo un macrófago con varios fagosomas. En conclusión, los hepatocitos claros y oscuros estaban presentes en el hígado de la gallina Taihe china. Estos diferían de electrones de alta densidad y organelos. Los hepatocitos de la gallina Taihe presentaron una apoptosis natural y capacidad de regeneración.

Análisis morfométrico de hepatoesteatosis microvesicular alcohólica de ratas / Morphometric analysis of alcoholic microvesicular hepatic steatosis in rats

Un total de 24 ratas hembras de 4 meses de vida con peso aproximado de 250 gramos fueron divididas en dos grupos de animales rotulados como A y B. El grupo A se mantuvo con pellet y agua ad libitum sirviendo como controles mientras que el grupo B conservaba el pellet y recibía una solución de alcohol 40% disuelto en agua lo cual derivó en una esteatosis alcohólica multivesicular. Ambos grupos se mantuvieron en estas condiciones por 60 días. Posteriormente las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado normal-control como de hígado graso para enseguida ser procesadas para microscopía electrónica de transmisión. De hepatocitos normales y esteatósicos se obtuvieron microfotografías electrónicas de transmisión con aumentos finales de 9.500X, las cuales fueron sometidas a estudios morfométricos para determinar fracciones volumétricas de los siguientes componentes celulares: Retículo endoplasmático rugoso, mitocondrias, inclusiones lipídicas y de glicógeno, eu y heterocromatina. De igual manera se cuantificaron las áreas celulares y nucleares. Del análisis de los resultados entre hepatocitos normales y alcohólicos se visualiza que existen notables diferencias en todos los componentes celulares cuantificados. Se concluye que los efectos de la ingesta diaria de alcohol provoca en los hepatocitos una esteatosis microvesicular que genera una drástica transformación en su ultraestructura y en su morfología, situación que se traduciría, por ende, en una variación funcional, representando de esta manera el efecto que esta droga provoca en los hepatocitos.

A total of 24 female rats, aged 4 months and weighing approximately 250 g, were divided into two groups, called A and B. The group A animals were kept on pellets and water ad libitum and served as controls, while group B animals were fed pellets and given a solution of 40% alcohol dissolved in water, leading to alcoholic multivesicular steatosis. Both groups were kept under these conditions for 60 days. The rats were then euthanized and samples of normal-control and fatty liver were taken and processed for examination by transmission electron microscope. Transmission electron microscope microphotographs of normal and steatotic hepatocytes were obtained with final magnification of 9,500 X. They were subjected to morphometric studies to determine the volumetric fractions of the following cell components: Rough Endoplasmic Reticulum, mitochondria, lipid and glycogen inclusions, and eu- and heterochromatin. In addition, the cell and nucleus areas were quantified and the nucleo cytoplasmic ratio of each cell type was determined. Analysis of the results between normal and alcoholic hepatocytes shows that noticeable differences exist in all the cell components quantified. It is concluded that the effects of the daily consumption of alcohol provoke microvesicular steatosis in the hepatocytes, generating a drastic transformation of their ultrastructure and morphology. This finally leads to functional variations, representing the effects produced by this drug in the hepatocytes.

Protective effects of lycopene on oxidative stress, proliferation and autophagy in iron supplementation rats

Lycopene is common in diet and known for its antioxidant activities. However, the impact of lycopene on iron metabolism is poorly investigated. In this study, we hypothesize that lycopene can prevent iron-mediated oxidative stress, proliferation and autophagy in liver and use a rat model of nutritional iron supplementation to confirm its intervention in these defence mechanisms. We found that iron supplementation induced cell proliferation predominantly in non parenchymal cells compared with hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggered autophagy as evidenced by the formation of autophagic vesicles detected by LC3-B staining. Iron supplementation also induced morphologic alterations of the mitochondrial membranes probably due to increased lipid peroxidation as indicated by elevated iron and malondialdehyde concentrations in serum and tissues. Lycopene reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and enhancing the total superoxide dismutase activities in serum and tissues. The result suggest that lycopene prevents iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

Persistencia de los efectos del láser infrarrojo 6 J/cm² en hepatocitos de rata / Persistence of the effects of 6 J/cm2 infrared laser in rat hepatocytes

Hígados de ratas Sprague Dawley fueron irradiados con dosis diarias de 6 J/cm2 emitida por el láser AsGa equivalente a 904 nm durante 15 días De estos animales previamente anestesiados fueron sacrificados transcurridos 5, 10, 30, 45 y 60 días post irradiación para posteriormente obtener quirúrgicamente muestras de hígado y ser procesadas para microscopía electrónica de transmisión, aplicando técnicas morfométricas utilizando aumentos de 8.500 X con especial énfasis en cuantificar fracciones volumétricas de componentes celulares con el objetivo de precisar la duración de las estimulaciones infrarrojas. El análisis de los resultados entre hepatocitos controles e irradiados con dosis de 6 J/cm2 y tiempo de estimulación infrarroja revela que existen marcadas diferencias entre las fracciones volumétricas de componentes celulares determinantes de funcionalidad celular e involucrados en síntesis proteica, cuantificación que demuestra claramente que el efecto del láser infrarrojo persiste hasta los 30 días post estimulación, evidenciándose modificaciones de organelos que revelan alta funcionalidad, mientras que sobre este tiempo es observada una notable inhibición de dicha funcionalidad, concluyéndose entonces que los efectos de radiación infrarroja persisten en tiempos precisos provocando en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad. en estas células de elevado metabolismo.

Livers of Sprague Dawley rats were irradiated with daily doses of 6 J/cm2 emitted by a laser AsGa, equivalent to 904 nm during 15 days. Experiment animals were anaesthetised and killed after 5, 10, 30, 45 and 60 days post irradiation, in order to obtain samples of liver by surgery. These were processed for transmission electron microscopy, and morphometric techniques were applied using 8,500 X magnification with special emphasis on measuring the volumetric fractions of cell components in order to determine the duration of infrared stimulation. Analysis of the results between control hepatocytes and those irradiated with doses of 6 J/cm2 and by period after infra-red stimulation revealed the existence of marked differences between the volumetric fractions of cell components which determine cell function or are involved in protein synthesis. The measurements show clearly that the effect of the infrared laser persists up to 30 days post stimulation, with evidence of modifications of organelles revealing high functioning, while after 30 days a notable inhibition of this functioning is observed. It is therefore concluded that the effects of infrared radiation persist for precise times, provoking a drastic transformation in hepatocyte components, and thus the functioning of these high-metabolism cells.

Estudio Morfométrico del Efecto de Láser Infrarrojo 16 J/cm2 sobre Hepatocitos de Rata / Morphometric Study of Effect 16 J/cm2 Infrared Laser in Rat Hepatocytes

Hígados de ratas Sprague Dawley fueron irradiados durante 15 días con dosis diarias de 8 y 16 J/cm2 proveniente del láser AsGa equivalente a 904 nm. De estos animales previamente anestesiados, fueron quirúrgicamente obtenidas muestras de hígado y posteriormente procesadas para microscopía electrónica de transmisión, siendo estudiadas y sometidas a técnicas morfométricas utilizando aumentos de 8.500 X con especial énfasis en cuantificar fracciones volumétricas de componentes celulares. El análisis de los resultados entre hepatocitos irradiados con dosis de 8 y 16 J/cm2 revela que existen marcadas diferencias entre las fracciones volumétricas de componentes celulares determinantes de funcionalidad celular e involucrados en síntesis proteica, cuantificación que demuestra claramente que dosis de 8 J/cm2 logra un óptimo de estimulación para lograr alta funcionalidad, mientras que 16 J/cm2 evidencia notable inhibición de dicha funcionalidad, concluyéndose entonces que los efectos de dosis específicas de radiación infrarroja provoca en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad representando el efecto de estas estimulaciones sobre este tipo celular de elevado metabolismo.

Sprague Dawley rat livers were irradiated with 8 and 16 J/cm2 daily doses during 15 days from the AsGA laser, equivalent to 904 nm. Liver samples were surgically removed from rats previously anesthetized, then processed for electronic microscopic transmission and subsequently studied and subjected to morphometric techniques using 8.500 X enlargements with special emphasis on quantifying cellular component volumetric fractions. The result analysis of the hepatocytes irradiated with 8 and 16J/cm2 doses revealed that there were marked differences between the volumetric fractions of cellular components that determine cellular functionality and protein synthesis, a quantification that clearly demonstrates that with a dose of 8 J/cm2, the optimum stimuli is achieved in order to obtain high functionality, while with a dose of 16 J/cm2 a notable reduction of that functionality is noted, concluding that the effects of specific infrared radiation doses provoke a drastic transformation of its components in hepatocytes, and therefore its functionality, activating or inhibiting it, and representing the effect of these stimulations over this elevated metabolism cellular type.

Tnf-a production and apoptosis in hepatocytes after listeria monocytogenes and salmonella typhimurium invasion / Produção de tnf-a e apoptose em hepatócitos após invasão por listeria monocytogenes e salmonella typhimurium

Invasion of hepatocytes by Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) can stimulate tumor necrosis factor alpha (TNF-α) release and induce apoptosis. In this study, we compared the behavior of hepatocytes invaded by three L. monocytogenes serotypes (LM-4a, LM-4b and LM-1/2a) and by ST to understand which bacterium is more effective in the infectious process. We quantified TNF-α release by ELISA, apoptosis rates by annexin V (early apoptosis) and TUNEL (late apoptosis) techniques. The cell morphology was studied too. TNF-α release rate was highest in ST-invaded hepatocytes. ST and LM-1/2a induced the highest apoptosis production rates evaluated by TUNEL. LM-4b produced the highest apoptosis rate measured by annexin. Invaded hepatocytes presented various morphological alterations. Overall, LM-4b and LM-1/2a proved to be the most efficient at cell invasion, although ST adapted faster to the environment and induced earlier hepatocyte TNF-α release.

A invasão de hepatócitos por Listeria monocytogenes (LM) e Salmonella Typhimurium (ST) pode estimular a liberação do Fator de Necrose Tumoral (TNF-α) e induzir a apoptose celular. Neste estudo comparamos o comportamento de hepatócitos invadidos por três sorotipos de L. monocytogenes (LM-4a, LM-4b e LM-1/2a) e por ST para entender qual bacteria é mais efetiva no processo infeccioso. Nós quantificamos a liberação de TNF-α pelos hepatócitos por ELISA e as taxas de apoptose pelas técnicas de anexina V (apoptose precoce) e TUNEL (apoptose tardia). A morfologia das células foi estudada também. A taxa de liberação de TNF-α foi mais alta em hepatócitos invadidos por ST. ST e LM-1/2a induziram as maiores taxas de apoptose pelo método TUNEL, enquanto LM-4b produziu as maiores taxas de apoptose por anexina V. Os hepatócitos invadidos apresentaram várias alterações morfológicas. Na análise do conjunto de dados, os sorotipos LM-4b e LM-1/2a provaram ser os mais eficientes na invasão celular, enquanto que ST adaptou-se mais rápido ao meio e induziu a liberação precoce de TNF-α pelos hepatócitos.

Análisis morfométrico del efecto de estimulaciones de láser infrarrojo sobre núcleos de hepatocitos de rata / Morphometric analysis of the effect of infrared laser stimulation on hepatocyte nuclei in rats

De ratas Sprague Dawley tanto normales como irradiadas con dosis diarias de 1, 2, 4, 8, y 16 J/cm2 durante 15 días emitidas por el láser AsGa equivalente a 904 nm previamente anestesiadas, fueron quirúrgicamente obtenidas muestras de hígado, las que posteriormente fueron procesadas para microscopía óptica, siendo estudiadas y sometidas a técnicas morfométricas utilizando aumentos de 1000X, con especial énfasis en cuantificar áreas de núcleos y nucleolos. El análisis de los resultados entre hepatocitos normales e irradiados revela que existen marcadas diferencias entre sus áreas tanto nucleares como nucleolares, concluyéndose que los efectos de estas dosis de radiación infrarroja provoca en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad, principalmente en la relativa a la síntesis proteica, representando el efecto de estas estimulaciones sobre este tipo celular de elevado metabolismo.

Liver samples were taken from previously anaesthetised Sprague Dawley rats, both normal and irradiated with daily doses of 1, 2, 4, 8, and 16 J/cm2 applied over 15 days by AsGa laser equivalent to 904 nm. These samples were then processed for optical microscopy. They were studied and subjected to morphometric techniques using 1000X magnification, placing special emphasis on the quantification of the areas of nuclei and nucleoli. An analytic comparison of the results between normal and irradiated hepatocytes reveals the existence of significant differences between both the nuclear and nucleolar areas studied, from which it is concluded that the effect of these doses of infrared radiation is to provoke a drastic transformation in the components of the hepatocytes, and therefore in their functioning, principally with respect to protein synthesis, and that this would be the effect of stimulation of this nature on this type of high-metabolism cell.

Análisis morfométrico del efecto del láser infrarrojo sobre hepatocitos de rata / Morphometric analisys of infrared laser effect on rat hepatocyte

El láser infrarrojo emitido por el diodo Arsenurio de Galio (904 nm) proporciona terapia a lesiones articulares por su acción analgésica, cicatrizante y antiinflamatoria, promoviendo a nivel celular síntesis de ATP mitocondrial, modulación de canales de calcio, activando el proceso mitótico e incremento en la síntesis de DNA y de proteínas. Para determinar las dosis que estimulen componentes celulares involucrados en síntesis proteica, del hígado de ratas fueron tomadas muestras de tejido normal e irradiado mediante láser infrarrojo con 1, 2, 4, 8 y 16 Joules/cm2 durante 15 días consecutivos. Fueron tratadas para microscopía electrónica de transmisión y se obtuvieron micrografías con aumentos de 10.000 X. Se realizaron estudios morfométricos, cuantificándose las fracciones volumétricas de núcleos, citoplasma, retículo endoplasmático rugoso (RER), inclusiones de glicógeno, nucleolos, eucromatina y heterocromatina, relación núcleo- citoplasmática y las áreas celulares y nucleares. Los resultados del presente estudio que compara hepatocitos normales e irradiados, indican que existen diferencias significativas en todos los parámetros evaluados. Se concluye que los hepatocitos estimulados alteran su morfología y por ende sus componentes celulares, modificando la función celular determinándose con exactitud la dosis de estimulación infrarroja donde estas células presentan un mayor desarrollo de su maquinaria citoplasmática involucrada en síntesis de proteínas.

The infrared lasser emitted by the Gallium Arsenide diode provides an adequate therapy for articular lessions due to their healing, analgesic, and anti-inflammatory powers. It also promotes at cellular level mitochondrial ATP synthesis, modulates Calcium channels and activate mitotic processes by increasing DNA and protein synthesis. To determine the effective doses which stimulates rat liver protein synthesis, several samples from normal and irradiated tissues to intensities of 1, 2, 4, 8, and 16 Joules/cm2 by 15 consecutive days were taken. These samples were later prepared and observed under transmission E.M. (10000X) and analyzed by morphometric studies, where volume and organelle distribution, such as nucleus, cytoplasm, endoplasmic reticulum, glycogen inclussions, nucleolus, eu and heterochromatin were accounted, together at nuclear-cytoplasmic relationships and the cellular and nuclear areas. Under comparison normal and irradiated hepatocytes presented a significative difference in all evaluated parameters. It can be concluded that at certain specific level of infrared irradiation, hepatocytes alter their morphology by modifyng those cellular components involved in protein síntesis.

Role of vitamin-E on rat liver-amiodarone: an ultrastructural study

Amiodarone, a class III antiarrhythmic drug, has been found to be effective in the management of patients with life-threatening ventricular arrhythmias. The aim of this study was to test whether the co administration of vitamin-E with amiodarone can reduce amiodarone-induced liver damage. Twelve male albino rats were divided into three groups [ml vegetable oil/day by oral gavages daily for 2 weeks and were used as control group. The rats of the second group received 5.4 mg amiodarone/100 gm rat dissolved in vegetable oil daily by oral gavages for 2 weeks. In the third group, the rats received 5.4 mg amiodarone and 5 mg vitamin-E/100 gram rat dissolved in 2 ml vegetable oil by oral gavages daily for 2 weeks. Two weeks after treatment, the rats were sacrificed and liver specimens were immediately taken and processed for transmission electron microscopic examinations. Sections from the rat liver receiving amiodarone examined by electron microscopy showed disrupted hepatocytes with increased vacuolations. Degenerated organelles and disrupted nuclei were observed. The microvilli of bile canaliculi were disrupted and the hepatocytes showed increased lipid contents. Both endothelial cells and Kupffer cells were damaged. Phospholipids inside the mitochondria showed a loss of cristae. Sections from the liver of rats received amiodarone and vitamin-E showed lesser effects, especially in depositions of phospholipids in the mitochondria and the whole organelles and the nucleus showed minor damage in comparison to the previous group. Milder hepatotoxic effects are seen in rats administered amiodarone and vitamin E simultaneously suggesting that vitamin-E may play a role in amelioration of the effects of amiodarone

Ultrastructural changes of the renal tubular cells and hepatocytes of neonatal rats suckled milk from mothers subjected to subchronic toxicity of cadmium chloride

Neonatal off springs from female rats administered cadmium chloride by the oral route [0.5 mg.kg[-1]. day[-1] throughout their entire gestation and lactation periods. The pups were nursed their mothers until they weaned at the age of 28 days. At the end of the experiment, neonatal rats were anaesthetized by ether, then dissected and liver and kidney tissues were rapidly removed and processed for transmission electron microscopic examination. The uItrastructural observations of the proximal convoluted tubules were the absent of the apical microvilli, basal striation and endocytotic-lysosomal apparatus. Meanwhile hepatocytes showed few, sporadic mitochondria in concomitant with ill developed rough and smooth endoplasmic reticula. In addition to the apoptosis of hepatocytes. In conclusion, cadmium was transmitted via milk to the rat pups and potentially toxic to proximal convoluted tubular cells and to less extent to hepatocytes with multifactor mechanisms of cadmium toxicity