Štefan Mozeš

Developmental Changes of Gut Microflora and Enzyme Activity in Rat Pups Exposed to Fat-rich Diet

The aim of this study was to investigate the effect of a high‐fat (HF)/energy diet on the intestinal microbiota, the alkaline phosphatase (AP) activity, and related parameters of growth and obesity during the suckling and weaning periods in male Sprague‐Dawley rats. From birth, nutrition in suckling pups was manipulated by feeding rat dams either HF or a standard diet, and then after weaning, by exposure of experimental pups to the HF, and control rats to normal diet. On days 15, 20, 40 the numbers of 2 microbial groups, i.e., Bacteroides/Prevotella (BAC) and the Lactobacillus/Enterococcus (LAB) in the jejunum, were determined by fluorescent in situ hybridization technique, and the AP activity was assayed histochemically. During all investigated periods HF pups gained body fat more rapidly than control animals, but from weaning they displayed significantly stunted growth resulting in final body weight loss. Obesity in HF rats was also accompanied by higher LAB and lower numbers of BAC and with permanently higher AP activity. Correlation of these data showed significant negative correlation between LAB, AP, and weight gain and energy efficiency, and significant positive correlation of BAC and AP activity with body fat. These data support the concept that postnatal nutritional experience represents an important factor affecting the ontogeny of intestinal microbial communities and intestinal function. These acquired changes could be a component of regulatory mechanisms involved in adverse and/or positive consequences of HF diet for adiposity, body weight, and energy‐balance control in later life.

Effect of Atropa belladonna L. on skin wound healing: biomechanical and histological study in rats and in vitro study in keratinocytes, 3T3 fibroblasts, and human umbilical vein endothelial cells.

The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague–Dawley rats subjected to two parallel full‐thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT‐assay. Results of the in vivo experiments showed in AB‐treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research.

Intestinal microflora and obesity in rats

The relationship was evaluated between early nutritional experiences, the intestinal microflora and the small intestinal functions in the mechanism of predisposition to obesity development. Male Sprague-Dawley rats were used in which the quantity of nutrition was manipulated from birth to weaning (day 30) by adjusting the number of pups in the nest to 4 small litters (SL) and 10 normal litters (NL) and fed a standard diet from days 30 to 40 of age. After 40 d, the postnatally overfed SL pups became heavier, displayed significantly enhanced adiposity, body mass gain and food intake as well as a significantly higher jejunal alkaline phosphatase and maltase activity than in rats nursed in NL nests. The effect of different early nutrition was also accompanied by the appearance of significantly decreased Bacteroides and significantly increased enterococci and lactobacilli of obese rats than in lean NL rats. The amounts of Bacteroides were negatively correlated with fat pad mass, body mass, body-mass gain and food intake whereas enterococci and lactobacilli were correlated positively with the same parameters. Our results demonstrate that postnatal nutritional experience may represent a predisposing factor influencing ontogeny of small intestine function and development of intestinal microbial communities. The acquired changes and associated alterations in food digestion could be a component of regulatory mechanisms contributing to the development of obesity and its maintenance in later life.

Body fat and RNA content of the VMH cells in rats neonatally treated with monosodium glutamate

The RNA content of the ventromedial hypothalamus (VMH), the lateral hypothalamic area (LHA), and the cortical neurons of male and female rats, neonatally treated with monosodium glutamate (MSG), were investigated. MSG (2 g/kg b.wt.) was injected subcutaneously to male and female rat pups daily for 5 days after birth. At 12 weeks of age a significant decrease of RNA content in the VMH cells and significantly increased body fat in neonatally MSG-treated animals were found. Correlation of these data showed a significant negative correlation between the body fat content and the RNA content in VMH neurons. The results a) confirm a closed relationship between the body fat content and the functional activity of VMH, b) indicate that obesity of neonatally MSG-treated animals should be due to decreased functional activity of the VMH cells.

In Vivo Monitoring the Changes of Interstitial pH and FAD/NADH Ratio by Fluorescence Spectroscopy in Healing Skin Wounds

Abstract The aim of our study was to evaluate the changes of interstitial pH and flavin adenine dinucleotide (FAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio in healing skin wounds using fluorescence spectroscopy in Sprague Dawley rats. In the experiment, excisional and incisional models of wound healing were used. The florescein as the pH-sensitive probe using excitation spectra (λEm = 535 nm) was used for the measurement of pH changes, and synchronous fluorescence spectra (Δλ = 60 nm) for the monitoring of FAD/NADH ratio changes were measured from the surfaces of healing wounds. Increase of interstitial pH and FAD/NADH ratio was recorded during the time interval from the 15th to the 65th minute after surgery. The decrease of pH between the 48th and the 72nd hour after surgery as well as the increase of FAD/NADH ratio between the 72nd and the 96th hour of wound healing were recorded. The results indicate that the use of fluorescence spectroscopy may be considered as a valuable tool for noninvasive in vivo monitoring of selected redox parameters in the early phases of wound healing.

Development of gut microflora in obese and lean rats

The influence was evaluated of post-weaning normal nutrition and over-nutrition upon the development of the intestinal microbiota, the alkaline phosphatase activity (AP) and occurrence of obesity in male Sprague-Dawley rats (from days 21 to 40 the control rats were submitted to ad libitum intake of a standard laboratory diet whereas overfed rats received the same diet supplemented with milk-based high fat liquid diet). The jejunal numbers of two dominant divisions of bacteria, i.e. Firmicutes (Lactobacillus/ Enterococcus — LAB) and the Bacteroidetes (Bacteroides/Prevotella — BAC), were determined using the fluorescent in situ hybridization (FISH) method, and the jejunal AP activity was assayed histochemically. On day 40, the overfed rats in comparison with control animals displayed increased adiposity accompanied by enhanced AP activity, abundance of LAB, lower amounts of BAC and, thereafter, higher LAB/BAC ratio (L/B). The numbers of LAB and L/B index positively correlated with body fat, energy intake and AP activity, whereas numbers of BAC showed an opposite tendency. These results revealed the significance of nutritional imprint upon the post-weaning development of intestinal microbial and functional axis and contribute to better understanding of their involvement in energy-balance control and in adverse and/or positive regulation of adiposity.

Postsurgical Administration of Estradiol Benzoate Decreases Tensile Strength of Healing Skin Wounds in Ovariectomized Rats

BACKGROUND The most effective method of increasing the level of estrogen in the wounds of post-menopausal women undergoing routine surgical procedures is by long-term preoperative administration. However, in the case of acute surgery or trauma, the most effective method of increasing the level of estrogen is administration immediately pre- or postsurgery. This study, therefore, was aimed at assessing the effect of postsurgical administration of estradiol benzoate on wound healing in ovariectomized (OVX) Sprague Dawley rats. MATERIALS AND METHODS Three months prior to the wound healing experiment, 16 rats were anesthetized and underwent ovariectomy, while the other eight rats were sham operated. Two parallel full thickness skin incisions and two round full thickness skin excisions were performed on the dorsum of each rat. Dose of 10 microg/d of estradiol benzoate was administered to eight OVX rats for 6 d postoperatively, whereas the other animals received a placebo. After 6 d, all animals were sacrificed and samples removed for biomechanical and histological evaluation. RESULTS The mean wound tensile strength of OVX estrogen treated rats (9.54 +/- 3.24 g/mm(2)) was significantly lower compared with vehicle-treated OVX animals (14.57 +/- 4.12 g/mm(2)) as well as with control rats subjected to sham-OVX surgery (11.71 +/- 3.33 g/mm(2)). Nevertheless, the histological evaluation in OVX estrogen treated rats showed a significantly increased process of neo-angiogenesis associated with slightly decreased collagen deposition. CONCLUSION Our results indicate that the question of the clinical significance of this type of hormone replacement therapy remains open and requires further research.

Effect of antibiotic treatment on intestinal microbial and enzymatic development in postnatally overfed obese rats.

To investigate the effect of the microbiota‐induced changes and early overfeeding after amoxicillin administration (a) in suckling pups via their dams up to 15 days of lactation and (b) in weaned pups on intestinal microbial/functional adaptability and obesity development in male Sprague‐Dawley rats.

Experimental Study on Predicting Skin Flap Necrosis by Fluorescence in the FAD and NADH Bands During Surgery

The objective of the present study was to assess the feasibility of using endogenous fluorescence in intraoperative prediction of skin flap necrosis. The investigation was carried out in 10 Sprague–Dawley rats in which proximally based over‐dimensioned random pattern skin flaps were dissected on the back and thereafter fixed into position. Immediately after surgery on each rat, synchronous fluorescence spectra (Δλ = 90 nm) from five parts of the skin flap surface were measured. The presence of necrosis was evaluated on postoperative day 7. In flap parts designated as necrotic (n = 18), a significantly lower (P < 0.001) fluorescent signal from the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) bands (360–380 and 440–460 nm, respectively) was detected in comparison with the vital parts (n = 32) (for FAD:1767 ± 39 versus 2579 ± 65 auxiliary units [A.U]. and for NADH:11724 ± 340 versus 16810 ± 473 A.U.). The results suggested a close relationship between the fluorescent signals from the FAD and NADH bands on one side and flap necrosis or survival on the other side. Thus, the use of fluorescence spectroscopy may be considered as a valuable noninvasive tool for the prediction of skin flap necrosis.

RNA content of neurons in the ventromedial nuclei and lateral hypothalamic area relative to feeding status

The total RNA content of hypothalamic and cortex neurons in relation to the feeding status of adult male Wistar rats was studied. Experimental conditions including food deprivation (12 and 24 hours) and relative satiation (short-term refeeding, glucose or glycerol administration) changed in different ways the total RNA content of the neurons in the ventromedial hypothalamic nuclei (VMH) and in the lateral hypothalamic area (LHA) with respect to fasting or satiety. Only the long-term absence of food (24 hours) significantly increased the total RNA content of the VMH cells, while the RNA content of the LHA neurons significantly decreased in both the 12 and 24 hr fasted rats compared with those fed ad lib. The sixty minute free access to food after 12 or 24 hours of fasting fully reversed these changes. The short-term food intake significantly increased the RNA content of the LHA cells of the 12 and 24 hr fasted animals while the total RNA content of the VMH neurons significantly decreased only in the 24 hr fasted rats. The effect of glucose and glycerol administration on the RNA content of the LHA neurons (in 12 hr fasted rats) was similar to the effect of refeeding. One hour after giving glucose (1 g/kg b.wt.) or glycerol (300 mg/kg b.wt.) the total RNA content in the LHA neurons significantly increased. No changes in RNA content were observed in the neurons of the cortex when comparing the experimental and control rats. The results demonstrated the close relationship between the RNA content of the hypothalamic neurons and the feeding status.(ABSTRACT TRUNCATED AT 250 WORDS)