Liraglutide modulates gut microbiota and reduces NAFLD in obese mice.

Metabolic disorders such as insulin resistance and diabetes are associated with obesity and nonalcoholic fatty liver disease (NAFLD). The aggressive form of a fatty liver disease may progress to cirrhosis and hepatocellular carcinoma. Furthermore, recent studies demonstrated that there is a dysbiosis in the gut microbiota associated with early stages of metabolic disease. Therefore, the identification and repurposing of drugs already used to treat insulin resistance may be an excellent option for other disorders. We evaluated the effect of liraglutide on obesity, NAFLD and gut microbiota modulation in two different animal models of obesity: the ob/ob mice and the high-fat diet (HFD)-fed mice. Liraglutide treatment induced significant weight loss in both obesity models, showed improvements in glycemic parameters and reduced inflammatory cell infiltration in the cecum and the liver. In ob/ob mice, the liraglutide treatment was able to reduce the accumulation of liver fat by 78% and reversed steatosis in the HFD mice. The gut microbiota analysis showed that liraglutide changed the overall composition as well as the relative abundance of weight-relevant phylotypes such as a reduction of Proteobacteria and an increase of Akkermansia muciniphila in the treated HFD group. We show that liraglutide can lead to weight loss and gut microbiota modulations, and is associated with an improvement of NAFLD. Furthermore, by generating a profile of the intestinal microbiota, we compiled a list of potential bacterial targets that may modulate metabolism and induce a metabolic profile that is considered normal or clinically controlled.

Walker-256 tumor alters morphology of intestinal myenteric plexus in rats.

BACKGROUND: Gastrointestinal (GI) dysmotility is common in patients with cancer. There are a few studies about the myenteric plexus in the presence of anatomically remote tumors. METHODS: Forty-eight male Wistar rats were divided into a control (CT) or Walker-256 (TW) group. Tumor cells were subcutaneously injected and saline was injected in the CT group. After 14 days, the small and large intestines were removed for histochemical analysis. The macroscopic morphology of the intestines and the fecal excretion were also observed. KEY RESULTS: The upper GI transit and weight of fecal pellets were reduced and the walls of the large intestine in tumor-bearing rats showed multiple constrictions. In the capsules' constitution of the myenteric plexus of the TW group, there were type III collagen fibers in addition to type I fibers, and the thin septa inside the capsule were absent. The large intestine in the TW group exhibited smaller neurons and the number of nitrergic-positive neurons was also reduced in the myenteric plexus, compared to the CT group. In the TW group, the neuronal numbers and the staining intensity of acetylcholinesterase (AChE) were reduced in the large intestine. Staining was not different in the small intestine. CONCLUSIONS AND INFERENCES: This study showed that the Walker-256 tumor induced alterations in the morphology of nitrergic and cholinergic neurons in the myenteric plexus and decreased the upper GI transit with the presence of multiple constrictions in the colon. Therefore, these alterations can interfere on neurotransmission and can be related to the intestinal motility alterations observed in tumor-bearing rats.

Changes in food intake, metabolic parameters and insulin resistance are induced by an isoenergetic, medium-chain fatty acid diet and are associated with modifications in insulin signalling in isolated rat pancreatic islets.

Long-chain fatty acids are capable of inducing alterations in the homoeostasis of glucose-stimulated insulin secretion (GSIS), but the effect of medium-chain fatty acids (MCFA) is poorly elucidated. In the present study, we fed a normoenergetic MCFA diet to male rats from the age of 1 month to the age of 4 months in order to analyse the effect of MCFA on body growth, insulin sensitivity and GSIS. The 45% MCFA substitution of whole fatty acids in the normoenergetic diet impaired whole body growth and resulted in increased body adiposity and hyperinsulinaemia, and reduced insulin-mediated glucose uptake in skeletal muscle. In addition, the isolated pancreatic islets from the MCFA-fed rats showed impaired GSIS and reduced protein kinase Ba (AKT1) protein expression and extracellular signal-related kinase isoforms 1 and 2 (ERK(1/2)) phosphorylation, which were accompanied by increased cellular death. Furthermore, there was a mildly increased cholinergic sensitivity to GSIS. We discuss these findings in further detail, and advocate that they might have a role in the mechanistic pathway leading to the compensatory hyperinsulinaemic status found in this animal model.

Morphological characteristics of the rat thymus duringperinatal protein deprivation and early refeeding: a qualitative and quantitative study

Protein malnutrition is particularly deleterious in young individuals. An immunodeficient state is a well‑known functional consequence but alterations in thymic morphology remain unknown. Our aim is to analyze morphological characteristics of the rat thymus in a perinatal undernutrition and renutrition model – we hypothesize these morphological alterations are reversible with early refeeding. Ninety-day-old Wistar rats were allowed to mate and divided into three groups: nourished (N – normal 20% protein diet), undernourished (UN – pre- and postnatal 5% protein diet until post-natal day 60 – PND 60) and renourished (RN – as UN but normal diet from PND 21 to 60). The thymi of 10 pups/group were submitted to macroscopic, histology, morphometry and scanning electron microscopy analyses. Body weight was highest in N and lowest in UN animals as expected but the thymic/body weight ratio remained similar in N and UN; this ratio was significantly higher in the RN group. UN thymi had a prevalence of type I collagen fibers, atrophic lobules and absence of a clear corticomedullary boundary. Thymic cortical component was decreased in UN. Apoptotic thymocytes were more frequently visualized in the UN thymi. N and RN thymi exhibited very similar morphology. Perinatal protein malnutrition induces drastic morphological alterations in rat thymi but these could be largely reversed with early renutrition. Functional studies are needed to assess if organ function mimics morphology in its recovery.

Effects of protein malnutrition on muscle fibers of the brachial biceps and medial pterygoid of Wistar rats

Protein malnutrition is a public health problem, and in childhood, it can lead to muscle deficits. Here, our objective was to evaluate the effects of malnutrition upon the muscle fibers in the medial pterygoid and braquial biceps. Ten just weaned rat pups that had been born to parents fed a nourished or malnourished diet (N = 5 per group) were studied. The medial pterygoid and braquial biceps muscles were removed and crosssectioned, and histological staining with picrosirius and histochemistry reaction with nicotinamide adenine dinucleotide - tetrazolium reductase (NADH-tr) were performed. The samples stained with picrosirius were observed under polarized light, and from the qualitative analysis, we observed that type I collagen fibers were only present in the braquial biceps muscles of the nourished animals. The NADH-tr reaction indicated that the pterygoid muscle specimens from the malnourished pups lacked intermediate muscle fibers. The crosssectional area of the muscle was lower in the malnourished group than in the nourished group. The density of muscle fibers was higher in the malnourished group than in the nourished group. The consequences of malnutrition were visible when comparing the muscles. We concluded that the differences in daily muscle action along with the differences in embryological origin are instrumental in establishing the results.

Structural and ultra-structural features of the first mandibular molars of young rats submitted to pre and postnatal protein deficiencies.

The effects of protein malnutrition, both in utero and prior to weaning, on formation of the first mandibular molars were evaluated by phase-contrast and electron microscopy in rats. The nourished group (GI) received a diet that included 20% casein, while the malnourished group (GII) received 5% casein. The first mandibular molars from GII exhibited low density of cells and odontoblasts, which lacked regular organization compared with molars from GI. In addition, a difference in collagen type was observed between the groups, with a prevalence of Type III collagen fibers detected in the dentin, periodontal ligament, and alveolar bone of GII, and a prevalence of Type I collagen fibers in GI. Finally, examination of surface area in molar sagittal sections indicated 30% less dentin in GII, compared with GI. Our results suggest that structural and ultra-structural features of the dentin-pulp complex and periodontal components of rat molars are affected by protein deficiency.

Ultrastructural study of the temporomandibular joint after unilateral meniscectomy in Wistar rats.

Meniscectomy of the temporomandibular joint (TMJ) was frequently performed until a few years ago but now is seldom employed. This procedure induces important articular modifications but the complete extent of ultrastructural changes is still unknown. Twenty-one Wistar rats were submitted to unilateral meniscectomy. Animals were randomly divided into three groups and followed for 15, 35 or 60 days. After killing both meniscectomized and contralateral TMJ specimens were sectioned sagittally and prepared for light and scanning electron microscopy studies. Normal TMJs are characterized by glenoid fossa and condylar process with distinct conjunctive layers in which regularly arranged type 1 collagen fibres predominate. Meniscectomized animals initially exhibit a different tissue covering the eroded articular surface of the condyle with the prevalence of type 3 collagen fibres but type 1 fibres predominate in the late postoperative period. Subchondral cysts are clearly visible. A complex remodelling process of the TMJ after meniscectomy is evident with important ultrastructural modifications that may correlate to unsatisfactory clinical results. The dynamic nature of this process is also observed when specimens from different postoperative periods are compared. Surgeons should always bear in mind these alterations when indicating this procedure.

Trypanosoma cruzi: preliminary investigation of NADH-positive and somatostatin-immunoreactive neurons in the myenteric plexus of the mouse colon during the infection.

In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.

Thyroxine-induced cardiac hypertrophy: influence of adrenergic nervous system versus renin-angiotensin system on myocyte remodeling.

The present study assessed the possible involvement of the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) in thyroxine (T4)-induced cardiac hypertrophy. Hemodynamic parameters, heart weight (HW), ratio of HW to body weight (HW/BW), and myocyte width were evaluated in absence of thyroid hormone (hypothyroidism) and after T4 administration. Male Wistar rats were used. Some were subjected to thyroidectomies, whereas hyperthyroidism was induced in others via daily intraperitoneal injection of T4 (25 or 100 microg x 100 g BW(-1) x day(-1)) for 7 days. In some cases, T4 administration was combined with the angiotensin I-converting enzyme inhibitor enalapril (Ena), with the angiotensin type 1 (AT1) receptor blocker losartan (Los) or with the beta-adrenergic blocker propanolol (Prop). Hemodynamics and morphology were then evaluated. Systolic blood pressure (SBP) was not altered by administration of either T4 alone or T4 in combination with the specific inhibitors. However, SBP decreased significantly in hypothyroid rats. An increased heart rate was seen after administration of either T4 alone or T4 in combination with either Los or Ena. Although the higher dose of T4 significantly increased HW, HW/BW increased in both T4-treated groups. Ena and Prop inhibited the increase in HW or HW/BW in hyperthyroid rats. Morphologically, both T4 dose levels significantly increased myocyte width, an occurrence prevented by RAS or SNS blockers. There was a good correlation between changes in HW/BW and myocyte width. These results indicate that T4-induced cardiac hypertrophy is associated with both the SNS and the RAS.

Cardiac denervation in mice infected with Trypanosoma cruzi.

The neuronal features of the hearts of mice that were acutely or chronically infected with the Y strain of Trypanosoma cruzi were compared with those of control hearts from uninfected mice. Whole-mount preparations of the murine atria, isolated by microdissection, were stained to reveal neurons with NADH-diaphorase activity. Counts, by a microscopist who was blind to the infection status of the donor mouse, revealed that there were significantly (38%) fewer such neurons in the atria from the acutely infected mice than in the atria from the control hearts. The ganglia of the infected mice were also irregularly distributed, severely damaged ganglia being found beside slightly degenerated or morphologically normal ones. Although the ganglia contained small, medium and large neurons, the apparent destruction caused by T. cruzi was confined to the large ones. As neuron counts in preparations of hearts from mice with chronic infections were 32% lower than those in the control hearts, there appears to be no additional loss of cardiac neurons as the acute infection in mice progresses to the chronic phase.

Structure and ultrastructure of the celiac-mesenteric ganglion complex in the domestic dog (Canis familiaris).

A number of neurons of the autonomic nervous system are situated in the ganglia and can be systematically divided into pre-vertebrals, paravertebrals, intramural and para-viscerals. The celiac-mesenteric ganglion, an important pre-vertebral ganglion, is located together with the abdominal aorta and links the central nervous system to the peripheral system, participating in the coordination of peripheral reflexes and principally innervating the stomach, intestines, accessory glands (liver and pancreas). In addition, the celiac-mesenteric ganglion also contributes to the innervation of the spleen and has a role in gastrointestinal motility control. This study examined the structural and ultrastructural aspects of 40 celiac-mesenteric ganglia from domestic dogs. For light microscopy ganglia were included in paraplast and stained with haematoxylin-eosin, picrosirius, toluidine blue, Calleja's and Masson's trichrome. For examination by electron microscopy, the ganglia were submitted to cryofracture, enzyme digestion, hydrolysis and fixed in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The celiac-mesenteric ganglion was observed as a ganglionic complex composed of various ganglionic units separated by types I and III collagen fibres, predominantly unmyelinated nerve fibres and continuous capillaries. This complex is surrounded by a double-layer capsule (internal and external). The principal ganglion cells had eccentric nuclei with two nucleoli, the nucleolemma was double and presented nuclear pores. In the cytoplasm there were vesicles of the Golgi apparatus, electron-dense vacuoles, mitochondrias, smooth and granulated endoplasmic reticulum and free ribosomes. In conclusion, this ganglionic complex, in contrast to similar structures in the enteric nervous system, presents separate ganglionic units in a systematic arrangement related to the extrinsic and specific innervation of the target organs.

The structure of the bilaminar zone in the human temporomandibular joint: a light and scanning electron microscopy study in young and elderly subjects.

The bilaminar zone (BZ) in the human temporomandibular joint (TMJ) of toothed adults (GI) and toothless, elderly humans (GII) were analysed using light and scanning electron microscopy (SEM). In both groups the BZ consists of an upper and a lower stratum of connective tissue separated by a vascularized middle region. The superior stratum contains bundles of collagen fibres disposed in different directions. The fibres are fairly thick and transversely oriented immediately posterior to the TMJ disc. The initial part of the inferior stratum contains curved bundles of collagen fibres oriented anterio-posteriorly. From the middle to the posterior part of the inferior stratum, the fibres are right-aligned in GI and clearly sinuous in nature in GII. In both groups, the middle and posterior portions of the middle region are distinguished by the presence of vessels and vascular spaces. Loosely arranged connective and adipose tissues are also evident. The vascular spaces are wider in GII than in GI. The predominance of type I collagen fibres is clear in all regions of the BZ in both groups. The elastic fibres lie parallel to the collagen fibres in both groups and they are thicker and more abundant in GI, apparently decreasing in GII.

[Morphologic evaluation of the bonding between adhesive/composite resin and dentin irradiated with Er:YAG and Nd:YAG lasers: comparative study using scanning microscopy]. / Avaliação morfológica da união entre adesivo/resina composta e dentina irradiada com laser Er:YAG e laser Nd:YAG: estudo comparativo por microscopia de varredura.

Since bonding systems were introduced in the restorative procedures carried out with esthetic materials, the treatment of dentin surfaces has been widely studied in order to establish the ideal technique. The application of 37% phosphoric acid on dentin is still the best known method. However, alternative methods for treating the dentin surface have been discussed in the literature, including the utilization of some kinds of laser irradiation. The purpose of this research was to morphologically evaluate the bond between adhesive materials and the dentin treated with Er:YAG and Nd:YAG lasers, in a comparative study by means of scanning electron microscopy (SEM). Irradiation either substituted acid etching, or was associated to it. Recently extracted bovine incisors were utilized. They received class V cavity preparations and were restored with a bonding system and a light-cured composite resin. Meanwhile, some of the teeth underwent irradiation with Er:YAG laser or Nd:YAG laser before the application of the bonding agent and the composite resin. The samples were selected, prepared for SEM and submitted to morphological analysis. Data were registered in photomicrographs. Based on the microscopic observations, we concluded that only in the dentin surfaces submitted to irradiation with Er:YAG laser and to acid conditioning there was penetration of resin into the dentine. With the Nd:YAG laser treatment, there was only visual superposition of resin over the dentin surface, which suggests that there was only occlusion of the tubules, with characteristics of fusion in the superficial dentine.

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats.

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8%; 12 animals) or a low-salt diet (LS; 0.15%; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 +/- 11 vs 119 +/- 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8 percent; 12 animals) or a low-salt diet (LS; 0.15 percent; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 +/- 11 vs 119 +/- 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.

The arrangement of the trabecular bone in the vestibular surface of the human fetus mandible. A scanning electron microscopy study.

The characteristics of trabecular bone in human fetuses at seven to nine months of intra-uterine life were studied in the vestibular surface of the mental and body portions of the mandible. In the mental portion, many circular trabeculae joined by "bridges" were observed in the alveolar region (AR). In the 8-month old fetus are trabeculae with tortuous disposition in the medium region (MR). Oblique trabeculae with superior and inferior dispositions defining a triangular area (future mental fossae) are verified in 9-month old fetuses. Trabeculae circularly disposed and vascular foramina are present close to the apex of this area. In the basilar region (BR) some trabeculae obliquely oriented are observed disposed near to the mandibular symphysis. The longitudinal bundles of collagen fibers assume an antero-posterior direction. At 9-month age the vascular foramina range from 6 to 20 microns and the osteocyte lacunae ranging from 1 to 4 microns diameter, are present in mandibles from all ages examined. In the body portion the presence of circular trabeculae joined by "bridges" is the main characteristic of the AR. In the MR most of the trabeculae are circular and the longitudinal trabeculae of this region assume an antero-posterior direction. The laminar, antero-posterior and juxtaposed trabeculae lying parallel to mandible base are a characteristic of the BR. The bundles of collagen fibers exhibit the same pattern of the trabeculae. The vascular foramina of the BR are smaller than that of the precedent regions. In the body portion of the mandible from all ages examined the osteocyte lacunae range from 1 to 4 microns in diameter.

Fall in the number of intracardiac neurons in aging rats.

The neurons of whole cardiac atria were stained using a NADH-diaphorase technique in young adult (3 months old) (GI) and aging rats (20 months old) (GII). Light microscopy revealed differences in the appearance of the neurons in the two groups. In GI, most ganglia contained 50-100 neurons while in GII, most ganglia usually contained 20 neurons. The mean total number of neurons in the atria of GII was 245+/-31, i.e. only 23% of the mean value in GI (1086+/-203). The mean size of the ganglionic neurons (area of maximum cell profile) was 702 microm2 in GI and 1065 microm2 in GII. Histological sections of the ganglia revealed that a capsule of collagen fibers sheaths each ganglion in both groups. In GII, the density of collagen fibers increases in the capsule and in the septa within the ganglia; yellow or red, type I collagen fibers predominate in this group. No elastic fibers were present in the cardiac ganglia of either group. It is suggested that in aging rats, structural changes and reorganization of the remnant neurons accompany neuron reduction.

Blood supply to the human sternocleidomastoid muscle by the sternocleidomastoid branch of the occipital artery.

The contribution of the sternocleidomastoid branch of the occipital artery, superior arterial pedicle (SAP), to the irrigation of the sternocleidomastoid muscle (SCM) was evaluated in fresh human cadavers by injecting Neoprene-latex for minute dissection. From its insertion in the mastoid process of the temporal bone, the SCM was divided into six levels. The Neoprene-latex injected into the SAP reached the upper three levels in all SCMs studied, which corresponds to the middle part of the studied SCM. In 43.3% of the SCM, this branch reached level four and in 36.6%, it reached level five.

Vasoactive-intestinal-peptide- and substance-P-immunoreactive nerve fibres in the myenteric plexus of mouse colon during the chronic phase of Trypanosoma cruzi infection.

The distribution of a tachykinin (substance P) and vasoactive intestinal peptide (VIP) and the number and morphology of the large granular vesicles (LGV) in the myenteric plexus of the colons of mice were investigated. Six of the 12 young, male, Swiss mice studied had been inoculated with the Y strain of Trypanosoma cruzi 2 months previously whereas the others were uninfected controls. Substance P (SP) and VIP were localized by light microscopy, using an immunohistochemical method, and LGV were counted in sections studied by electron microscopy. There were far fewer LGV and less intensely staining varicose VIP- and SP-positive nerve fibres in the infected mice than in the controls. Denervation of the myenteric plexus may decrease the content of tachykinins (TK) and VIP in animals infected with T. cruzi. Such reduction in TK and VIP activity could be related to the disturbances in intestinal motility observed in the chronic phase of Chagas disease.