Triheptanoin, an odd-medium-chain triglyceride, impacts brain cognitive function in young and aged mice.

ABSTRACTThe brain aging process triggers cognitive function impairment, such as memory loss and compromised quality of life. Cognitive impairment is based on bioenergetic status, with reduced glucose uptake and metabolism in aged brains. Anaplerotic substrates are reported to promote mitochondrial ATP generation, having been tested in clinical trials for the treatment of neurological disorders and metabolic diseases.Objectives and Methods: To assess whether the improvement in oxidative capacity ameliorates cognitive function in adults (12 weeks), and aged (22-month-old) C57/6BJ mice, they received (1) a ketogenic diet, (2) a ketogenic diet supplemented with the anaplerotic substance, triheptanoin, or (3) a control diet for 12 weeks. Spontaneous alternation and time spent in a previously closed arm in the Y-maze test and time interacting with an unknown object in the novel object recognition test (NORT) were used to evaluate working memory. Acetylcholinesterase (AChE) activity in the prefrontal lobe, brain left hemisphere, and cerebellum was also evaluated. Glucose transporter 3 (GLUT3) expression in the prefrontal lobe was analyzed by western blotting.Results: The ketogenic diet (KD) reduced spontaneous alternation in aged mice, leading to lower AChE activity in the aged prefrontal lobe and cerebellum, and in the parieto-temporal-occipital lobe of adult mice. Furthermore, KD decreased GLUT3 protein expression in the frontal lobe of the adults.Discussion: Supplementation of KD with triheptanoin prevented memory impairment and showed similar values of AChE activity and GLUT3 expression compared to the controls. Our data suggest that triheptanoin has a potential role in the bioenergetic capacity of the brain, improving cognitive function.

Determination of drug-polymer solubility from supersaturated spray-dried amorphous solid dispersions: A case study with Efavirenz and Soluplus®.

Amorphous solid dispersions (ASDs) are found to be a well-established strategy for overcoming limited aqueous solubility and poor oral bioavailability of active pharmaceutical ingredients (APIs). One of the main parameters affecting ASDs physical stability is the API solubility in the carrier, because this value determines the maximal API load without a risk of phase separation and recrystallization. Phase-diagrams can be experimentally obtained by following the recrystallization of the API from a supersaturated homogeneous API-polymer solid solution, commonly produced by processes as solvent casting or comilling, which are very time-consuming (hours). The work deals with the construction of a temperature-composition EFV-Soluplus® phase diagram, from a thermal study of recrystallization of a supersaturated solid solution (85 wt% in EFV) generated by spray drying. This supersaturated solution is kept at a given annealing temperature to reach the equilibrium state and the amount that still remains dispersed in the polymer carrier at this equilibrium temperature is determined by means of the new glass transition temperature of the binary mixture. From our knowledge, this is the first study employing a fast process (spray drying) to prepare a supersaturated solid solution of an API in a polymer aiming to determine a temperature-composition phase diagram. The EFV solubility in Soluplus ranges from 20 wt% at 25 °C to 30 wt% at 40 °C. It can be a very useful preformulation tool for researchers studying amorphous solid dispersions of Efavirenz in Soluplus, to assist for predicting the stability of EFV-Soluplus ASDs at different EFV loadings and under different thermal conditions.

Malnutrition increases NO production and induces changes in inflammatory and oxidative status in the distal colon of lactating rats.

BACKGROUND: Epidemiological studies have indicated the lack of breast feeding as a risk factor associated with later development of inflammatory bowel disease. Nevertheless, the repercussion of little feeding during suckling on large intestine inflammatory response and anti-oxidant resources has not yet been completely understood. This study hypothesized that unfavorable lactation is able to induce oxidative stress and release of inflammatory mediators modifying the integrity of the colon epithelium in weanling rats. METHODS: Wistar rats were reared under different early nutritional conditions according to litter size in two groups: N6 (6 pups/dam) and N15 (15 pups/dam) until the 25th postnatal day. The distal colon was removed and processed for biochemical, morphometric, and immunohistochemical analyzes. Lipoperoxidation, nitric oxide (NO), reduced (GSH) and oxidized (GSSG) glutathione, tumor necrosis factor-alpha (TNF-α), interleukins-1ß, 4 and 10 (IL-1ß; IL-4; IL-10) levels, and total superoxide dismutase (tSOD), and catalase (CAT) activities were assessed. Morphometric analysis was carried out using paraffin sections and wholemount myenteric plexus preparations. KEY RESULTS: Increased lipoperoxidation, NO, TNF-α and IL-1b levels, reduced tSOD and increased CAT activities were found in the N15 compared to N6 group. No intergroup difference was detected for IL-10, while lower levels of IL-4, GSH and GSSG and lower neuronal size and density were induced by undernutrition. CONCLUSIONS & INFERENCES: Reduced feeding during suckling changed the inflammatory response and oxidative status in the colon of weanling rats. These data suggest potential mechanisms by which malnutrition early in life may increase the vulnerability of the large intestine to insults.

A critical period for omega-3 nutritional supplementation in the development of the rodent visual system.

Retinocollicular connections form precise topographical maps that are normally completed through the selective elimination of misplaced axons and the stabilization of topographically ordered axon terminals during early development. Omega-3 fatty acids, acquired exclusively through the diet, and its main metabolite, docosahexaenoic acid (DHA), are involved in brain development and synaptic maturation. We have previously shown that the nutritional restriction of omega-3/DHA results in abnormal retinocollicular topographical fine-tuning. Therefore, we studied the role of omega-3 fatty acids nutritional supplementation and the developmental time windows during which this postnatal supplementation would restore normal topographical maps in the visual system. Female rats and their litters were chronically fed with either control (soy oil) or restricted omega-3 (coconut oil) diets. Fish oil supplementation was introduced between either postnatal day (PND) 7-13, PND7-28 or PND21-42. At PND13, PND28 or PND42, animals received an anterograde eye injection of a neuronal tracer to visualize retinocollicular axons. Confirming previous observations we found that an omega-3/DHA deficiency resulted in an abnormally high innervation density of retinal axons at the visual layers of the superior colliculus (SC). Although a short-term fish oil supplementation between PND7-13 could not restore normal retinocollicular topography, an extended treatment between PND7-28 completely recovered normal innervation densities of retinotectal axons. However, a late onset supplementation protocol, between PND28-42, was no longer effective in the restoration of the abnormal topographical pattern induced by an early omega-3 nutritional malnutrition. The results suggest a critical period for omega3/DHA dietary intake for the proper development of visual topographical maps.

Stimulation of prostaglandin EP2 receptors on RGC-5 cells in culture blunts the negative effect of serum withdrawal.

Reduced neurotrophic support is one possible cause for retinal ganglion cells dying in glaucoma. Experiments were designed to investigate the effect of EP2 receptor agonist butaprost on transformed retinal ganglion (RGC-5) cells where reduced neurotrophic support was simulated by serum withdrawal. Cultures were analysed for cell viability, flow cytometry, reactive oxygen species and apoptosis. Western blot and immunohistochemistry were used to provide information for the occurrence of PGE(2) receptor-types. We demonstrated the existence of all four types of PGE(2) receptors in RGC-5 cells and exposure of cultures to butaprost resulted in an elevation of cAMP. Serum deprivation induced RGC-5 cell death was significantly attenuated by butaprost as well as by rolipram and forskolin where intracellular cAMP levels were increased. These data are of value in relation to the possible use of EP2 receptor agonists to reduce both elevated intraocular pressure and retinal ganglion cell death as occurs in glaucoma.

Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform.

An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression.

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.

Differential vulnerability of the rat retina, suprachiasmatic nucleus and intergeniculate leaflet to malnutrition induced during brain development.

We investigated in young rats the effects of malnutrition on the main structures of the circadian timing system: retina, hypothalamic suprachiasmatic nuclei (SCN), thalamic intergeniculate leaflet, retinohypothalamic- and geniculohypothalamic tracts. Control rats were born from mothers fed a commercial diet since gestation, and malnourished rats from mothers fed a multideficient diet since gestation (GLA group) or lactation (LA group). After weaning, pups received the same diet as their mothers, and were analysed at postnatal days 27, 30-33 and 60-63. Brain sections were processed to visualise in the SCN neuropeptide Y immunoreactivity and terminal labeling after intraocular tracer injections. Nissl staining was used to assess cytoarchitectonic boundaries of the SCN and cell features in retinal whole mounts. Cell counts, morphometric and densitometric analysis were performed. Compared with controls, the total retinal surface was reduced and the topographical distribution of retinal ganglion cells was altered in malnourished rats, with changes in their density. Alterations were also detected in the SCN dimensions in the GLA and LA groups at one and two postnatal months, as well as in the SCN portion occupied by the retinal input in the GLA group at days 30-33, but not in the NPY-containing geniculohypothalamic tract. The present data point to subtle changes, with a low and differential vulnerability to early malnutrition, of structures involved in circadian timing regulation. Furthermore, the present findings suggest that the altered circadian rhythmicity previously documented in malnourished rats cannot be ascribed to impaired development of the retino- and geniculohypothalamic projections to the SCN.

Avaliação da qualidade de matérias-primas de ruibarbo utilizadas em formulações farmacêuticas

As plantas denominadas de ruibarbo sintetizam antraquinonas e taninos, que são responsáveis pelos efeitos laxante e adstringente, respectivamente. Análises da qualidade de cinco matérias-primas de ruibarbo foram realizadas a fim de detectar adulterações. As reações para antraquinonas e taninos foram positivas. Os constituintes rapônticos foram observados na amostra de Rheum palmatum (2). As medidas de cinzas totais para Rheum palmatum (2) e Ferraria cathartica estão acima do esperado. Conforme resultados, as amostras de Rheum palmatum (2) e Ferraria cathartica devem estar adulteradas.

Plants known as rhubarb synthesize anthraquinones and tannins. Theses compounds have medicinal uses as laxative and astringent, respectivity. The quality of five raw materials was analysed to detect adulterant. Anthraquinones and tannins reactions were positive. Derivatives rhapontocoside were observed in the sample of Rheum palmatum (2). Total ashes were increased to Rheum palmatum (2) and Ferraria cathartica. According to the results, samples of Rheum palmatum (2) and Ferraria cathartica shoulde are adulterated.

Estudo farmacognóstico de galhos de Vanillosmopsis erythropappa Schult. Bip. - Asteraceae

Vanillosmopsis erythropappa é um vegetal rico em óleos essenciais, especialmente o á-bisabolol. O objetivo deste estudo foi pesquisar a variação sazonal qualitativa de substâncias naturais, teores de óleos essenciais, cinzas totais, umidade e pH. Os resultados mostraram uma variação de flavonóides, taninos, triterpenóides, esteróides, saponinas, óleos essenciais, cinzas totais, umidade e pH, demonstrando que os aspectos ambientais influenciam o metabolismo dessa espécie. As médias anuais dos valores obtidos foram: óleos essenciais= 0,29 ± 0,09 por cento; cinzas totais= 3,13 ± 0,49 por cento; umidade= 6,56 ± 0,80 por cento e pH= 5,40 ± 0,24.

Vanillosmopsis erythropappa is a plant rich in essential oils, especially á-bisabolol. The aim of this study was research the qualitative seasonal variation of natural substances, tenor of essential oils, total ashes, dampness and pH. The results showed a variation of the flavonoids, tannins, triterpenoids, steroids, saponins, essential oils, total ashes, dampness and pH, demonstrating that environmental aspects influence on the metabolism of this specie. The annual averages of the obtained values were: essential oils = 0,29 ± 0,09 percent; total ashes = 3,13 ± 0,49 percent; dampness= 6,56 ± 0,80 percent and pH = 5,40 ± 0,24.

Comparative study of glutamate mediated gamma-aminobutyric acid release from nitric oxide synthase and tyrosine hydroxylase immunoreactive cells of the Cebus apella retina.

The effects of excitatory amino acids (EAAs) upon transporter-mediated gamma-aminobutyric acid (GABA) release were investigated in cells containing tyrosine hydroxylase (TH) or nitric oxide synthase (NOS) in retina of the primate Cebus apella. Retinas were treated in vitro with 50 microM Kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C, in an Mg2+-free Locke's solution with or without Ca2+. The effects of EAAs were measured immunocytochemically by determining the GABA content in TH or NOS-immunoreactive cells in the inner retina, after stimulation. L-Glu and KA induced a Ca2+-independent GABA release from most GABA-immunoreactive cells of the inner retina. Double label experiments indicated that this release occurs in NOS+/GABA+ cells, but not in TH+/GABA+ cells suggesting that these cell subpopulations may be differentiated in some functional aspects.

Photoreceptor topography of the retina in the New World monkey Cebus apella.

The number and topographical distribution of photoreceptors was studied in whole-mounted retinas of Cebus apella. It was estimated a total of 48 million rods and 3.8 million cones. The average peak foveal cone density and the Nyquist Limit at the foveola were estimated as 169, 127 cells/mm(2) and 46.77+/-7.98 cyc/deg, respectively. A cone-enriched rim was found near the ora serrata, more noticeable in the nasal retina. Rod distribution was asymmetrical along horizontal and vertical meridians with a higher density in the dorsal retina. The rod/cone ratio was variable and asymmetrical along both meridians.

Transporter-mediated GABA release induced by excitatory amino acid agonist is associated with GAD-67 but not GAD-65 immunoreactive cells of the primate retina.

The release of GABA from amacrine and interplexiform cells after exposure to excitatory amino acids (EAAs) agonists was investigated by immunohistochemistry. Cebus monkey retinas were treated in vitro with 50 microM kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C. The effects of the EAAs were measured by detecting immunocytochemically the GABA remaining in the tissue after stimulation. L-Glu and KA reduced the number of GABA-immunoreactive perikarya in the innermost part of the inner nuclear layer by approximately 60% and 80%, respectively, as compared to controls. The cell processes in the inner plexiform layer (IPL) were restricted to only three defined bands in the strata 1, 3 and 5, as compared to an intense and homogeneous labeling in the IPL of the untreated retinas. The effect of KA was inhibited by 100 microM CNQX, 100 microM NNC-711, or when Na(+) was replaced by choline. The release of GABA was Ca(2+)-independent, suggesting the mobilization of GABA from the cytoplasmic pool of this neurotransmitter. At least two subsets of retinal neurons including amacrine and interplexiform cells retained GABA-immunoreactivity after stimulation with EAAs, as revealed by glutamic acid decarboxylase (GAD) immunocytochemistry. Our results suggest that non-NMDA receptor activation by KA and glutamate are associated with the efflux of GABA from cells of the inner retina (amacrine and interplexiform cells). The data also show that cells containing GAD-67 released GABA via its transporter, while cells containing exclusively GAD-65 apparently did not release the neurotransmitter by the reversal of the transporter.

Retinal ganglion cell depletion alters the phenotypic expression of GABA and GAD in the rat retina.

We have looked at the phenotypic expression of gamma-aminobutyric acid (GABA) and the two isoforms of its synthetic enzyme [glutamic acid decarboxylase (GAD)-65 and -67] in adult rat retinas that had the superior colliculus, pretectum and optic tract lesioned unilaterally at birth. It has been shown previously that this type of manipulation induces retrograde degeneration of retinal ganglion cells presumably without affecting other intraretinal neurons. We present evidence that GABAergic amacrine cells are affected by such manipulation. The number of cells immunoreactive for GABA, GAD-65 and GAD-67 decreased in the inner nuclear layer. In the retinal ganglion cell layer, however, the number of GABA- and GAD-65-labelled cells increased, while the number of GAD-67-labelled cells did not change. Biochemical assay showed that overall GAD activity was not altered in retinas of lesioned animals. Our results support the notion that, while neonatal lesion reorganizes the expression of GABA and GAD in the retina, enzyme activity is maintained within normal levels.

GABAergic retinocollicular projection in the New World monkey Cebus apella.

GABA immunoreactivity was examined in the retina of the New World monkey Cebus apella. Labeled cell bodies were identified as horizontal, bipolar, interplexiform, amacrine and a population of putative ganglion cells. To determine whether ganglion cells were immunoreactive to GABA, double-labeling experiments were performed using Fast Blue as retrograde neuronal tracer injected into the superior colliculus. Retinas containing FB-labeled ganglion cells were subsequently incubated with antiserum against GABA. Although retinocollicular ganglion cells were found in three different layers (ganglion cell layer, inner nuclear layer and inner plexiform layer), our experiments revealed GABA-positive ganglion cells only in the outer half of the ganglion cell layer.

Ganglion cell size and distribution in the retina of the two-toed sloth (Choloepus didactylus L.).

The distribution of ganglion cell densities and sizes was studied in Nissl-stained flat-mount retinae of the two-toed sloth. The area centralis, a weak specialization with low ganglion cell density, is located in the temporal retina close to the center of the eye. The presence of a visual streak was noted. The distribution of different ganglion cell sizes was approximately equal throughout the retina. Although the retinal organization differs from that of the closely related three-toed sloth, the presumed function of retinal specializations in both species is to guide limb movements by permitting visualization of the branch along which the animal is climbing.

Ganglion cell size and distribution in the retina of the two-toed sloth (Choloepus didactylus L.)

The distribution of ganglion cell densities and sizes was studied in Nissl-stained flat-mount retinae fo the two-toed sloth. The area centrailis, a weak specializtion with low ganglion cell density, is located in the temporal retina close to the center of the eye. The presence of a visual treak was nsoted. The distribution of different ganglion cell sizes was approximately equal throughout the retina. Although the retinal organization differs from that of the closely related three-toed sloth, presumed function of retinl specializations in both species is to guide limb movements by permiting visualization of the branch along which the animal is climbing

Unusual distribution of ganglion cells in the retina of the three-toed sloth (Bradypus variegatus).

1. The distribution and size of retinal ganglion cells labelled with horseradish peroxidase (HRP) were studied in flat-mounted retinas of three-toed sloths. 2. Massive injections of HRP solution were made throughout the thalamus and midbrain in anesthetized sloths in order to retrogradely label the retinal ganglion cell population. Twenty to thirty h later the eyes were excised and the retinas flat-mounted and reacted with phenylenediamine-HCl and H2O2 to label ganglion cells, thus distinguishing them from other cells in the same retinal layer. 3. Ganglion cell density gradually increased from about 500 cells/mm2 at the far periphery to a peak of about 1,500 cells/mm2 in an area, termed the area centralis, deep in the inferior temporal retina. The presence of a vertical visual streak was also noted. 4. The area centralis contained a higher frequency of small ganglion cells than the peripheral retina where large cells predominated. 5. The unusual position of the area centralis and visual streak in the retina can be explained by the sloth's unique ability to rotate its head 180 degrees while climbing upside-down along horizontal branches so that the head is right-side-up. If it is assumed that the branch directly above the sloth's head needs to be visualized for accurate claw placement then the branch would be imaged on the inferior temporal retina in an area corresponding to the maximum density region.

Unusual distribution of ganglion cells in the retina of the three-toed sloth (Bradypus variegatus)

1. The distribution ans size of retinal ganglion cells labelled with horseradish preoxidase (HRP) were studied in flat-mounted retinas of three-toed sloths. 2. Massive injections of HRP solution were made throughout the thalamus and midbrain in anesthetized sloths in order to retrogradely label the retinal ganglion cell population. Twenty to thirty h later the eyes were and the retinas flat-mounted and reacted with phenylenediamine-HCL and H2O2 to label ganglion cells,thus distinguishing then from other cells int he same retinal layer. 3. Ganglion cell density graually increased from about 500 cell/mm2 at the far periphery to a peak of about 1,500 cells/mm2 in an area, termed the area centralis, deep in the inferior temporal retina. The presence of a vertical visual steak was also noted. 4. The area centralis contained a higher frequency of small ganglion cells than the peripheral retina where large cells preominated. 5. /the unusual postion of the area centralis and cisual streak in the retina can be explained by the slot's unique ability to rotate its head 180- while climbing upside-dow along horizontal branches so that the head is right-side-up. If it is assumed that the branch directly above the sloth's head needs to be visualized for accurate claw placement then the branch would be imaged on the inferior temporal retina in an area corresponding to the maximum density region