Mariana Paula Cid

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

INTRODUCTION the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. OBJECTIVE to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. METHODS a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. RESULTS the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde´s staining demonstrated the conservationof reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson´s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. CONCLUSIONS the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

Acute stress or systemic insulin injection increases flunitrazepam sensitive-GABAA receptor density in synaptosomes of chick forebrain: Modulation by systemic epinephrine

Interactions between acute stress and systemic insulin and epinephrine on GABAA receptor density in the forebrain were studied. Here, 10 day-old chicks were intraperitoneally injected with insulin, epinephrine or vehicle and then immediately stressed by partial water immersion for 15 min and killed by decapitation. Non-stressed controls were similarly injected, then returned to their rearing boxes for 15 min and then killed. Forebrains were dissected and GABAA receptor density was measured ex vivo in synaptosomes by 3[H]-flunitrazepam binding assay. In non-stressed chicks, insulin at 1.25, 2.50 and 5.00 IU/kg of body weight (non-hypoglycemic doses) increased Bmax by 33, 53 and 44% compared to saline, respectively. A similar increase of 41% was observed in receptor density after stress. However, the insulin effect was not additive to the stress-induced increase suggesting that both effects occur through similar mechanisms. In contrast, epinephrine, at 0.25 and 0.5 mg/kg did not induce any changes in Bmax in non-stressed chicks. Nevertheless, after stress these doses increased the receptor density by about 13 and 27%, respectively. Similarly, the same epinephrine doses co-administered with insulin (2.50 IU/kg), increased the receptor density by about 20% compared to insulin alone. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulates the increase in forebrain GABAA receptor binding induced by both insulin and stress.

Neonatal acute stress by novelty in the absence of social isolation decreases fearfulness in young chicks.

Two hours after hatching (Day 0), groups of chicks from both sexes were housed either individually (IND) or socially in pairs (SOC) for 24 h. On Day 1, for each of the two conditions, half of the chicks were individually exposed to early novelty for 10 min, which comprised being placed in a novel-cage with small pebbles glued to the floor. The other half (controls) remained in the home-cage (IND-C and SOC-C). Thus, the IND-N group was exposed to early novelty, and the SOC-N+I group was exposed to early novelty and social isolation. Subsequently, all groups were mixed and socially reared until reaching 15 days of age. At this time, chicks were exposed to open field (OF) and tonic immobility (TI) tests. The IND-N group showed a shorter latency to ambulate in the OF test, shorter immobility duration in the TI test, a reduced plasma corticosterone concentration and increased flunitrazepam sensitive-GABAA receptor basal forebrain density compared with other groups, indicating that a neonatal novelty induced lower fearfulness in young chicks. In contrast, the effect of neonatal novelty was abolished by a simultaneous effect of social isolation in the SOC-N+I group. Thus, early post-hatch life events such as early novelty could improve a birds later ability to cope with new stressful events. In addition, it is possible that both novelty and social isolation act on different neurobiological processes.

Sedative effect of central administration of Coriandrum sativum essential oil and its major component linalool in neonatal chicks.

Abstract Context Coriandrum sativum L. (Apiaceae) (coriander) is an herb grown throughout the world as a culinary, medicinal or essential crop. In traditional medicine, it is used for the relief of anxiety and insomnia. Systemic hydro-alcoholic and aqueous extract from aerial parts and seeds had anxiolytic and sedative action in rodents, but little is known about its central effect in chicks. Objective To study the effects of intracerebroventricular administration of essential oil from coriander seeds and its major component linalool on locomotor activity and emotionality of neonatal chicks. Materials and methods The chemical composition of coriander essential oil was determined by a gas-chromatographic analysis (> 80% linalool). Behavioural effects of central administration of coriander oil and linalool (both at doses of 0.86, 8.6 and 86 μg/chick) versus saline and a sedative diazepam dose (17.5 μg/chick, standard drug) in an open field test for 10 min were observed. Results Doses of 8.6 and 86 μg from coriander oil and linalool significantly decreased (p < 0.05) squares crossed number, attempted escapes, defecation number and distress calls, and significantly increased (p < 0.05) the sleeping posture on an open field compared with saline and were similar to the diazepam group. Discussion and conclusion The results indicate that intracerebroventricular injection of essential oil from Coriandrum sativum seeds induced a sedative effect at 8.6 and 86 μg doses. This effect may be due to monoterpene linalool, which also induced a similar sedative effect, and, therefore, could be considered as a potential therapeutic agent similar to diazepam.

Recruitment of GABAA receptors and fearfulness in chicks: Modulation by systemic insulin and/or epinephrine

One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation fearfulness.

Cytotoxicity of hydroxyapatite and morphology in composites with Ti

Hydroxyapatite (Ca10(PO4)6(OH)2 is the material used to improve osseointegration of implants in humans. Traditionally Ti-based implant is coated with hydroxyapatite, but this presents several problems such as, low fracture toughness of hydroxyapatite, generation of interfacial cracks during the deposition step and low adhesion ceramic /metal. Furthermore some studies reported that some excellent Ti¿HA bio-composites could be fabricated by the powder metallurgy (PM) technology. In present study it was investigate the cytotoxicity of coralline and bovine hydroxyapatite. Topography of Ti-HA composites obtained by powder metallurgy was evaluated by scanning electron microscopy. The results showed that HA samples analyzed did not affect cell viability. Furthermore, a good integration of HA to the matrix of Ti was observed by microscopy studies.

Bicuculline, a GABAA-receptor antagonist, blocked HPA axis activation induced by ghrelin under an acute stress

HighlightsCentral administration of ghrelin in chicks induced anxiogenic like effect.Ghrelin significantly increased plasma ACTH and corticosterone level.Bicuculline methiodide blocks the behavioral and physiological effect of ghrelin.Ghrelin, GABAAR and HPA axis interacts a complex way to regulate anxiogenic response. ABSTRACT Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic‐like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABAA receptor competitive antagonist) but not by diazepam (a GABAA receptor allosteric agonist) in neonatal meat‐type chicks (Cobb). Numerous studies have indicated that hypothalamic–pituitary–adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra‐IMM ghrelin and mediated through GABAA receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic‐like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30 pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABAA receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABAA receptor.

Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility

Background In clinical orthopedics, a critical problem is the bone tissue loss produced by a disease or injury. The use of composites from titanium and hydroxyapatite for biomedical applications has increased due to the resulting advantageous combination of hydroxyapatite bioactivity and favorable mechanical properties of titanium. Powder metallurgy is a simple and lower-cost method that uses powder from titanium and hydroxyapatite to obtain composites having hydroxyapatite phases in a metallic matrix. However, this method has certain limitations arising from thermal decomposition of hydroxyapatite in the titanium-hydroxyapatite system above 800°C. We obtained a composite from titanium and bovine hydroxyapatite powders sintered at 800°C and evaluated its bioactivity and cytocompatibility according to the ISO 10993 standard. Methods Surface analysis and bioactivity of the composite was evaluated by X-ray diffraction and SEM. MTT assay was carried out to assess cytotoxicity on Vero and NIH3T3 cells. Cell morphology and cell adhesion on the composite surface were analyzed using fluorescence and SEM. Results We obtained a porous composite with hydroxyapatite particles well integrated in titanium matrix which presented excellent bioactivity. Our data did not reveal any toxicity of titanium-hydroxyapatite composite on Vero or NIH3T3 cells. Moreover, extracts from composite did not affect cell morphology or density. Finally, NIH3T3 cells were capable of adhering to and proliferating on the composite surface. Conclusions The composite obtained displayed promising biomedical applications through the simple method of powder metallurgy. Additionally, these findings provide an in vitro proof for adequate biocompatibility of titanium-hydroxyapatite composite sintered at 800°C.