5 results
Effects of antifreeze protein from Lolium perenne L. (LpAFP) in the vitrification of in vitro-produced bovine embryos
- R.A. Silva Júnior, R. Desenzi, M.M.S. Ramires, A.F. Souza, M.A.M. Donato, C.A. Peixoto, T. Nascimento, C.C. Bartolomeu, A.M. Batista
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In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.
Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue
- T.J.S. Macedo, V.G. Menezes, R.S. Barberino, R.L.S. Silva, B.B. Gouveia, A.P.O. Monte, T.L.B.G. Lins, J.M.S. Santos, M.É.S. Bezerra, A. Wischral, M.A.A. Queiroz, G.G.L. Araújo, A.M. Batista, M.H.T. Matos
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This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.
Corticobasal Degeneration Presenting with Depression and Dystonia: A Case Report
- J. Cerejeira, R. André, P. Batista, P. Carriço, A.M. Ferreira, H. Firmino, C. Januário
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- European Psychiatry / Volume 24 / Issue S1 / January 2009
- Published online by Cambridge University Press:
- 16 April 2020, 24-E854
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Corticobasal degeneration is a rare neurodegenerative disorder affecting both cortex and basal ganglia with clinical and underlying pathological heterogeneity. Although motor features of CBD were emphasized in earlier descriptions psychiatric symptoms, including cognitive impairment and mood disorders, have been consistently reported during the course of the disease. Clinical diagnosis of CBD is challenging and can be difficult to differentiate from other neuropsychiatric disorders with overlapping features. This can lead to significant underdiagnosis of CBD particularly during its early stages.
We report a case of a 48 year-old female patient presenting with insidious orofacial dystonia co-occurring with depression which remained controlled for five years. Later, while experiencing major psycho-social stress factors, she presented with a rapidly progressive clinical syndrome compatible with the diagnosis of cortico-basal degeneration with severe motor, cognitive and behavioural symptoms, including alien limb phenomenon, nonfluent aphasia and personality changes. Neuropsychological assessment revealed significant frontal lobe dysfunction and SPECT imaging showed asymetrical fronto-parietal hypoperfusion.
This case illustrates the difficulties in the clinical diagnosis of CBD both in early and late stages due to its clinical overlap with mood and movement disorders as well as with Fronto-Temporal Dementia. At the same time, it highlights the influence of psycho-social stress factors in the manifestation of degenerative disorders.
Electroconvulsive therapy in elderly - a preliminary study
- C. Agostinho, M. Duarte, R. Alves, I. Cunha, A.M. Batista
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- Journal:
- European Psychiatry / Volume 33 / Issue S1 / March 2016
- Published online by Cambridge University Press:
- 23 March 2020, p. s230
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Introduction
Studies with electroconvulsive therapy (ECT) in elderly focus mainly on the assessment of possible side effects on the cognitive functioning; there are few studies that evaluate the effectiveness.
ObjectiveEvaluate the effectiveness of this treatment in the population over 65 years.
AimsPerform a preliminary study to evaluate the response to ECT of ≥ 65 years patients with depression.
MethodsWe carry out a descriptive study based on patients treated in the last 10 years in the ECT Unit of Centro Hospitalar Psiquiátrico de Lisboa.
ResultsOur initial sample consisted of 457 patients. We select patients aged ≥ 65 years with depression, and with complete data, including electroconvulsive parameters, and initial and final Hamilton Rating Scale for Depression (HRSD) scores (n = 59). Of this, 81.36% (n = 48) had unipolar depression, and 18.64% (n = 11) had bipolar depression. In the first group, the mean variation between the initial and final scores in HRSD was 13.88 points, and 27.10% (n = 13) of the patients ended the treatment in the normal range of HRSD score. In the second group, the mean variation was 12.82, and 63.60% (n = 7) ended the treatment in the normal range of HRSD. Considering the initial and final HRSD scores, it appears that unipolar depression group presents higher values (severe depression) (P < 0.05). When we compare the mean variation between the initial and final HRSD scores, we didn’t observe a statistically significant difference between the two groups. There was a clinical improvement in both.
ConclusionsThe acute treatment with ECT appears to improve depressive symptoms in bipolar and unipolar depression, when considering an elderly population.
Disclosure of interestThe authors have not supplied their declaration of competing interest.
Corneal Metabolic State Assessment by Fluorescence Lifetime Imaging Microscopy
- A. Batista, C. Loureiro, J. Domingues, J.S. Silva, A.M. Morgado
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 7-8
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- August 2013
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A long time objective of ophthalmologists is to diagnose corneal cells dysfunction prior to its pathologic expression. With this motivation, we are currently developing a new instrument for in vivo metabolic imaging of corneal tissues.
Metabolic alterations are known to be the first sign of several corneal pathologies and can be assessed through non-invasive monitoring of metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The quantification of the relative proportions between free and protein-bound NADH and FAD can be achieved using fluorescence lifetime-resolved methods. This approach has already been applied in age-related macular degeneration, diabetic retinopathy and epithelial cancer.
FAD and NADH imaging can be performed by one-photon excitation (1PE) and two-photon excitation (2PE) techniques. The latest has the advantage of allowing simultaneous excitation of both metabolic co-factors. However, there are still safety concerns when considering in vivo ocular studies in humans using 2PE.
Due to these concerns we used, as a first approach, a 1PE system for evaluating the feasibility of corneal FAD imaging. The use of FAD has advantages over NADH. It can be excited over longer excitation wavelengths, is more resistant to photo-bleaching and is located exclusively in the mitochondrial space.
A PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan) was used to monitor FAD autofluorescence. The instrument uses a 440 nm pulsed diode laser (330 ps) running at a pulse rate of 40 MHz. The instrument was modified by us to allow the acquisition of both fluorescence lifetime and reflectance images and to enhance scattered light rejection.
Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH, Berlin, Germany). The fluorescence decay times were obtained after applying a non-linear least square fit to the decay data and the goodness of fit was evaluated by the analysis of the residuals and the chi-squared (χ2).
We have acquired fluorescence lifetime images of ex vivo healthy Wistar rat corneas (Fig.1) using two different instrument setups: 1- using the emission filters provided by the manufacturer; 2- placing extra emission filters to fully reject the scattered excitation light. In both setups, FAD fluorescence data presented a bi-exponential decay with a short (protein-bound FAD) and a longer (free FAD) lifetime component.
While both setups provide FAD fluorescence decays, only the second retrieves valid metabolic information. We obtained two lifetime components, one of 0.118 (0.028) ns and another of 2.11 (0.16) ns, with a relative contributions of 39.4 (2.2) and 60.6 (2.2), respectively. These values are in accordance with the literature.
Corneal layer discrimination is possible based on morphologic characteristics. However, the fluorescence lifetime images do not provide morphological detail (Fig.1), possibly because FAD is only present in the mitochondria. These organelles are small and tend to accumulate around the nuclei.
So, we modified the instrument’s optical setup to allow the acquisition of both fluorescence lifetime images and reflectance images. Figure 2 shows an example of the corneal epithelial layer.
The image resolution and depth penetration are still not ideal. Since the assessment of corneal endothelial layer metabolic function is also within our goals, we are currently implementing further modifications to improve both the instrument’s resolution and depth penetration.
The characterization of FAD fluorescence lifetime in unhealthy corneas is important to detect corneal dysfunctions prior to its pathologic expression. Therefore, we intend to study metabolic altered Wistar rat corneas. The alterations will be induced by potassium cyanide, which is a reversible inhibitor of the fourth complex of the mitochondrial electron transport chain.
Financial support received from the Fundação para a Ciência e a Tecnologia under the research projects PTDC/SAU-BEB/104183/2008 and PTDC/SAU-ENB/122128/2010.