Manual and semi-automatic quantification of in vivo ¹H-MRS data for the classification of human primary brain tumors

In vivo proton magnetic resonance spectroscopy (¹H-MRS) is a technique capable of assessing biochemical content and pathways in normal and pathological tissue. In the brain, ¹H-MRS complements the information given by magnetic resonance images. The main goal of the present study was to assess the accuracy of ¹H-MRS for the classification of brain tumors in a pilot study comparing results obtained by manual and semi-automatic quantification of metabolites. In vivo single-voxel ¹H-MRS was performed in 24 control subjects and 26 patients with brain neoplasms that included meningiomas, high-grade neuroglial tumors and pilocytic astrocytomas. Seven metabolite groups (lactate, lipids, N-acetyl-aspartate, glutamate and glutamine group, total creatine, total choline, myo-inositol) were evaluated in all spectra by two methods: a manual one consisting of integration of manually defined peak areas, and the advanced method for accurate, robust and efficient spectral fitting (AMARES), a semi-automatic quantification method implemented in the jMRUI software. Statistical methods included discriminant analysis and the leave-one-out cross-validation method. Both manual and semi-automatic analyses detected differences in metabolite content between tumor groups and controls (P < 0.005). The classification accuracy obtained with the manual method was 75% for high-grade neuroglial tumors, 55% for meningiomas and 56% for pilocytic astrocytomas, while for the semi-automatic method it was 78, 70, and 98%, respectively. Both methods classified all control subjects correctly. The study demonstrated that ¹H-MRS accurately differentiated normal from tumoral brain tissue and confirmed the superiority of the semi-automatic quantification method.

In vivo photorelease of GABA in the mouse cortex

Electrical stimulation has been used for more than 100 years in neuroscientific and biomedical research as a powerful tool for controlled perturbations of neural activity. Despite quickly driving neuronal activity, this technique presents some important limitations, such as the impossibility to activate or deactivate specific neuronal populations within a single stimulation site. This problem can be avoided by pharmacological methods based on the administration of receptor ligands able to cause specific changes in neuronal activity. However, intracerebral injections of neuroactive molecules inherently confound the dynamics of drug diffusion with receptor activation. Caged compounds have been proposed to circumvent this problem, for spatially and temporally controlled release of molecules. Caged compounds consist of a protecting group and a ligand made inactive by the bond between the two parts. By breaking this bond with light of an appropriate wavelength, the ligand recovers its activity within milliseconds. To test these compounds in vivo, we recorded local field potentials (LFPs) from the cerebral cortex of anesthetized female mice (CF1, 60-70 days, 20-30 g) before and after infusion with caged γ-amino-butyric-acid (GABA). After 30 min, we irradiated the cortical surface with pulses of blue light in order to photorelease the caged GABA and measure its effect on global brain activity. Laser pulses significantly and consistently decreased LFP power in four different frequency bands with a precision of few milliseconds (P < 0.000001); however, the inhibitory effects lasted several minutes (P < 0.0043). The technical difficulties and limitations of neurotransmitter photorelease are presented, and perspectives for future in vivo applications of the method are discussed.

In vitro and in vivo studies of pirarubicin-loaded SWNT for the treatment of bladder cancer

Intravesical chemotherapy is an important part of the treatment for superficial bladder cancer. However, the response to it is limited and its side effects are extensive. Functional single-walled carbon nanotubes (SWNT) have shown promise for tumor-targeted accumulation and low toxicity. In the present study, we performed in vivo and in vitro investigations to determine whether SWNT-based drug delivery could induce high tumor depression in rat bladder cancer and could decrease the side effects of pirarubicin (tetrahydropyranyl-adriamycin, THP). We modified SWNT with phospholipid-branched polyethylene glycol and constructed an SWNT-THP conjugate via a cleavable ester bond. The cytotoxicity of SWNT-THP against the human bladder cancer cell line BIU-87 was evaluated in vitro. Rat bladder cancer in situ models constructed by N-methyl-N-nitrosourea intravesical installation (1 g/L, 2 mg/rat once every 2 weeks for 8 weeks) were used for in vivo evaluation of the cytotoxicity of SWNT and SWNT-THP. Specific side effects in the THP group including urinary frequency (N = 12), macroscopic hematuria (N = 1), and vomiting (N = 7) were identified; however, no side effects were observed with SWNT-THP treatment. Flow cytometry was used to assess the cytotoxicity in vitro and in vivo. Results showed that SWNT alone did not yield significant tumor depression compared to saline (1.74 ± 0.56 and 1.23 ± 0.42%) in vitro. SWNT-THP exhibited higher tumor depression than THP-saline in vitro (74.35 ± 2.56 and 51.24 ± 1.45%) and in vivo (52.46 ± 2.41 and 96.85 ± 0.85%). The present findings indicate that SWNT delivery of THP for the treatment of bladder cancer leads to minimal side effects without loss of therapeutic efficacy. Therefore, this nanotechnology may play a crucial role in the improvement of intravesical treatment of bladder cancer.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

Ultrasound method applied to characterize healthy femoral diaphysis of Wistar rats in vivo

A simple experimental protocol applying a quantitative ultrasound (QUS) pulse-echo technique was used to measure the acoustic parameters of healthy femoral diaphyses of Wistar rats in vivo. Five quantitative parameters [apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), time slope of apparent backscatter (TSAB), integrated reflection coefficient (IRC), and frequency slope of integrated reflection (FSIR)] were calculated using the echoes from cortical and trabecular bone in the femurs of 14 Wistar rats. Signal acquisition was performed three times in each rat, with the ultrasound signal acquired along the femur's central region from three positions 1 mm apart from each other. The parameters estimated for the three positions were averaged to represent the femur diaphysis. The results showed that AIB, FSAB, TSAB, and IRC values were statistically similar, but the FSIR values from Experiments 1 and 3 were different. Furthermore, Pearson's correlation coefficient showed, in general, strong correlations among the parameters. The proposed protocol and calculated parameters demonstrated the potential to characterize the femur diaphysis of rats in vivo. The results are relevant because rats have a bone structure very similar to humans, and thus are an important step toward preclinical trials and subsequent application of QUS in humans.

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

Alterações estruturais in vivo dos isômeros todo-trans, 9-cis e 13-cis do beta-caroteno

Com o objetivo de verificar alterações estruturais nos isômeros todo-trans, 9- e 13-cis do beta-caroteno foi realizado um ensaio biológico baseado no modelo de esgotamento das reservas hepáticas de carotenóides em ratos. Animais depletados desses carotenóides receberam, durante quinze dias, os isômeros puros todo-trans, 9-cis e 13-cis do beta-caroteno. Ao final deste período, verificou-se a ocorrência de re-isomerização in vivo desses isômeros, a partir da quantificação dos mesmos depositados no fígado dos animais. Foi observada re-isomerização do 9-cis em todo-trans, do todo-trans em 9-cis, do 13-cis em 9-cis e todo-trans. O 13-cis foi mais susceptível à isomerização que o 9-cis, pois este último passou a todo-trans e nunca a 13-cis. Já o 13-cis, tanto pode se transformar em 9-cis quanto em todo-trans.

Antioxidant activity in vivo and in vitro of Halimeda incrassata aqueous extracts

The aim of the present paper was to provide the evidences for the antioxidant activity in Halimeda incrassata (Ellis) Lamouroux aqueous extracts obtained after simple water extraction of the fresh algae at room temperature (23°C). Previously in the literature, only antioxidant activity associated to carotenoids fractions of seaweeds has been reported. From different species of seaweeds, Halimeda incrassata aqueous extract exhibited the highest antioxidant activity on the inhibition of TBARS formed during the spontaneous lipid peroxidation of rat brain homogenates with an IC50 of 0.340mg.mL-1. Halimeda incrassata aqueous extract (0.5mg.mL-1), was also capable of decreasing the in vitro generation of hydrogen peroxide by two distinct metabolic pathways involving glutamic and malonic acids. Also, Halimeda incrassata (at doses of 50, 100 and 200mg.Kg-1) showed a neuroprotective effect in vivo on the gerbil model of bilateral carotid occlusion because of decreasing the locomotor and exploratory activity induced by ischemia. In summary, Halimeda incrassata aqueous extracts exhibit antioxidant properties in different in vitro as well as in vivo models which could be explained by the presence of several hydrosoluble compounds. Further studies on this way are necessary to elucidate the precise structure of these compounds. Low toxicity of most seaweeds to humans, but particularly of Halimeda genus may favor its use as functional food.

Isolamento da globulina majoritária, digestibilidade in vivo e in vitro das proteínas do tremoço-doce (Lupinus albus L.), var. Multolupa

Os objetivos do trabalho foram isolar, purificar e estudar algumas propriedades da fração globulina majoritária de tremoço-doce, var. Multolupa; assim como avaliar as características de digestibilidade da farinha e frações isoladas. As frações protéicas foram separadas por fracionamento diferencial com uso de diferentes solventes. A globulina majoritária de tremoço-doce foi isolada, purificada por cromatografia em Q-Sepharose, revelando um único pico de proteína. Apresentou um peso molecular de 162,5 ± 10,0 kDa, determinado por cromatografia em Sephacryl S-300, e subunidades entre 20-70 kDa em PAGE-SDS. A solubilidade em função do pH e concentrações de NaCl revelaram curva típica dessa fração. A digestibilidade da proteína da farinha e das frações albumina, globulina e glutelina foi avaliada por experimentos in vitro e in vivo e se revelou alta para a fração globulina, seguida pela glutelina, albumina e farinha. A digestibilidade in vivo da fração globulina, tanto aparente quanto verdadeira, não diferiu significativamente daquela determinada para a caseína. Apesar da alta digestibilidade da fração majoritária e frações protéicas, a utilização como única fonte de proteína nas dietas revelou valores baixos de RPLR (razão protéica líquida relativa), indicando ser insuficiente para sustentar o crescimento dos animais, comparativamente à caseína.