Luís Antunes

Comparison of Anesthetic Depth Indexes Based on Thalamocortical Local Field Potentials in Rats

Background:Local field potentials may allow a more precise analysis of the brain electrical activity than the electroencephalogram. In this study, local field potentials were recorded in the thalamocortical axis of rats to (i) compare the performance of several indexes of anesthetic depth and (ii) investigate the existence of thalamocortical correlated or disrupted activity during isoflurane steady-state anesthesia. Methods:Five rats chronically implanted with microelectrodes were used to record local field potentials in the primary somatosensory cortex and ventroposterolateral thalamic nuclei at six periods: before induction of anesthesia; in the last 5 min of randomized 20-min steady-state end-tidal 0.8, 1.1, 1.4, and 1.7% isoflurane concentrations; and after recovery. The approximate entropy, the index of consciousness, the spectral edge frequency, and the permutation entropy were estimated using epochs of 8 s. A correction factor for burst suppression was applied to the spectral edge frequency and to the permutation entropy. The correlation between the derived indexes and the end-tidal isoflurane was calculated and compared for the two studied brain regions indexes. Coherence analysis was also performed. Results:The burst suppression–corrected permutation entropy showed the highest correlation with the end-tidal isoflurane concentration, and a high coherence was obtained between the two studied areas. Conclusions:The permutation entropy corrected with the classic burst suppression ratio is a promising alternative to other indexes of anesthetic depth. Furthermore, high coherence level of activity exists between the somatosensory cortical and thalamic regions, even at deep isoflurane stages.

Lower Isoflurane Concentration Affects Spatial Learning and Neurodegeneration in Adult Mice Compared with Higher Concentrations

Background:Volatile anesthetics such as isoflurane are widely used in clinical and research contexts. Concerns have been raised that the effects of these drugs on the central nervous system may result in long-term impairment after surgery or general anesthesia. Hence, this study aimed to detect how different isoflurane concentrations influence spatial learning and cell death in adult mice. Methods:Fifty-two C57BL/6 mice were randomly divided in four groups. Mice in three groups were exposed to different concentrations of isoflurane (1, 1.5, and 2%) for 1 h; the control group was not exposed to anesthesia. Five mice per group were killed 3 h after anesthesia to perform histopathologic and immunohistochemical analyses (hematoxylin-eosin staining; caspase-3 activation). Eight mice per group were used for behavioral tests (open field, T-maze spontaneous alternation, and water maze) on subsequent days. Results:There were no differences between groups in the T-maze spontaneous alternation test or in the open field (no confounding effects of stress or locomotion). The group anesthetized with 1% isoflurane performed worse in the water maze task on day 1 (550.4 ±162.78 cm) compared with the control group (400.1 ± 112.88 cm), 1.5% isoflurane (351.9 ± 150.67 cm), and 2% isoflurane (364.5 ± 113.70 cm; P ≤ 0.05) and on day 3 (305.0 ± 81.75 cm) compared with control group (175.13 ± 77.00 cm) and 2% isoflurane (204.11 ± 85.75 cm; P ≤ 0.038). In the pyramidal cell layer of the region cornu ammonis 1 of the hippocampus, 1% isoflurane showed a tendency to cause more neurodegeneration (apoptosis) (61.4 ± 26.40, profiles/mm2) than the group with 2% of isoflurane (20.6 ± 17.77, profiles/mm2; P = 0.051). Conclusion:Low isoflurane concentration (1%) caused spatial learning impairment and more neurodegeneration compared with higher isoflurane concentrations. Results for mice receiving the latter concentrations were similar to those of control mice.

Clustering Fetal Heart Rate Tracings by Compression

Fetal heart rate (FHR) monitoring is widely used regarding the detection of fetuses in danger of death or damage. Thirty one FHR tracings acquired in the antepartum period were clustered by compression in order to identify abnormal ones. A recently introduced approach based on algorithmic information theory was used. The new method can mine patterns in completely different areas, without domain-specific parameters to set, and does not require specific background knowledge. At the highest level the FHR tracings were clustered according to an unanticipated feature, namely the technology used in signal acquisition. At the lower levels all tracings with abnormal or suspicious patterns were clustered together, independently of the technology used

MicroRNA profile: a promising ancillary tool for accurate renal cell tumour diagnosis

Background:Renal cell tumours (RCTs) are clinically, morphologically and genetically heterogeneous. Accurate identification of renal cell carcinomas (RCCs) and its discrimination from normal tissue and benign tumours is mandatory. We, thus, aimed to define a panel of microRNAs that might aid in the diagnostic workup of RCTs.Methods:Fresh-frozen tissues from 120 RCTs (clear-cell RCC, papillary RCC, chromophobe RCC (chRCC) and oncocytomas: 30 cases each), 10 normal renal tissues and 60 cases of ex-vivo fine-needle aspiration biopsies from RCTs (15 of each subtype validation set) were collected. Expression levels of miR-21, miR-141, miR-155, miR-183 and miR-200b were assessed by quantitative reverse transcription–PCR. Receiver operator characteristic curves were constructed and the areas under the curve were calculated to assess diagnostic performance. Disease-specific survival curves and a Cox regression model comprising all significant variables were computed.Results:Renal cell tumours displayed significantly lower expression levels of miR-21, miR-141 and miR-200b compared with that of normal tissues, and expression levels of all miRs differed significantly between malignant and benign RCTs. Expression analysis of miR-141 or miR-200b accurately distinguished RCTs from normal renal tissues, oncocytoma from RCC and chRCC from oncocytoma. The diagnostic performance was confirmed in the validation set. Interestingly, miR-21, miR-141 and miR-155 expression levels showed prognostic significance in a univariate analysis.Conclusion:The miR-141 or miR-200b panel accurately distinguishes RCC from normal kidney and oncocytoma in tissue samples, discriminating from normal kidney and oncocytoma, whereas miR-21, miR-141 and miR-155 convey prognostic information. This approach is feasible in fine-needle aspiration biopsies and might provide an ancillary tool for routine diagnosis.

Deregulated expression of selected histone methylases and demethylases in prostate carcinoma.

Prostate cancer (PCa), a leading cause of cancer-related morbidity and mortality, arises through the acquisition of genetic and epigenetic alterations. Deregulation of histone methyltransferases (HMTs) or demethylases (HDMs) has been associated with PCa development and progression. However, the precise influence of altered HMTs or HDMs expression and respective histone marks in PCa onset and progression remains largely unknown. To clarify the role of HMTs and HDMs in prostate carcinogenesis, expression levels of 37 HMTs and 20 HDMs were assessed in normal prostate and PCa tissue samples by RT-qPCR. SMYD3, SUV39H2, PRMT6, KDM5A, and KDM6A were upregulated, whereas KMT2A-E (MLL1-5) and KDM4B were downregulated in PCa, compared with normal prostate tissues. Remarkably, PRMT6 was the histone modifier that best discriminated normal from tumorous tissue samples. Interestingly, EZH2 and SMYD3 expression levels significantly correlated with less differentiated and more aggressive tumors. Remarkably, SMYD3 expression levels were of independent prognostic value for the prediction of disease-specific survival of PCa patients with clinically localized disease submitted to radical prostatectomy. We concluded that expression profiling of HMTs and HDMs, especially SMYD3, might be of clinical usefulness for the assessment of PCa patients and assist in pre-therapeutic decision-making.

Anaesthesia with ketamine/medetomidine in the rabbit: influence of route of administration and the effect of combination with butorphanol

OBJECTIVE To compare the characteristics of anaesthesia induced with ketamine/medetomidine administered by the subcutaneous and intramuscular routes and to assess the effects of the addition of butorphanol to this combination. STUDY DESIGN Prospective randomised study. ANIMALS Six female New Zealand White rabbits. METHODS Rabbits were given one of four combinations of ketamine and medetomidine (K/M) either subcutaneously (SC) or intramuscularly (IM) on four successive occasions with a 7-day interval between treatments. The dose combinations were; 15/0.25 mg kg-1 SC; 15/0.25 mg kg-1 IM; 15/0.5 mg kg-1 SC, and 15/0.25 mg kg-1 together with 0.4 mg kg-1 butorphanol (K/M/B) SC. The effects of anaesthesia on arterial blood gas values and cardiovascular variables were recorded at predetermined time points. Toe and ear pinch reflexes were judged to determine the duration of surgical anaesthesia. Loss of the righting reflex was used to measure the duration of sleep time. Analyses used repeated measures analysis of variance. RESULTS All groups lost the righting reflex and ear pinch response. Three animals in the groups that received K/M alone lost their toe pinch reflex, whereas four lost this reflex when given K/M/B. Time of onset of loss of the righting, toe and ear pinch reflexes did not differ significantly among the groups. The higher dose combination of medetomidine with ketamine and the combination of K/M/B produced a greater duration of loss of the ear pinch response than the lower dose of K/M administered by either route. No significant differences were found among the groups in the duration of loss of the toe pinch reflex. All animals developed a moderate bradycardia (mean heart rate <166 beats minute-1) and moderate hypoxaemia (mean PaO2 < 6.0 kPa). Animals given butorphanol showed the greatest reduction in respiratory rate (31 ± 13 breaths minute-1, p < 0.05) but this was not reflected in any significant differences in arterial PCO2, PO2 or pH among the groups. CONCLUSIONS Administration of K/M by the SC route produced equivalent effects in comparison to intramuscular administration. The addition of butorphanol increased the duration of anaesthesia, but produced a slight increase in the degree of respiratory depression. All dose rates resulted in hypoxaemia so oxygen should be administered when these combinations are used in rabbits. CLINICAL RELEVANCE Subcutaneous administration is both technically simpler and may cause less discomfort to the animal than IM injection, and so is preferred. The combination of K/M with butorphanol has relatively minor effects on the depth and duration of anaesthesia, so offers little advantage to the use of K/M alone.

Ketamine NMDA receptor-independent toxicity during zebrafish (Danio rerio) embryonic development

Concerns have been raised that the effect of anaesthetic drugs on the central nervous system may result in long-term impairment, namely when ketamine is used during embryogenesis. In addition, the cell and molecular basis of anaesthetics teratology and toxicity are still uncertain and its implications in the development remain to be clarified. More recently, the potential risks for human, and animal, exposure through environmental contamination also became an important question. In this study, the effects of sub- and over anaesthetic doses of ketamine were investigated during zebrafish (Danio rerio) embryonic development by exposing zebrafish embryos to ketamine concentrations (0.2, 0.4 and 0.8 mg mL(-1)) for a period of 20 min during the blastula stage. Ethanol 2% was used as a positive control. Morphological parameters, the overall pattern of cell death using acridine orange and overall degree of oxidative stress levels by 2,7-dichlorodihydrofluorescein-diacetate were determined. Lethality and/or developmental anomalies were measured based on specific time endpoints until 144 h post fertilisation. Results showed a concentration-dependent increase in anomalies and mortality. Cephalic disorders, enlarged organs and tail/spine anomalies were the most prominent deformities observed at 144 hpf. Acridine orange images revealed no differences in cellular death pattern in exposed embryos at 24 hpf. At the same time point, the cellular redox processes were found to be similar among groups. In summary, this study shows that ketamine is teratogen and toxic, interfering with the normal developmental pathways of embryogenesis, suggesting that ketamine exerts an independent NMDA receptor action during the zebrafish blastula stage.

Incidence rates and trends of lip, oral and oro-pharyngeal cancers in Portugal

OBJECTIVES To analyse the trends and patterns of lip, oral and oro-pharyngeal cancer incidence in Portugal between 1998 and 2007. PATIENTS AND METHODS Data on lip, oral and oro-pharyngeal cancers was collected from the databases maintained at the three main Regional Cancer Registries of Portugal (1998-2007). The data were analysed by gender, age and by site. Incidence rates were age standardized by the direct method, and joinpoint regression was used to estimate trends in incidence. RESULTS During this 10-year period, a total of 9623 cases of lip, oral and oropharynx cancers were reported, 7565 (78.6%) in males and 2058 (21.4%) in females. There was an increase in the age-standardized incidence of oral cancers by 1.96% per year for both sexes grouped together and an increase of 4.34% per year for the female group. Oro-pharyngeal cancer showed an increase incidence trend of 3.49% per year for both sexes grouped together and an increase of 3.49% per year for male group among the sites analysed. Lip cancer showed a decrease in its incidence rate. CONCLUSION In view of rising trends, it is necessary to implement policies on oral cancer control by initiating campaigns on oral cancer awareness and screening and to harness political measures on tobacco and alcohol control for the Portuguese population.

Performance of Anesthetic Depth Indexes in Rabbits under Propofol Anesthesia Prediction Probabilities and Concentration-effect Relations

Background:The permutation entropy, the approximate entropy, and the index of consciousness are some of the most recently studied electroencephalogram-derived indexes. In this work, a thorough comparison of these indexes was performed using propofol anesthesia in a rabbit model. Methods:Six rabbits were anesthetized with three propofol infusion rates: 70, 100, and 130 mg · kg −1 · h−1, each maintained for 30 min, in a random order for each animal. Data recording was performed in the awake animals 20, 25, and 30 min after each infusion rate was begun in the recovered animals and consisted of electroencephalogram recordings, evaluation of depth of anesthesia according to a clinical scale, and arterial blood samples for plasma propofol determination. Median and spectral edge frequencies were analyzed for single-scale permutation entropy and composite multiscale permutation entropy, approximate entropy, index of consciousness, and the spectral parameters. The spectral parameters and single-scale and multiscale permutation entropies were corrected for the presence of burst suppression. Performance of the indexes was compared by prediction probability and pharmacodynamic analysis. Results:The single-scale and composite multiscale permutation entropies with a burst suppression correction showed better prediction probabilities than did the other electroencephalogram-derived parameters but not better than the electromyographic activity. Conclusion:Single-scale and multiscale permutation entropies may be promising measures of propofol anesthetic depth when corrected for burst suppression. Additional studies should investigate the information measured by electromyography algorithms from commercial monitors of anesthetic depth. The rabbit may be a promising animal model for electroencephalographic studies because it provides a good-quality signal.

Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation

AIM OF THE STUDY Upper tract urothelial carcinoma (UTUC) accounts for 5-10% of all urothelial tumours. It is mostly diagnosed at advanced stages, entailing a worse prognosis, owing to the lack of early and specific symptoms as well as of effective diagnostic tools. We previously identified a panel of epigenetic biomarkers (GDF15, TMEFF2 and VIM promoter methylation) that accurately identifies bladder cancer in urine. Herein, we assessed the performance of the same panel for UTUC detection and prognosis, in tissue and urine. MATERIAL AND METHODS Methylation levels of reference and target genes were determined using real-time quantitative methylation-specific polymerase chain reaction (MSP) in bisulphite-modified DNA of 57 UTUC tissues, 36 normal upper tract urothelium (NUTUs), 22 urines from UTUC suspects and 20 urines from controls. Receiver operator characteristics (ROC)-curve analysis was performed to determine the performance of the biomarker panel and survival analyses were conducted to evaluate their prognostic value. RESULTS Methylation levels of GDF15, TMEFF2 and VIM were significantly higher in UTUC compared to NUTUs (P=0.022; P<0.001; P<0.001, respectively). The panel accurately identified UTUC with 100% and 91% sensitivity, corresponding to an area under the curve of 1.000 and 0.923 in tissue and urines, respectively, with 100% specificity. Low VIM promoter methylation levels independently predicted poor disease-specific survival. CONCLUSIONS GDF15, TMEFF2 and VIM promoter methylation allows for accurate identification of UTUC, in tissue and urine and VIM methylation provides relevant prognostic information, especially in high-stage disease. This assay may improve the clinical management of UTUC patients.