Detection of visual activation in the rat brain using 2-deoxy-2-[(18)F]fluoro-D: -glucose and statistical parametric mapping (SPM).

PURPOSE: This study was designed to assess changes in brain glucose metabolism in rats after visual stimulation. MATERIALS AND METHODS: We sought to determine whether visual activation in the rat brain could be detected using a small-animal positron emission tomography (PET) scanner and 2-deoxy-2-[(18)F]fluoro-D: -glucose (FDG). Eleven rats were divided into two groups: (a) five animals exposed to ambient light and (b) six animals stimulated by stroboscopic light (10 Hz) with one eye covered. Rats were injected with FDG and, after 45 min of visual stimulation, were sacrificed and scanned for 90 min in a dedicated PET tomograph. Images were reconstructed by a three-dimensional ordered subset expectation maximization algorithm (1.8 mm full width at half maximum). A region-of-interest (ROI) analysis was performed on 14 brain structures drawn on coronal sections. Statistical parametric mapping (SPM) adapted for small animals was also carried out. Additionally, the brains of three rats were sliced into 20-microm sections for autoradiography. RESULTS: Analysis of ROI data revealed significant differences between groups in the right superior colliculus, right thalamus, and brainstem (p < or = 0.05). SPM detected the same areas as the ROI approach. Autoradiographs confirmed the existence of hyperactivation in the left superior colliculus and auditory cortex. CONCLUSIONS: To our knowledge, this is the first report that uses FDG-PET and SPM analysis to show changes in rat brain glucose metabolism after a visual stimulus.

Extraction of the respiratory signal from small-animal CT projections for a retrospective gating method.

We propose a retrospective respiratory gating algorithm to generate dynamic CT studies. To this end, we compared three different methods of extracting the respiratory signal from the projections of small-animal cone-beam computed tomography (CBCT) scanners. Given a set of frames acquired from a certain axial angle, subtraction of their average image from each individual frame produces a set of difference images. Pixels in these images have positive or negative values (according to the respiratory phase) in those areas where there is lung movement. The respiratory signals were extracted by analysing the shape of the histogram of these difference images: we calculated the first four central and non-central moments. However, only odd-order moments produced the desired breathing signal, as the even-order moments lacked information about the phase. Each of these curves was compared to a reference signal recorded by means of a pneumatic pillow. Given the similar correlation coefficients yielded by all of them, we selected the mean to implement our retrospective protocol. Respiratory phase bins were separated, reconstructed independently and included in a dynamic sequence, suitable for cine playback. We validated our method in five adult rat studies by comparing profiles drawn across the diaphragm dome, with and without retrospective respiratory gating. Results showed a sharper transition in the gated reconstruction, with an average slope improvement of 60.7%.

[Research and surgery]. / Investigación y cirugía.

From the Second World War onwards an amazing development in science and clinical & surgery practice has taken place: antibiotics, blood storage, cardiac surgery, organ and tissue transplant, complete joint replacement, total intravenous nutrition or minimally invasive surgery. Paradoxically, during the last two decades of the XXth century, social popularity of doctors has decreased at the same time of some important changes: increases in costs of medical attention, trials against medical mistakes, some doubts on the real role of Academic Public Hospitals and rising in the importance of alternative medicine. Increasing complexity of biomedical research in the continuous changing age of molecular biology has promoted an increasing scepticism regarding clinics and surgeons are able to keep on contributing to medical advances. The study of the contemporaneous History of Medicine demonstrates that some of the more significant achievements have been accomplished by surgeons. Undoubtedly Science and Clinics must get adapted to these times of change and persist in generating important findings. Current Clinical and surgical practice is completely determined by yesterdays' scientific research; tomorrow won't be different.

[Design of experimental models in surgical investigation]. / Diseño de modelos experimentales en investigación quirúrgica.

INTRODUCTION: Research in Medicine is essentially based on three knowledge resources: diseased people (natural and primary), cadaveric bodies (Pathology primary resource) and experimental animals, whom constitutes physiopathologic knowledge resource. Experimental advances reached in the last century have determined the change of the concept "experimental animal" to a wider term: "experimental model". OBJECTIVES: This paper tryes to clarify this concept. To attain this goal, we must define previous considerations in the meaning of the concept "experimental models" and its two key-elements: statistics and design of experiments.

Investigación y cirugía / Research and surgery

A partir de la Segunda Guerra Mundial se ha producido un desarrollo sin precedentes en la ciencia y en la práctica médico-quirúrgicas: antibióticos, bancos de sangre, cirugía cardiaca, órganos artificiales, trasplante de órganos y de tejidos, reemplazamiento articular completo, alimentación parenteral total o cirugía mínimamente invasiva. Paradójicamente, durante las últimas dos décadas del siglo pasado, la apreciación pública de los médicos ha disminuido, a la vez que ha tenido lugar una serie de distorsiones: escalada de costes en la atención de la salud, cuestionamiento de los centros médicos académicos, pleitos por errores médicos o alza de las medicinas alternativas. La creciente complejidad de la investigación biomédica en la era continuamente cambiante de la biología molecular, ha promovido una actitud de escepticismo respecto a si los clínicos y en especial los cirujanos, pueden seguir contribuyendo a los avances médicos. El estudio de la historia de la medicina contemporánea pone de manifiesto que varios de los logros más significativos se deben a cirujanos. No cabe duda de que la ciencia y la clínica sabrán acomodarse a estos tiempos de cambio y continuarán generando importantes hallazgos. La práctica médico-quirúrgica de hoy está determinada por la investigación científica de ayer; mañana no será diferente

From the Second World War onwards an amazing development in science and clinical & surgery practice has taken place: antibiotics, blood storage, cardiac surgery, organ and tissue transplant, complete joint replacement, total intravenous nutrition or minimally invasive surgery. Paradoxically, during the last two decades of the XXth century, social popularity of doctors has decreased at the same time of some important changes: increases in costs of medical attention, trials against medical mistakes, some doubts on the real role of Academic Public Hospitals and rising in the importance of alternative medicine. Increasing complexity of biomedical research in the continuous changing age of molecular biology has promoted an increasing scepticism regarding clinics and surgeons are able to keep on contributing to medical advances. The study of the contemporaneous History of Medicine demonstrates that some of the more significant achievements have been accomplished by surgeons. Undoubtedly Science and Clinics must get adapted to these times of change and persist in generating important findings. Current Clinical and surgical practice is completely determined by yesterdayscientific research; tomorrow won´t be different

Diseño de modelos experimentales en investigación quirúrgica / Design of experimental models in surgical investigation

Introducción: La investigación en Medicina se basa fundamentalmente en tres fuentes de conocimiento: En primer lugar, el hombre enfermo que es la fuente natural del conocimiento en la Clínica, en segundo lugar el cadáver, que es la fuente de conocimiento de la Anatomía Patológica y, por último, el animal de experimentación que es la fuente del conocimiento de la Fisiopatología. Los avances experimentados en el último siglo han determinado que el concepto 'animal de experimentación' deba sustituirse por un concepto mucho más amplio, el de 'modelo experimental'. Objetivos: En este artículo trataremos de aclarar éste último concepto, para lo cual deberemos hacer una serie de consideraciones previas sobre el método experimental y las dos herramientas de que dispone, la estadística y el diseño de experimentos

Introduction: Research in Medicine is essentially based on three knowledge resources: diseased people (natural and primary), cadaveric bodies (Pathology primary resource) and experimental animals, whom constitutes physiopathologic knowledge resource. Experimental advances reached in the last century have determined the change of the concept 'experimental animal' to a wider term: 'experimental model'. Objetives: This paper tryes to clarify this concept. To attain this goal, we must define previous considerations in the meaning of the concept 'experimental models' and its two key-elements: statistics and design of experiments

Impact of ventricular enlargement on the measurement of metabolic activity in spatially normalized PET.

In pathologic brains with morphological alterations, the process of spatial normalization, as performed by SPM methods, may introduce a confounding effect in the measurement of metabolic activity data. To investigate the effect of the spatial normalization of PET images, we analyzed MRI and PET studies of 20 schizophrenic patients and 18 controls. Using a Talairach-based segmentation procedure and manual segmentation, we measured regional metabolic activity in the untransformed brains and after their spatial normalization. The effect of spatial normalization seems minimal for large ROIs like the main brain lobes, even in brains showing pronounced morphological abnormalities. However, the caudate nucleus shows a considerable change in metabolic activity values after normalization. This normalization effect is much larger in patients than in controls, and leads to artifactual differences between them. We obtained incorrect results (SPM analysis) regarding functional differences between patients and controls in the caudate due to this bias introduced by the spatial normalization. There was a significant correlation between the size of the lateral ventricles and the underestimation of metabolic activity of the caudate. Normalization bias seems to arise from a misalignment of the caudate in the normalized space, pixel overlap between the normalized caudate, and the caudate of the template being on average lower than 50% in both groups. Spatial normalization of the PET images of pathologic brains may introduce a potential source of error that should be taken into account in the analysis of functional data, in particular, in the study of small brain nuclei like the caudate.

Effects of MDMA on blood glucose levels and brain glucose metabolism.

PURPOSE: This study was designed to assess changes in glucose metabolism in rats administered single or repeated doses of MDMA. METHODS: Two different experiments were performed: (1) A single-dose study with four groups receiving 20 mg/kg, 40 mg/kg, saline or heat, and (2) a repeated-dose study with two groups receiving three doses, at intervals of 2 h, of 5 mg/kg or saline. Rats were imaged using a dedicated small-animal PET scanner 1 h after single-dose administration or 7 days after repeated doses. Glucose metabolism was measured in 12 cerebral regions of interest. Rectal temperature and blood glucose were monitored. RESULTS: Peak body temperature was reached 1 h after MDMA administration. Blood glucose levels decreased significantly after MDMA administration. In the single-dose experiment, brain glucose metabolism showed hyperactivation in cerebellum and hypo-activation in the hippocampus, amygdala and auditory cortex. In the repeated-dose experiment, brain glucose metabolism did not show any significant change at day 7. CONCLUSION: These results are the first to indicate that MDMA has the potential to produce significant hypoglycaemia. In addition, they show that MDMA alters glucose metabolism in components of the motor, limbic and somatosensory systems acutely but not on a long-term basis.

Peripheral leukocyte response to oncological radiotherapy: expression of heat shock proteins.

PURPOSE: To study Heat Shock Proteins (HSP) expression in patients subjected to radiotherapy and their potential use as biomarkers for radiation tolerance. An evaluation is also made of whether irradiated volume is critical to the outcome of normal tissue injury using polymorphonuclear neutrophils as biosensors, and whether HSP antibodies (Ab) may be involved in post-radiotherapy disease. MATERIAL AND METHODS: Twelve patients receiving the same total dose of radiotherapy, but in three different volumes, and four healthy volunteers used as controls were analysed. hsp27 and 70i mRNA were determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Southern-blot, HSP by flow cytometry, and HSP-Ab by Enzyme-linked Immnoadsorbent Assay (ELISA). The clinical protocol included radiation related toxicity based on clinical and analytical scales. RESULTS: Radiotherapy caused hsp downregulation, maximum in patients with the largest irradiated volumes, and a decrease in intracellular HSP content. Patients with greatest intraleukocyte HSP levels before treatment suffered more severe radiation morbidity. Patients with endocrine neoplasms presented the highest HSP-Ab titers. CONCLUSIONS: Radiotherapy downregulates hsp27 and 70i, which would enhance radiosensitivity. HSP content prior to treatment is suggested as a prognostic biomarker for radiation tolerance, with circulating leukocytes as biosensors. HSP-Ab may be biomarkers of tumor disease, but do not seem to be involved in the morbidity of acute post-radiotherapy disease, which is closely related to the volumes irradiated.

Fluorescent and compositional changes in crystallin supramolecular structures in pig lens during development.

Water soluble proteins (WSPs) in Sus scrofa lenses from pigs in different developmental stages: (young (GI), young adult (GII), and middle-aged (GIII)) were separated using GF-HPLC, yielding fractions of different molecular weights. Non-tryptophan (345/420 nm) and tryptophan (280/345 nm) fluorescence was measured in these fractions. Relative non-tryptophan fluorescence increased with age at a rate directly correlated to the molecular weight of aggregates forming the different chromatographic fractions, while tryptophan fluorescence tended to decrease. The crystallins constituting each fraction were separated using 2D-electrophoresis and after development with Coomassie blue they were identified using MS-TOF. Also, the protein content of each spot was quantified by subsequent scanning and integration. The proportions of unchanged crystallins characteristically changed with age in chromatographic fractions of different molecular weights. Thus it was possible to relate these changes with those occurring in the fluorescent properties and molecular weight of supramolecular structures.

Hydrodynamic and biochemical effects of isolated hypothermic renal perfusion depending on the pump model and perfusion solution.

OBJECTIVES: To evaluate hydrodynamic, biochemical, and histological consequences of hypothermic isolated renal perfusion using a new computerized perfusion system. MATERIALS AND METHODS: The device that allowed us to obtain on renal hydrodynamics during perfusion included multiple parts. The organ was perfused at 4 degrees C with a constant flow either using a classic roller pump or a pump designed in our laboratory to employ vacuum or atmospheric pressure sequentially to achieve a truly pulsatile wave (vacuum-powered tubular pump). The study included 16 minipigs with Eurocollins or Belzer perfusion solutions sampled at predefined interval and histological studies of the organs performed. RESULTS: There was a significant difference in weight increase between the two types of pumps; those perfused with Eurocollins showed greater values than those with Belzer solution. Onset of nitric oxide (NO) in the perfusion solution increased inversely with the renal vascular resistance. The highest NO levels were observed with the Belzer solution and vacuum pump. CONCLUSIONS: Changes in renal hydrodynamics, as induced by perfusion wave form and solution type, may be recorded in real time using a computerized system. A vacuum pump with the Belzer solution achieved the best experimental results.

Influence of the normalization template on the outcome of statistical parametric mapping of PET scans.

Spatial normalization is an essential preprocessing step in statistical parametric mapping (SPM)-based analysis of PET scans. The standard template provided with the SPM99 software package was originally constructed using (15)O-H(2)O PET scans and is commonly applied regardless of the tracer actually used in the scans being analyzed. This work studies the effect of using three different normalization templates in the outcome of the statistical analysis of PET scans: (1) the standard SPM99 PET template; (2) an (18)F-FDG PET template, constructed by averaging PET scans previously normalized to the standard template; and (3) an MRI-aided (18)F-FDG PET template, constructed by averaging PET scans normalized according to the deformation parameters obtained from MRI scans. A strictly anatomical MRI normalization of each PET was used as a reference, under the rationale that a normalization based only upon MRI should provide higher spatial accuracy. The potential bias involved in the normalization process was estimated in a clinical SPM study comparing schizophrenic patients with control subjects. For each between-group comparison, three SPM maps were obtained, one for each template. To evaluate the influence of the template, these SPM maps were compared to the reference SPM map achieved using the anatomical normalization. SPMs obtained by MRI-aided normalization showed the highest spatial specificity, and also higher sensitivity when compared to the standard normalization using the SPM99 (15)O-H(2)O template. These results show that the use of the standard template under inappropriate conditions (different tracer or mental state) may lead to inconsistent interpretations of the statistical analysis.

Automatic quantification of viability in epithelial cell cultures by texture analysis.

Quantification of live cells in phase contrast microscopy images allows in vivo assessment of the viability of cultured cells. An automatic screening procedure seems advisable because of the large number of cells that must be counted to achieve reasonable accuracy. This paper presents a method that quantifies necrosis in cell cultures by texture analysis of microscope images. The image is divided into regions of equal size that are classified by means of a segmentation algorithm based on texture analysis into three categories: live cells, necrotic cells and background. The classification uses three discriminant functions, built from parameters derived from the histogram and the co-occurrence matrix and calculated by performing an initial stepwise discriminant analysis on 21 sample images from a training set. The areas occupied by live and necrotic cells and number of live cells have been obtained for primary cellular cultures in intervals of 48 h during 2 weeks. The results have been compared with those obtained by an experienced observer, showing a very good correlation (Pearson's coefficient 0.95, kappa 0.87, N= 1600). A method has been developed that provides an accuracy similar to that provided by an expert, while allowing a much higher number of fields to be counted.

[Statistical parametric mapping (SPM) in nuclear medicine]. / Mapas de estadísticos paramétricos (SPM) en medicina nuclear.

In the scope of medical research, functional neuroimaging analysis permits the study of pathological or cognitive cerebral processes by using statistical quantification techniques. A tool of increasing use is the SPM (Statistical Parametric Mapping) software due to its wide availability and the variety of statistical studies that can be made. Nevertheless, being unaware of the theoretical background on which it is based may easily lead to inaccurate results and even to the reaching of erroneous conclusions. The present article summarizes these theoretical principles and discusses the main key points of the method without requiring advanced mathematical knowledge.

Mapas de estadísticos paramétricos (SPM) en medicina nuclear / Statistical parametric mapping (SPM) in nuclear medicine

En el ámbito de la investigación médica, el análisis de neuroimágenes funcionales (PET, SPECT y fMRI) mediante técnicas de cuantificación estadística permite el estudio de diversos procesos cerebrales, patológicos o cognitivos. Una herramienta de creciente uso para este fin es el software SPM (Statistical Parametric Mapping) gracias a su amplia disponibilidad y el gran abanico de estudios estadísticos que permite realizar. Sin embargo, el desconocimiento de los fundamentos teóricos en los que se basa puede conducir fácilmente a resultados imprecisos e incluso a conclusiones erróneas. Este artículo presenta brevemente dichos principios teóricos y discute los principales puntos críticos en la utilización del método sin exigir conocimientos matemáticos avanzados del lector. (AU)

Role of elastic fibers in cooling-induced relaxation.

The objective of this work was to confirm the main role of elastic fibers in differing responses of certain vessels during cooling from 37 to 8 degrees C. Previous results have shown that the nature of the vessel (conduit vessel vs muscular vessel) determines the different behavior (dilatation vs contraction) of isolated vessel segments when temperature decreases from 37 to 8 degrees C. In this work, it has been demonstrated that vessels with a great amount of elastic fibers show a dilatation when cooling. On the other hand, muscular vessels with fewer elastic fibers, such as the renal artery, undergo a contraction. The output of calcium from intracellular stores causes contraction of the renal artery during cooling. In this vessel, vasodilatation occurs only when mechanisms of smooth muscle contraction are inactive, as is the case with vessels that have undergone a cold storage period of 48 h. The results presented in this work confirm that there are two main effects, which directly depend on the vessel origin. In conduit arteries, the decrease of temperature induces a vascular relaxation, dependent on the elastic component of the vessel wall. In muscular vessels, the predominant effect is cooling-induced contraction due to an increase of intracellular calcium. This cooling-induced contraction needs the vessel to be in optimal conditions with an active metabolism of the muscular cells. These results are a crucial issue in the sense of explaining several biomedical mechanisms where hypothermia is implicated. The type of vessel implicated in procedures, such as isolated organ perfusion, extracorporeal circulation, and bypass surgery, must be taken into account.

Perfusate lactate dehydrogenase level and intrarenal resistance could not be adequate markers of perfusion quality during isolated kidney perfusion.

The main goal of this work was to study the influence of perfusion pressure and flow waveform during kidney perfusion, and the relationship between renal vascular resistance (RVR) and lactate dehydrogenase (LDH) concentration in the perfusate. Simultaneous constant pressure kidney perfusions were performed with either pulsatile or continuous flow at either 30 or 80 mm Hg of constant perfusion pressure. Mean flow, pressure, and RVR were displayed online during perfusion. Perfusate samples for LDH, creatine phosphatase kinase (CPK), and alkaline phosphatase (AP) determinations were taken. At the end of the perfusion, 2 ml of Evans blue was injected into the circuit to obtain images of perfusate distribution, and the kidneys were weighed. Also, hematoxylin/eosine studies were performed, showing more Bowman's space and tubular dilation in kidneys perfused with high pressure. We did not find differences in RVR between kidneys perfused at 30 and 80 mm Hg; nevertheless, perfusate distribution was better in the 80 mm Hg perfusions. We did not find any correlation between enzyme release and RVR in kidneys perfused with different mean pressures. These findings suggest that vascular resistance and LDH concentration cannot be independently considered as adequate markers of perfusate distribution.

Paradoxical effects of temperature on vascular tone.

Temperature may have significant influence on vascular tone in such cases as organ preservation, coronary bypass surgery, and extracorporeal circulation. The aim of this research was to study the direct effect of temperature variation on vascular tone in an attempt to elucidate the mechanisms involved. In a first series of experiments, the isometric tension of two different vessels (rat thoracic aorta and pig renal branch artery) was studied at different temperatures. To study the role of calcium in this response, a second series of experiments was performed. In this the vessels were incubated with the intracellular chelator BAPTA/AM. Further experiments were performed to test the effect of cold storage. Our results show that changes in temperature lead to different results in pig renal artery and rat aorta. A decrease in temperature induced a highly reproducible relaxation in rat aorta, whereas pig renal artery presented cooling-induced contraction. Moreover, whereas calcium depletion failed to inhibit cooling-induced relaxation in rat aorta, it did not provoke cooling-induced contraction in pig renal artery. Similar responses were obtained with cold storage and calcium depletion. We intend to demonstrate that, just as the effect of temperature variation on pig renal artery is due to a metabolic mechanism, its effect on rat aorta may be due to structural factors. This hypothesis is supported by the result of histological studies which demonstrate a higher proportion of elastin fibres in rat aorta than in pig renal artery.

[Integration of multimodal medical images]. / Integración de imágenes médicas multimodalidad.

Multimodality analysis is becoming a required tool in medical practice due to the development of different image techniques and new imaging processing tools. This paper is a review of the basis of medical image integration, image fusion techniques, and their clinical applications, especially addressing our experience with brain tomographic studies in nuclear medicine and radiology.

Isolated kidney controlled perfusion with true physiological pulsatile waveform.

A computer controlled perfusion system has been developed to study the behavior of perfused kidneys in several conditions. The system is designed to perform kidney perfusions at constant pressure and low temperature (about 4 degrees C). We compared 2 types of perfusion pumps, a classical roller pump widely used in hemodialysis circuits and a vacuum powered tubular pump with active valves developed by our group and able to produce a flow pattern very similar to the pulse wave in the human circulatory system. In this preliminary study, we show the hydrodynamics obtained with both pumps in isolated hypothermic kidneys perfused with this system. The different flow patterns with both pumps seem to determine differences in the preservation conditions of the kidney.