Lactoferrin/sialic acid prevents adverse effects of intrauterine growth restriction on neurite length: investigations in an in vitro rabbit neurosphere model.

Introduction: Intrauterine growth restriction (IUGR) is a well-known cause of impaired neurodevelopment during life. In this study, we aimed to characterize alterations in neuronal development underlying IUGR and discover strategies to ameliorate adverse neurodevelopment effects by using a recently established rabbit in vitro neurosphere culture. Methods: IUGR was surgically induced in pregnant rabbits by ligation of placental vessels in one uterine horn, while the contralateral horn remained unaffected for normal growth (control). At this time point, rabbits were randomly assigned to receive either no treatment, docosahexaenoic acid (DHA), melatonin (MEL), or lactoferrin (LF) until c-section. Neurospheres consisting of neural progenitor cells were obtained from control and IUGR pup's whole brain and comparatively analyzed for the ability to differentiate into neurons, extend neurite length, and form dendritic branching or pre-synapses. We established for the very first time a protocol to cultivate control and IUGR rabbit neurospheres not only for 5 days but under long-term conditions up to 14 days under differentiation conditions. Additionally, an in vitro evaluation of these therapies was evaluated by exposing neurospheres from non-treated rabbits to DHA, MEL, and SA (sialic acid, which is the major lactoferrin compound) and by assessing the ability to differentiate neurons, extend neurite length, and form dendritic branching or pre-synapses. Results: We revealed that IUGR significantly increased the neurite length after 5 days of cultivation in vitro, a result in good agreement with previous in vivo findings in IUGR rabbits presenting more complex dendritic arborization of neurons in the frontal cortex. MEL, DHA, and SA decreased the IUGR-induced length of primary dendrites in vitro, however, only SA was able to reduce the total neurite length to control level in IUGR neurospheres. After prenatal in vivo administration of SAs parent compound LF with subsequent evaluation in vitro, LF was able to prevent abnormal neurite extension. Discussion: We established for the first time the maintenance of the rabbit neurosphere culture for 14 days under differentiation conditions with increasing complexity of neuronal length and branching up to pre-synaptic formation. From the therapies tested, LF or its major compound, SA, prevents abnormal neurite extension and was therefore identified as the most promising therapy against IUGR-induced changes in neuronal development.

Application of the adverse outcome pathway to identify molecular changes in prenatal brain programming induced by IUGR: Discoveries after EGCG exposure.

Following a multi-disciplinary approach integrating information from several experimental models we have collected new evidence supporting, expanding and redesigning the AOP "Disrupted laminin/int-ß1 interaction leading to decreased cognitive function". Investigations in vitro in rabbit and rat neurospheres and in vivo in mice exposed to EGCG (epigallocatechin-gallate) during neurodevelopment are combined with in vitro evaluations in neural progenitor cells overexpressing int-ß1 and literature information from int-ß1 deficiency models. We have discovered for the first time that neural progenitor cells from intrauterine growth restricted (IUGR) animals overexpress int-ß1 at gene and protein level and due to this change in prenatal brain programming they respond differently than control neurospheres to the exposure of EGCG, a compound triggering neural progenitor cell migration alterations. We have also identified that EGCG developmental exposure has deleterious effects on neuronal branching and arborization in vitro and in vivo. Our results warn that a thorough developmental neurotoxicity characterization of this and other catechin-based food supplements is needed before recommending their consumption during pregnancy.

Protocols for the Evaluation of Neurodevelopmental Alterations in Rabbit Models In Vitro and In Vivo.

The rabbit model is gaining importance in the field of neurodevelopmental evaluation due to its higher similarity to humans in terms of brain development and maturation than rodents. In this publication, we detailed 14 protocols covering toxicological relevant endpoints for the assessment of neurodevelopmental adverse effects in the rabbit species. These protocols include both in vitro and in vivo techniques, which also cover different evaluation time-points, the neonatal period, and long-term examinations at postnatal days (PNDs) 50-70. Specifically, the protocols (P) included are as follows: neurosphere preparation (GD30/PND0; P2) and neurosphere assay (P3), behavioral ontogeny (PND1; P4), brain obtaining and brain weight measurement at two different ages: PND1 (P5) and PND70 (P12), neurohistopathological evaluations after immersion fixation for neurons, astrocytes, oligodendrocytes and microglia (PND1; P6-9) or perfusion fixation (PND70; P12), motor activity (P11, open field), memory and sensory function (P11, object recognition test), learning (P10, Skinner box), and histological evaluation of plasticity (P13 and P14) through dendritic spines and perineuronal nets. The expected control values and their variabilities are presented together with the information on how to troubleshoot the most common issues related to each protocol. To sum up, this publication offers a comprehensive compilation of reliable protocols adapted to the rabbit model for neurodevelopmental assessment in toxicology.

Psychometric properties of the oral feeding assessment in premature infants scale.

Professionals that work in neonatal units need to identify the strengths and weaknesses of the premature infant who is in the transition process from feeding through a gastric tube to oral feeding. The main aim of this study was to validate the Oral FEeding Assessment in premaTure INfants (OFEATINg) instrument. A psychometric validity and reliability study was conducted in Neonatal Intensive Care Units of two public, metropolitan, university hospitals. The study population were premature infants at a postconceptional age of 31-35 weeks. The study included evaluation of the reliability, convergent, discriminant and construct validity, sensitivity and specificity of the OFEATINg instrument. A total of 621 feedings of 56 preterm infants were evaluated. Confirmatory factor analysis identified 3 factors and 13 indicators with a good fit to the model. Cronbach's alpha coefficient was 0.78. The instrument showed high indices of inter-rater reliability (Pearson 0.9 and intraclass correlation coefficient 0.95). The OFEATINg scale is a valid and reliable instrument for evaluating the readiness for oral feeding of preterm infants. It may enable clinicians to evaluate the physiological and behavioral abilities involved in the oral feeding process and help them make decisions related to the transition to full oral feeding.Clinical trial registration: This study was prospectively registered at the two Institutional review boards.

Docosahexaenoic Acid and Melatonin Prevent Impaired Oligodendrogenesis Induced by Intrauterine Growth Restriction (IUGR).

In this study, our aims were to characterize oligodendrogenesis alterations in fetuses with intrauterine growth restriction (IUGR) and to find therapeutic strategies to prevent/treat them using a novel rabbit in vitro neurosphere culture. IUGR was surgically induced in one uterine horn of pregnant rabbits, while the contralateral horn served as a control. Neural progenitor cells (NPCs) were obtained from pup's whole brain and cultured as neurospheres mimicking the basic processes of brain development including migration and cell differentiation. Five substances, chosen based on evidence provided in the literature, were screened in vitro in neurospheres from untreated rabbits: Docosahexaenoic acid (DHA), melatonin (MEL), zinc, 3,3',5-Triiodo-L-thyronine (T3), and lactoferrin (LF) or its metabolite sialic acid (SA). DHA, MEL and LF were further selected for in vivo administration and subsequent evaluation in the Neurosphere Assay. In the IUGR culture, we observed a significantly reduced percentage of oligodendrocytes (OLs) which correlated with clinical findings indicating white matter injury in IUGR infants. We identified DHA and MEL as the most effective therapies. In all cases, our in vitro rabbit neurosphere assay predicted the outcome of the in vivo administration of the therapies and confirmed the reliability of the model, making it a powerful and consistent tool to select new neuroprotective therapies.

Fetal ischemia monitoring with in vivo implanted electrochemical multiparametric microsensors.

Under intrauterine growth restriction (IUGR), abnormal attainment of the nutrients and oxygen by the fetus restricts the normal evolution of the prenatal causing in many cases high morbidity being one of the top-ten causes of neonatal death. The current gold standards in hospitals to detect this relevant problem is the clinical observation by echography, cardiotocography and Doppler. These qualitative techniques are not conclusive and requires risky invasive fetal scalp blood testing and/or amniocentesis. We developed micro-implantable multiparametric electrochemical sensors for measuring ischemia in real time in fetal tissue and vascular. This implantable technology is designed to continuous monitoring for an early detection of ischemia to avoid potential fetal injury. Two miniaturized electrochemical sensors were developed based on oxygen and pH detection. The sensors were optimized in vitro under controlled concentration, to assess the selectivity and sensitivity required. The sensors were then validated in vivo in the ewe fetus model, by means of their insertion in the muscle leg and inside the iliac artery of the fetus. Ischemia was achieved by gradually obstructing the umbilical cord to regulate the amount of blood reaching the fetus. An important challenge in fetal monitoring is the detection of low levels of oxygen and pH changes under ischemic conditions, requiring high sensitivity sensors. Significant differences were observed in both; pH and pO2 sensors under changes from normoxia to hypoxia states in the fetus tissue and vascular with both sensors. Herein, we demonstrate the feasibility of the developed sensors for future fetal monitoring in medical applications.

Miniaturized Electrochemical Sensors to Monitor Fetal Hypoxia and Acidosis in a Pregnant Sheep Model.

Perinatal asphyxia is a major cause of severe brain damage and death. For its prenatal identification, Doppler ultrasound has been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. We have evaluated the performance of a miniaturized multiparametric sensor aiming to evaluate tissular oxygen and pH changes continuously in an umbilical cord occlusion (UCO) sheep model. The electrochemical sensors were inserted in fetal hindlimb skeletal muscle and electrochemical signals were recorded. Fetal hemodynamic changes and metabolic status were also monitored during the experiment. Additionally, histological assessment of the tissue surrounding the sensors was performed. Both electrochemical sensors detected the pO2 and pH changes induced by the UCO and these changes were correlated with hemodynamic parameters as well as with pH and oxygen content in the blood. Finally, histological assessment revealed no signs of alteration on the same day of insertion. This study provides the first evidence showing the application of miniaturized multiparametric electrochemical sensors detecting changes in oxygen and pH in skeletal muscular tissue in a fetal sheep model.

in vivo Monitoring with micro-implantable hypoxia sensor based on tissue acidosis.

Hypoxia is a common medical problem, sometimes difficult to detect and caused by different situations. Control of hypoxia is of great medical importance and early detection is essential to prevent life threatening complications. However, the few current methods are invasive, expensive, and risky. Thus, the development of reliable and accurate sensors for the continuous monitoring of hypoxia is of vital importance for clinical monitoring. Herein, we report an implantable sensor to address these needs. The developed device is a low-cost, miniaturised implantable electrochemical sensor for monitoring hypoxia in tissue by means of pH detection. This technology is based on protonation/deprotonation of polypyrrole conductive polymer. The sensor was optimized in vitro and tested in vivo intramuscularly and ex vivo in blood in adult rabbits with respiration-induced hypoxia and correlated with the standard device ePOCTM. The sensor demonstrated excellent sensitivity and reproducibility; 46.4 ± 0.4 mV/pH in the pH range of 4-9 and the selectivity coefficient exhibited low interference activity in vitro. The device was linear (R2 = 0.925) with a low dispersion of the values (n = 11) with a cut-off of 7.1 for hypoxia in vivo and ex vivo. Statistics with one-way ANOVA (α = 0.05), shows statistical differences between hypoxia and normoxia states and the good performance of the pH sensor, which demonstrated good agreement with the standard device. The sensor was stable and functional after 18 months. The excellent results demonstrated the feasibility of the sensors in real-time monitoring of intramuscular tissue and blood for medical applications.

Non-invasive monitoring of pH and oxygen using miniaturized electrochemical sensors in an animal model of acute hypoxia.

BACKGROUND: One of the most prevalent causes of fetal hypoxia leading to stillbirth is placental insufficiency. Hemodynamic changes evaluated with Doppler ultrasound have been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. As a first step, the present work aimed to evaluate the performance of miniaturized electrochemical sensors in the continuous monitoring of oxygen and pH changes in a model of acute hypoxia-acidosis. METHODS: pH and oxygen electrochemical sensors were evaluated in a ventilatory hypoxia rabbit model. The ventilator hypoxia protocol included 3 differential phases: basal (100% FiO2), the hypoxia-acidosis period (10% FiO2) and recovery (100% FiO2). Sensors were tested in blood tissue (ex vivo sensing) and in muscular tissue (in vivo sensing). pH electrochemical and oxygen sensors were evaluated on the day of insertion (short-term evaluation) and pH electrochemical sensors were also tested after 5 days of insertion (long-term evaluation). pH and oxygen sensing were registered throughout the ventilatory hypoxia protocol (basal, hypoxia-acidosis, and recovery) and were compared with blood gas metabolites results from carotid artery catheterization (obtained with the EPOC blood analyzer). Finally, histological assessment was performed on the sensor insertion site. One-way ANOVA was used for the analysis of the evolution of acid-based metabolites and electrochemical sensor signaling results; a t-test was used for pre- and post-calibration analyses; and chi-square analyses for categorical variables. RESULTS: At the short-term evaluation, both the pH and oxygen electrochemical sensors distinguished the basal and hypoxia-acidosis periods in both the in vivo and ex vivo sensing. However, only the ex vivo sensing detected the recovery period. In the long-term evaluation, the pH electrochemical sensor signal seemed to lose sensibility. Finally, histological assessment revealed no signs of alteration on the day of evaluation (short-term), whereas in the long-term evaluation a sub-acute inflammatory reaction adjacent to the implantation site was detected. CONCLUSIONS: Miniaturized electrochemical sensors represent a new generation of tools for the continuous monitoring of hypoxia-acidosis, which is especially indicated in high-risk pregnancies. Further studies including more tissue-compatible material would be required in order to improve long-term electrochemical sensing.

Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction.

The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at -80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level.

Micro-needle implantable electrochemical oxygen sensor: ex-vivo and in-vivo studies.

Oxygen is vital for energy metabolism in mammals and the variability of the concentration is considered a clinical alert for a wide range of metabolic malfunctions in medicine. In this article, we describe the development and application of a micro-needle implantable platinum-based electrochemical sensor for measuring partial pressure of oxygen in intramuscular tissue (in-vivo) and vascular blood (ex-vivo). The Pt-Nafion® sensor was characterized morphological and electrochemically showing a higher sensitivity of -2.496 nA/mmHg (-1.495 nA/µM) when comparing with its bare counterpart. Our sensor was able to discriminate states with different oxygen partial pressures (pO2) for ex-vivo (blood) following the same trend of the commercial gas analyzer used as standard. For in-vivo (intramuscular) experiments, since there is not a gold standard for measuring pO2 in tissue, it was not possible to correlate the obtained currents with the pO2 in tissue. However, our sensor was able to detect clear statistical differences of O2 between hyperoxia and hypoxia states in tissue.

Structural Brain Changes during the Neonatal Period in a Rabbit Model of Intrauterine Growth Restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) is associated with abnormal neurodevelopment, but the associated structural brain changes are poorly documented. The aim of this study was to describe in an animal model the brain changes at the cellular level in the gray and white matter induced by IUGR during the neonatal period. METHODS: The IUGR model was surgically induced in pregnant rabbits by ligating 40-50% of the uteroplacental vessels in 1 horn, whereas the uteroplacental vessels of the contralateral horn were not ligated. After 5 days, IUGR animals from the ligated horn and controls from the nonligated were delivered. On the day of delivery, perinatal data and placentas were collected. On postnatal day 1, functional changes were first evaluated, and thereafter, neuronal arborization in the frontal cortex and density of pre-oligodendrocytes, astrocytes, and microglia in the corpus callosum were evaluated. RESULTS: Higher stillbirth in IUGR fetuses together with a reduced birth weight as compared to controls was evidenced. IUGR animals showed poorer functional results, an altered neuronal arborization pattern, and a decrease in the pre-oligodendrocytes, with no differences in microglia and astrocyte densities. CONCLUSIONS: Overall, in the rabbit model used, IUGR is related to functional and brain changes evidenced already at birth, including changes in the neuronal arborization and abnormal oligodendrocyte maturation.

Nutritional intra-amniotic therapy increases survival in a rabbit model of fetal growth restriction.

OBJECTIVE: To evaluate the perinatal effects of a prenatal therapy based on intra-amniotic nutritional supplementation in a rabbit model of intrauterine growth restriction (IUGR). METHODS: IUGR was surgically induced in pregnant rabbits at gestational day 25 by ligating 40-50% of uteroplacental vessels of each gestational sac. At the same time, modified-parenteral nutrition solution (containing glucose, amino acids and electrolytes) was injected into the amniotic sac of nearly half of the IUGR fetuses (IUGR-T group n = 106), whereas sham injections were performed in the rest of fetuses (IUGR group n = 118). A control group without IUGR induction but sham injection was also included (n = 115). Five days after the ligation procedure, a cesarean section was performed to evaluate fetal cardiac function, survival and birth weight. RESULTS: Survival was significantly improved in the IUGR fetuses that were treated with intra-amniotic nutritional supplementation as compared to non-treated IUGR animals (survival rate: controls 71% vs. IUGR 44% p = 0.003 and IUGR-T 63% vs. IUGR 44% p = 0.02), whereas, birth weight (controls mean 43g ± SD 9 vs. IUGR 36g ± SD 9 vs. IUGR-T 35g ± SD 8, p = 0.001) and fetal cardiac function were similar among the IUGR groups. CONCLUSION: Intra-amniotic injection of a modified-parenteral nutrient solution appears to be a promising therapy for reducing mortality among IUGR. These results provide an opportunity to develop new intra-amniotic nutritional strategies to reach the fetus by bypassing the placental insufficiency.

Early Environmental Enrichment Enhances Abnormal Brain Connectivity in a Rabbit Model of Intrauterine Growth Restriction.

INTRODUCTION: The structural correspondence of neurodevelopmental impairments related to intrauterine growth restriction (IUGR) that persists later in life remains elusive. Moreover, early postnatal stimulation strategies have been proposed to mitigate these effects. Long-term brain connectivity abnormalities in an IUGR rabbit model and the effects of early postnatal environmental enrichment (EE) were explored. MATERIALS AND METHODS: IUGR was surgically induced in one horn, whereas the contralateral one produced the controls. Postnatally, a subgroup of IUGR animals was housed in an enriched environment. Functional assessment was performed at the neonatal and long-term periods. At the long-term period, structural brain connectivity was evaluated by means of diffusion-weighted brain magnetic resonance imaging and by histological assessment focused on the hippocampus. RESULTS: IUGR animals displayed poorer functional results and presented altered whole-brain networks and decreased median fractional anisotropy in the hippocampus. Reduced density of dendritic spines and perineuronal nets from hippocampal neurons were also observed. Of note, IUGR animals exposed to enriched environment presented an improvement in terms of both function and structure. CONCLUSIONS: IUGR is associated with altered brain connectivity at the global and cellular level. A strategy based on early EE has the potential to restore the neurodevelopmental consequences of IUGR.

Ultrasound-Guided Intrauterine Labeling of Rat Fetuses.

AIM: To compare intra-amniotic versus fetal subcutaneous injections for selective fetal labeling in multifetal rat pregnancies. METHODS: A total of 14 pregnant rats were randomized to receive intra-amniotic injections of dyes (including Fluorescein, Indigo Carmine, or Evans Blue) or fetal subcutaneous injections (of commercial tattoo ink) both guided by ultrasound at 15-17 days of gestation. Survival, injection, and labeling success rates of both techniques were compared. RESULTS: Survival rates (84.4% for intra-amniotic injections vs. 90.9% for fetal subcutaneous injections) and injection success rates (94% for intra-amniotic injections vs. 100% for fetal subcutaneous injections) were similar among both groups. None of the neonates from the intra-amniotic injections group were labeled at birth, while 93% of the neonates from fetal subcutaneous injections group were tagged, showing a visible spot in the skin at birth. CONCLUSION: Our results suggest that ultrasound-guided fetal subcutaneous injections might be an adequate strategy for selectively labeling fetuses in multifetal pregnant animals.

[What happens after the accident? Psychosocial needs of people with traumatic brain injury and their families]. / ¿Y después del accidente? Las necesidades psicosociales de las personas con traumatismo craneoencefálico y de sus familiares.

OBJECTIVE: To identify factors that people with a traumatic brain injury and their families perceived as helping to improve their quality of life. METHODS: Three focus groups and five interviews were conducted with a total of 37 participants: 14 persons with traumatic brain injury and 23 caregivers. A content analysis was conducted. The constant comparative method was applied. RESULTS: We detected five factors that improved the quality of life of persons with a traumatic brain and their families: 1) Informal support (family and friends); 2) formal support (counseling, employment, built and bureaucratic environment); 3) type of clinical characteristics; 4) social participation, and 5) social visibility. CONCLUSIONS: The needs expressed by our participants primarily focused on social and emotional factors. For persons with severe traumatic brain injury attempting to achieve the best possible community integration, a new semiology is required, not limited to medical care, but also involving social and psychological care tailored to the needs of each individual and family and their environment.

¿Y después del accidente? Las necesidades psicosociales de las personas con traumatismo craneoencefálico y de sus familiares / What happens after the accident? Psychosocial needs of people with traumatic brain injury and their families

Objetivo: Identificar los factores que las personas con traumatismo craneoencefálico (TCE) y sus familias perciben que contribuyen a mejorar su calidad de vida. Métodos: Se realizaron tres grupos focales y cinco entrevistas, con un total de 37 participantes: 14 personas con TCE y 23 familiares. Se realizó un análisis de contenido. Se aplicó el método de comparaciones constantes. Resultados: Tanto para las personas con TCE por accidente de tráfico como para sus familias se detectan cinco factores principales que mejoran su calidad de vida: 1) apoyo informal (familia y amigos); 2) apoyo formal (apoyo psicológico, inserción laboral, entorno construido y burocrático); 3) tipo de secuelas; 4) participación y 5) visibilización social. Conclusiones: Las necesidades expresadas por los participantes se centran sobre todo en aspectos sociales y emocionales. Para las personas con TCE y sus familias, que tratan de lograr la mejor integración posible en la comunidad, se requiere una nueva semiología, no sólo limitada a la atención médica sino también en materia de asistencia social y psicológica, que se adapte a las necesidades de cada familia y del contexto (AU)

Objective: To identify factors that people with a traumatic brain injury and their families perceived as helping to improve their quality of life. Methods: Three focus groups and five interviews were conducted with a total of 37 participants: 14 persons with traumatic brain injury and 23 caregivers. A content analysis was conducted. The constant comparative method was applied. Results: We detected five factors that improved the quality of life of persons with a traumatic brain and their families: 1) Informal support (family and friends); 2) formal support (counseling, employment, built and bureaucratic environment); 3) type of clinical characteristics; 4) social participation, and 5) social visibility. Conclusions: The needs expressed by our participants primarily focused on social and emotional factors. For persons with severe traumatic brain injury attempting to achieve the best possible community integration, a new semiology is required, not limited to medical care, but also involving social and psychological care tailored to the needs of each individual and family and their environment (AU)

Functional biogeography of oceanic islands and the scaling of functional diversity in the Azores.

Analyses of species-diversity patterns of remote islands have been crucial to the development of biogeographic theory, yet little is known about corresponding patterns in functional traits on islands and how, for example, they may be affected by the introduction of exotic species. We collated trait data for spiders and beetles and used a functional diversity index (FRic) to test for nonrandomness in the contribution of endemic, other native (also combined as indigenous), and exotic species to functional-trait space across the nine islands of the Azores. In general, for both taxa and for each distributional category, functional diversity increases with species richness, which, in turn scales with island area. Null simulations support the hypothesis that each distributional group contributes to functional diversity in proportion to their species richness. Exotic spiders have added novel trait space to a greater degree than have exotic beetles, likely indicating greater impact of the reduction of immigration filters and/or differential historical losses of indigenous species. Analyses of species occurring in native-forest remnants provide limited indications of the operation of habitat filtering of exotics for three islands, but only for beetles. Although the general linear (not saturating) pattern of trait-space increase with richness of exotics suggests an ongoing process of functional enrichment and accommodation, further work is urgently needed to determine how estimates of extinction debt of indigenous species should be adjusted in the light of these findings.

Knowledge discovery about quality of life changes of spinal cord injury patients: clustering based on rules by states.

In this paper, an integral Knowledge Discovery Methodology, named Clustering based on rules by States, which incorporates artificial intelligence (AI) and statistical methods as well as interpretation-oriented tools, is used for extracting knowledge patterns about the evolution over time of the Quality of Life (QoL) of patients with Spinal Cord Injury. The methodology incorporates the interaction with experts as a crucial element with the clustering methodology to guarantee usefulness of the results. Four typical patterns are discovered by taking into account prior expert knowledge. Several hypotheses are elaborated about the reasons for psychological distress or decreases in QoL of patients over time. The knowledge discovery from data (KDD) approach turns out, once again, to be a suitable formal framework for handling multidimensional complexity of the health domains.