Increased glomerular cell (podocyte) apoptosis in rats with streptozotocin-induced diabetes mellitus: role in the development of diabetic glomerular disease.

AIMS/HYPOTHESIS: Podocyte loss by apoptosis, in addition to favouring progression of established diabetic nephropathy, has been recently indicated as an early phenomenon triggering the initiation of glomerular lesions. This study aimed to assess the rate of glomerular cell death and its relationship with renal functional, structural and molecular changes in rats with experimental diabetes. METHODS: Male Sprague-Dawley rats with streptozotocin-induced diabetes and coeval non-diabetic control animals were killed at 7 days and at 2, 4 and 6 months for the assessment of apoptosis, renal function, renal structure and the expression of podocyte markers and apoptosis- and cell cycle-related proteins. RESULTS: Glomerular cell apoptosis was significantly increased in diabetic vs non-diabetic rats at 4 months and to an even greater extent at 6 months, with podocytes accounting for 70% of apoptosing cells. The increase in apoptosis was preceded by increases in proteinuria, albuminuria and mean glomerular and mesangial areas, and by reductions in glomerular cell density and content of synaptopodin and Wilms' tumour protein-1. It coincided with the development of mesangial expansion and glomerular sclerosis, and with the upregulation/activation both of tumour protein p53, which increased progressively throughout the study, and of p21 (also known as cyclin-dependent kinase inhibitor 1A, CIP1 and WAF1), which peaked at 4 months and decreased thereafter. CONCLUSIONS/INTERPRETATION: Glomerular cell (podocyte) apoptosis is not an early feature in the course of experimental diabetic glomerulopathy, since it is preceded by glomerular hypertrophy, which may decrease glomerular cell density to the point of inducing compensatory podocyte hypertrophy. This is associated with reduced podocyte protein expression (podocytopathy) and proteinuria, and ultimately results in apoptotic cell loss (podocytopenia), driving progression to mesangial expansion and glomerular sclerosis.

Development of ISFET array-based microsystems for bioelectrochemical measurements of cell populations.

Monitoring the bioelectrochemical activity of living cells with sensor array-based microsystems represents an emerging technique in a large area of biomedical applications, ranging from basic research to various fields of pharmacological analyses. The main appeal is the ability of these miniaturised microsystems to perform, in real time, non-invasive in-vitro investigations of the physiological state of a cell population. In this paper, we present two different microsystems designed for multisite monitoring of the physiological state of a cell population. The first microsystem, intended for cellular metabolism monitoring, consists of an array of 12 spatially distributed ISFETs to detect small pH variations induced by the cell population. The second microsystem consists of an array of 40 ISFETs and 20 gold microelectrodes and it has been designed to monitor the electrical activity of neurons. This is achieved by direct coupling of the neuronal culture with the ISFET sensitive layer and by utilising gold microelectrodes for neuronal electrical stimulation.

Insulin release at the molecular level: metabolic-electrophysiological modeling of the pancreatic beta-cells.

The role of pancreatic beta-cells is fundamental in the control endocrine system, maintaining the blood glucose homeostais in a physiological regime, via the glucose-induced release of insulin. An increasing amount of detailed experimental evidences at the cellular and molecular biology levels have been collected on the key factors determining the insulin release by the pancreatic beta-cells. The direct transposition of such experimental data into accurate mathematical descriptions might contribute to considerably clarify the impact of each cellular component on the global glucose metabolism. Under these perspectives, we model and computer-simulate the stimulus-secretion coupling in beta-cells by describing four interacting cellular subsystems, consisting in the glucose transport and metabolism, the excitable electrophysiological behavior, the dynamics of the intracellular calcium ions, and the exocytosis of granules containing insulin. We explicit the molecular nature of each subsystem, expressing the mutual relationships and the feedbacks that determine the metabolic-electrophysiological behavior of an isolated beta-cell. Finally, we discuss the simulation results of the behavior of isolated beta-cells as well as of population of electrically coupled beta-cells in Langerhans islets, under physiological and pathological conditions, including noninsulin-dependent diabetes mellitus (NIDDM) and hyperinsulinemic hypoglycaemia (PHHI).

Activity-driven computational strategies of a dynamically regulated integrate-and-fire model neuron.

Activity-dependent slow biochemical regulation processes, affecting intrinsic properties of a neuron, might play an important role in determining information processing strategies in the nervous system. We introduce second-order biochemical phenomena into a linear leaky integrate-and-fire model neuron together with a detailed kinetic description for synaptic signal transduction. In this framework, we investigate the membrane intrinsic electrical properties differentiation, showing the appearance of activity-dependent shifts between integration and temporal coincidence detection operating mode, for the single unit of a network.

Gallbladder perforation during laparoscopic cholecystectomy.

A matched-cohort analytic study was performed to assess the influence on postoperative morbidity and on long-term outcome of gallbladder perforation (GP) during laparoscopic cholecystectomy (LC) and to determine the existence of risk factors of GP. A total of 1127 consecutive patients who underwent successful LC were included. All patients received a preoperative dose of intravenous antibiotic. If GP occurred, free bile was aspirated, the abdominal cavity was irrigated, spilled stones were retrieved whenever possible, and antibiotic treatment was prolonged. Intraoperative GP occurred in 131 cases (11.6%). The Cox multivariate proportional hazards model showed that the surgeon's experience was the only factor associated with a higher risk of GP (p < 0.0001). Patients who had GP were retrospectively matched with 131 patients who did not have perforation. Statistical differences between the two matched groups were found for the median length of surgery: 74 minutes in the GP group versus 61 minutes, p < 0.01). No differences were found for (1) postoperative complications and reoperations (3. 8% in GP group vs. 6.1%, and 0% in GP group vs. 0.8%, respectively); or (2) mean postoperative hospital stay (2.9 +/- 2.3 days in GP group vs. 2.6 +/- 1.6 days). No late consequences occurred that could be attributed to intraoperative GP. The results suggest that the frequency of GP during LC tends to diminish as the surgeon gains experience with this type of surgery. This event does not cause complications if adequate prophylactic antibiotic therapy is administered; spilled stones are retrieved whenever possible, and the abdominal cavity is abundantly irrigated.

Fast calculation of short-term depressing synaptic conductances.

An efficient implementation of synaptic transmission models in realistic network simulations is an important theme of computational neuroscience. The amount of CPU time required to simulate synaptic interactions can increase as the square of the number of units of such networks, depending on the connectivity convergence. As a consequence, any realistic description of synaptic phenomena, incorporating biophysical details, is computationally highly demanding. We present a consolidating algorithm based on a biophysical extended model of ligand-gated postsynaptic channels, describing short-term plasticity such as synaptic depression. The considerable speed-up of simulation times makes this algorithm suitable for investigating emergent collective effects of short-term depression in large-scale networks of model neurons.

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

Langmuir-Blodgett films of antibodies as mediators of endothelial cell adhesion on polyurethanes.

The effect of endothelial cell adhesion on polyurethanes coated with Langmuir-Blodgett antibody films has been examined. The films were cross-linked with glutaraldehyde with the aim of providing a densely packed and covalently linked two-dimensional antibody network on the polyurethane surfaces. Our results demonstrate that although neither of the two polyurethanes examined were entirely suited to cellular adhesion, Langmuir-Blodgett antibody films, cross-linked with small concentrations of glutaraldehyde, are more suitable for endothelial cell adhesion than surfaces free of antibody.

A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates.

The purpose of this paper is to describe a low-cost simple technique based on the hydraulically driven deposition of adhesion molecules for patterning populations of neurons on silicon micromachined substrates. First, the design and fabrication process of the silicon micromachined substrates and the design of a flow-through chamber for the localised deposition of adhesive proteins are described. The experimental protocol for the deposition of the adhesive proteins is then presented. Finally, the results of experiments of 'entrapment' of chick embryo spinal cord neurons in microstructures of the silicon substrates and of formation of patterned biological neural networks are shown.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Analysis of the signals generated by networks of neurons coupled to planar arrays of microtransducers in simulated experiments.

Planar microelectrode arrays can be used to characterize the dynamics of networks of neurons reconstituted in vitro. In this paper simulations related to experiments of the electrical activity recording by means of planar arrays of microtransducers coupled to networks of neurons are described. First a detailed model of single and synaptically connected neurons is given, appropriate to computer simulate the action potentials of neuronal populations. Then 'realistic' signals are generated. These signals are intended to reproduce, both in shape and intensity, those recorded by a microelectrode array. Typical experimental conditions are considered, and a detailed analysis given, of the bioelectronic coupling and of its influence on the shape of the recorded signals. Finally, simulated experiments dealing with dorsal root ganglia neurons are described and analysed in comparison with experimental results reported in the literature and obtained in our own laboratory. The effectiveness of the planar microelectrode technique is briefly discussed.

Role of follow-up in management of local recurrences of colorectal cancer: a prospective, randomized study.

PURPOSE: This prospective, randomized, single-center study was designed to evaluate the influence of follow-up on detection and resectability of local recurrences and on survival after radical surgery for colorectal cancer. METHODS: Between 1987 and 1990, 207 consecutive patients who underwent curative resections for primary untreated large-bowel carcinoma were randomly assigned to a conventional follow-up group (Group A; n = 103) and to an intense follow-up group (Group B; n = 104). All the patients were followed up prospectively, and the outcome was known for all of them at five years. Patients in Group A were seen at six-month intervals for one year, and once a year thereafter. Patients in Group B were checked every three months during the first two years, at six-month intervals for the next three years, and once a year thereafter. RESULTS: Of the 103 patients in Group A, local recurrence was detected in 20; 9 (13 percent) of these patients had colon cancer, and 11 (29 percent) had rectal cancer. Of the 104 patients in Group B, local recurrence was detected in 26; 12 (16 percent) of these patients had colon cancer, and 14 (45 percent) had rectal cancer. Twelve cases (60 percent) of local recurrence in Group A and 24 cases (92 percent) in Group B were detected at scheduled visits (P < 0.05). Local recurrences were detected earlier in patients of Group B (10.3 +/- 2.7 vs. 20.2 +/- 6.1 months; P < 0.0003). Curative re-resection was possible in 2 patients (10 percent) in Group A, 1 with colon cancer and 1 with rectal cancer, and in 17 patients (65 percent) in Group B, 6 with colon cancer and 11 with rectal cancer (P < 0.01). Of the Group B patients who had curative re-resections of local recurrence, 8 (47 percent) were disease-free and long-term survivors as of the last follow-up, and 2 (11.7 percent) were alive, but with a new recurrence. The 2 patients in Group A who had curative re-resections died as a result of cancer. The five-year survival rate in Group A was 58.3 percent and in Group B was 73.1 percent. The difference is statistically significant (P < 0.02). CONCLUSIONS: Our data support use of an intense follow-up plan after primary resection of large-bowel cancer, at least in patients with rectal cancer.

Curative surgery for colorectal cancer: long-term results and life expectancy in the elderly.

PURPOSE: The long-term prognosis after curative surgery for colorectal cancer was evaluated in relation to age and life expectancy as a possible basis for assessing the risk to benefit ratios in the elderly. METHODS: Data relating to 1,256 patients operated on from 1976 to 1994 were stored in a computer database prospectively from 1987. Patients were subdivided into four age groups (A = <60 years; B = 60-69; C = 70-79; D = > or =80). Distribution of general contraindications to curative surgery was examined. In the 869 patients who underwent curative treatment (A = 206; B = 256; C = 289; D = 118), distribution of tumor stage and elective/emergency surgery and the operative mortality rate were evaluated. Crude and age-corrected survival curves were calculated in 794 patients. The median crude survival of each group was related by gender and tumor stage to demographic life expectancy, assuming as "relative median survival index" the ratio between the two values. RESULTS: General contraindications to curative surgery increased significantly with age. The operative mortality rate was higher in Group D than in Groups A, B, plus C over the total series (P < 0.001) and in both elective (P < 0.001) and emergency surgery (P < 0.05). Intergroup analysis of long-term survival rates showed significant differences between "crude" (P = 0.0057) but not age-corrected (P = 0.66) curves. The relative median survival index increased with age, up to approximately 1 in the local stages of Groups C and D. CONCLUSIONS: To evaluate long-term results, elderly patients should be compared with unaffected, same-age subjects. Because the risks may be very high, the surgical policy in the elderly should be carefully weighed and related to life expectancy and actual results.

Simulations of the behavior of synaptically driven neurons via time-invariant circuit models.

This paper describes computer simulations of the behavior of Hodgkin-Huxley neurons, based on a redefinition of membrane and synaptic connections as time-invariant circuit elements. Examples are given showing that this self-consistent equivalent circuit representation allows very efficient computer simulations and could facilitate the introduction of detailed biological neurons into formal neural networks.

Reduced postoperative morbidity after elective laparoscopic cholecystectomy: stratified matched case-control study.

To answer the question whether laparoscopic cholecystectomy (LC) or open cholecystectomy (OC) is safer in terms of complications and to what extent the "learning curve" influences the frequency of complications after LC, we conducted a matched case-control study. First, 200 patients undergoing LC (LC group A), and two groups of 200 patients undergoing LC at two different periods of the learning curve (LC groups B and C) were matched, taking into account sex, age, anesthesiologic risk, and surgical difficulties. We evaluated the frequency and grade of postoperative complications of these patients and of the last 200 patients undergoing OC before the introduction of LC, retrospectively matched with the LC groups. The total rate of complications in the OC group was 16.0% compared with 5.5% in the LC groups (p < 0.003); the difference was particularly significant for complications classified as grade I, in female patients, those younger than 70, those with low anesthesiologic risk (ASA), and those after cholecystectomy without surgical difficulties. Matched case-control analysis revealed that the complication rate in the LC group significantly decreases with experience (P < 0.01). We conclude that LC is today the treatment of choice for symptomatic cholelithiasis and is replacing OC as the gold standard against which new therapies should be compared.

In vitro 2-D networks of neurons characterized by processing the signals recorded with a planar microtransducer array.

The purpose of this paper is to extensively analyze and utilize the key features that characterize the recently available electrophysiological technique of growing selected populations of neurons on planar substrate microelectrode arrays. This experimental configuration is first simulated by modeling the signal transduction operated by an array of microtransducers coupled to a network of Hodgkin-Huxley-like neurons, connected to each other with given levels of synaptic strength. Signal processing tools are then described and validated by identifying the various degrees of connectivity previously introduced into the simulated network. Finally, these software tools are utilized to characterize the activity and identify the synaptic connectivity of networks of cultured neurons extracted from dorsal root ganglia (DRG) of chick embryos and exposed to synapse inhibiting/reinforcing ions. As a result, correlations between various regimens of electrophysiological activity and synaptic strength are obtained.

Mechanical and morphological properties of living 3T6 cells probed via scanning force microscopy.

Scanning Force Microscopy (SFM) is utilized to study living confluent 3T6 cells. Images based on mechanical contrast are obtained and related morphological details, mostly regarding the cell cytoskeleton, are analyzed. Moreover, numerical estimates of the local mechanical properties of the living cells are given, by extensive use of the "force-vs.-distance" operation mode. On the basis of the results obtained, the potentialities of SFM as an optimal new technique available for probing the cell cytoskeleton of unstained living cells, and assessing related models, are shortly discussed.

Characterization and interpretation of electrical signals from random networks of cultured neurons.

The main purpose of this paper is to describe several software tools specifically adapted to characterize the electrophysiological behavior of a population of neurons coupled to a planar array of microtransducers by analyzing the recorded signals. Both graphic and signal processing tools are considered. They include: image analysis of the neuronal network topology, peak detection procedure, multi-graphics visualization, spike classification, cross-correlation, interspike and interbursting interval analysis. These tools are utilized to characterize changes induced in the electrophysiological behavior of populations of cultured neurons by modifications to the medium-culture.

Silicon neuron simulation with SPICE: tool for neurobiology and neural networks.

The paper deals with computer simulations of 'silicon neurons', which are assemblies of CMOS circuits that generate the equivalents of the ionic currents and of the action potentials of real (biological) neurons. The circuit simulation program SPICE is used to simulate the generation of action potentials by a silicon neuron. Moreover, the equivalent circuits of silicon synapses are described and the behaviours of simple two- and three-neuron networks are analysed. Implications for the areas of neurobiology and formal neural networks are briefly considered.

Scanning force microscopy on live cultured cells: imaging and force-versus-distance investigations.

Extensive measurements with the scanning force microscope on living cells in their native liquid environment are described with the purpose of critically assessing the extent of the interaction between the SFM tip and the (soft) cell materials and the effect of such interaction on topographic information. Images are obtained under various force conditions and systematically correlated with force-versus-distance curves. As a result, detailed indications about tip indentation are given, thickness estimates deduced and identification of submembranous cytoplasmic structures suggested.