Robotic navigation to subcortical neural tissue for intracellular electrophysiology in vivo.

In vivo studies of neurophysiology using the whole cell patch-clamp technique enable exquisite access to both intracellular dynamics and cytosol of cells in the living brain but are underrepresented in deep subcortical nuclei because of fouling of the sensitive electrode tip. We have developed an autonomous method to navigate electrodes around obstacles such as blood vessels after identifying them as a source of contamination during regional pipette localization (RPL) in vivo. In mice, robotic navigation prevented fouling of the electrode tip, increasing RPL success probability 3 mm below the pial surface to 82% (n = 72/88) over traditional, linear localization (25%, n = 24/95), and resulted in high-quality thalamic whole cell recordings with average access resistance (32.0 MΩ) and resting membrane potential (-62.9 mV) similar to cortical recordings in isoflurane-anesthetized mice. Whole cell yield improved from 1% (n = 1/95) to 10% (n = 9/88) when robotic navigation was used during RPL. This method opens the door to whole cell studies in deep subcortical nuclei, including multimodal cell typing and studies of long-range circuits.NEW & NOTEWORTHY This work represents an automated method for accessing subcortical neural tissue for intracellular electrophysiology in vivo. We have implemented a novel algorithm to detect obstructions during regional pipette localization and move around them while minimizing lateral displacement within brain tissue. This approach leverages computer control of pressure, manipulator position, and impedance measurements to create a closed-loop platform for pipette navigation in vivo. This technique enables whole cell patching studies to be performed throughout the living brain.

Cleaning patch-clamp pipettes for immediate reuse.

Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.

A new model of primary human adipocytes reveals reduced early insulin signalling in type 2 diabetes.

The aim of this study was to establish a diabetic model of primary human adipocytes for investigating potential defects in early insulin signalling. Specimens of human subcutaneous adipose tissue were obtained during orthopaedic surgical procedures. Preadipocytes were isolated and differentiated to adipocytes. Western blot analysis and immunoprecipitation were performed to determine protein content of IRS-1, IRS-2, p85, phosphorylation of IRS-1, IRS-2, Akt and MAPK as well as association between p85 and IRS-1/IRS-2. In addition to short-term insulin stimulation, the effect of hyperinsulinaemia was investigated by treating cells with insulin over a period of 36 hours. We found a significantly reduced basal expression of IRS-1 (54 +/- 15%) in adipocytes from type 2 diabetic subjects compared to controls with a further significant reduction in expression after long-term treatment (30 +/- 12%) compared to short-term treatment. IRS-2 expression also showed a significant reduction under hyperinsulinaemic conditions (20 +/- 2%) in diabetics vs. controls. Furthermore, long-term treatment with insulin in diabetic adipocytes led to a significant reduction in the phosphorylation of IRS-1(68 +/- 11%), IRS-2 (82 +/- 11%), Akt (42 +/- 2%), and MAPK (92 +/- 12%) and in the subsequent association between p85 to IRS-1 and IRS-2 (100 +/- 16% and 96 +/- 12%) in comparison to controls. Investigating glucose uptake diabetic adipocytes revealed a significant reduction of 90 +/- 2%. In this study, we were able to establish a new diabetic model of primary human adipocytes. A defect in early insulin signalling in type 2 diabetic patients under hyperinsulinaemic conditions was determined. These results might help to give further insights in early insulin action; additionally, this human model represents a useful target for the study of new therapeutic approaches.

Prediction of colloid osmotic pressure in renal patients.

BACKGROUND: Colloid osmotic pressure (COP) plays a major role in transcapillary fluid shift, including in the glomerular capillary. However, COP is generally estimated by quadratic equations derived from total plasma protein and/or albumin concentrations. The aim of this study was to assess the accuracy of such equations, and to determine the potential role of liver-derived non-albumin proteins in the maintenance of COP, especially in patients presenting a nephrotic syndrome. METHODS: COP was directly assessed with an osmometer in 170 patients (347 samples), and the results compared with calculated COP, using 4 previously published formulas [Brenner 1972, Canaan-Kühl 1993, Landis-Pappenheimer 1963, Navar 1977]. RESULTS: The 4 calculated COP values were strongly correlated with measured COP (range r = 0.88 - 0.96). However, in absolute terms, measured COP differed significantly from each of the 4 calculated mean values of COP (p < 0.001). Fibrinogen exerted per se a weak oncotic effect as measured in vitro. However, fibrinogen was highly related to albumin and presumably reflected the oncotic effect of other liver-derived non-albumin proteins. Inclusion of albumin and fibrinogen in a linear model provided an excellent fit for predicted COP with a highly significant correlation (r = 0.96, p < 0.001) over a wide range of COP values. The predicted equation was: COP(mmHg) = 6.89 x (albumin + fibrinogen) (g/dl) - 5.68. CONCLUSION: None of the 4 most commonly used formulas correctly predicted COP, and direct measurement of COP is still preferable for research studies. The introduction of fibrinogen into the formula estimating COP leads to higher accuracy, and therefore represents a more convenient model for routine evaluation.

[Antihypertensive treatment in type II diabetes and diabetic nephropathy]. / Traitement antihypertenseur du diabète et de la néphropathie diabétique de type II.

Hypertension is very prevalent in patients with type II diabetes. Beside increasing the cardiovascular risk, hypertension has several deleterious effects on the kidney: hypertension promotes the development of diabetic nephropathy, the progression from microalbuminuria to overt diabetic nephropathy and progression to end stage renal disease. In this review, we analyze systematically the benefit of antihypertensive therapy in patients with type II diabetes, with either normo-albuminuria, microalbuminuria or overt nephropathy. General considerations are developed about the general use of antihypertensive drugs in this population. An approach based on the prevention of the global or absolute cardiovascular risk is further recommended due to the very high cardiovascular burden in this diabetic patients.

[Tubular hypokalemia of genetic origin]. / Hypokaliémies tubulaires d'origine génétique.

Recent progress in molecular genetics have improved our understanding of the pathophysiology of several inherited diseases characterized by a renal hypokalemia. Some of these diseases result from inactivating mutations on the main cotransports involved in the reabsorption of sodium, namely the Gitelman and Bartter syndromes, that clinically mimics diuretic abuse with mild hypovolemia and low or normal blood pressure. Conversely some affections eventually lead to an increase in sodium reabsorption with hypervolemia and arterial hypertension: Liddle syndrome, apparent mineralocorticoid excess and dexamethasone suppressible hyperaldosteronism.

[Antihypertensive treatment in type 1 diabetes and diabetic nephropathy]. / Traitement antihypertenseur du diabète et de la néphropathie diabétique de type 1.

In diabetic patient, hypertension is a major factor contributing to both cardiovascular morbidity-mortality and progression toward renal impairment. This review analyzes studies from the literature regarding the benefits of antihypertensive treatment at every stage of type 1 diabetes and diabetic nephropathy.

Digital radiography of interproximal caries: effect of different filters.

The aim of this study was to compare the effect of different image processing modes (filters) on reproducibility and validity of the assessment of interproximal carious lesions on digitized radiographic images. Standardized radiographs of 34 extracted teeth exhibiting interproximal caries were obtained. All radiographs were digitized and using the FRIACOM-software the central depth (CD) of each carious lesion was measured on the unchanged radiographic image and after use of five different filters with 7fold and 18fold magnification. All measurements were repeated after 1 week to estimate reproducibility. Histometric CD assessments provided a gold standard for comparison with the radiographic measurements (validity). Neither reproducibility nor validity of CD measurements were improved by one of the filters. Measurements of dentinal lesions showed a statistically higher variability than those of enamel lesions (p<0.001). 18fold magnification provided more valid CD measurements than 7fold magnification (p<0.001). In this study digital manipulations of radiographic images failed to result in statistically significantly improved reproducibility or validity of CD measurements.

Abysmal prognosis of patients with type 2 diabetes entering dialysis.

INTRODUCTION: The importance of non-insulin-dependent diabetes mellitus (type II diabetes) as a leading cause of end-stage renal disease is now widely recognized. The purpose of this study was to assess life-prognosis and its predictors in a cohort of patients newly entering dialysis. MATERIAL AND METHODS: Eighty-four consecutive type II diabetes patients (40% of all patients) starting dialysis between 01/01/95 and 31/12/96 were studied retrospectively, focusing on clinical data at inception and life-prognosis after a mean follow-up of 211 days. Patients were divided into three groups, according to onset of renal failure: acute 11% (9/84), chronic 61% (51/84) and acutely aggravated chronic renal failure 28% (25/84). RESULTS: Patients (mean age 67 years) had long-standing diabetes (mean duration approximately 15 years), heavy proteinuria (approximately 3 g/24h) and diabetic retinopathy (67%). The average creatinine clearance (Cockcroft's formula) was 13 ml/min. Cardiovascular diseases were highly prevalent at the start of dialysis: history of myocardial infarction (26%), angina (36%) and acute left ventricular dysfunction (67%). More than 80% of the patients underwent the first session dialysis under emergency conditions, a situation in part related to late referral to the nephrology division (63% for chronic patients). A great majority of the patients were overhydrated when starting dialysis, as evidenced by the average weight loss of 6 kg, during the first month of dialysis, required to reach dry weight. Nearly 64% of the patients presented high blood pressure (> 140/90 mmHg) when starting dialysis despite antihypertensive therapy (mean: 2.3 drugs). The outcome of this type II diabetes population was dramatic: 32% (27/84) died after a mean follow-up of 211 days, mostly from cardiovascular diseases. The rate of recovery of renal function was low in both the acute and the acutely aggravated renal failure group (30% and 24%, respectively). Of note, iatrogenic nephrotoxic agents accounted for renal function impairment in nearly 30% of patients. CONCLUSION: Our observational study illustrates the high burden of cardiovascular diseases contrasting with sub-optimal cardiovascular therapeutic interventions in type II diabetes patients entering dialysis. Factors aggravating renal failure were mainly iatrogenic, and therefore largely avoidable. Late referral generally implied a poor clinical condition at the start of dialysis.

Nodular glomerulosclerosis with deposition of monoclonal immunoglobulin heavy chains lacking C(H)1.

The objective of this study was to further characterize the clinical and immunopathologic features of heavy chain deposition disease (HCDD), a recently described entity. Four patients were diagnosed as having HCDD on a kidney biopsy. All presented with nodular glomerulosclerosis with deposition of gamma1 heavy chains lacking CH1 epitopes, but without light chains. Two different patterns were observed in the serum. First, patients 1 and 2 had a circulating monoclonal IgGlambda containing a short gamma1 heavy chain lacking CH1 epitopes, with an apparent molecular weight of 40 kD consistent with a complete CH1 deletion. Biosynthetic experiments also showed that the deleted heavy chain was produced in excess compared with light chains, and was secreted in vitro together with half Ig molecules, although these abnormal components were not detected by Western blot analysis of whole serum. Second, patients 3 and 4 had a circulating monoclonal IgG1lambda with an apparently normal, nondeleted heavy chain subunit, but serum fractionation followed by immunoblotting revealed an isolated monoclonal gamma1 chain lacking CH1 epitopes. These data strongly suggest that renal deposition of a CH1-deleted heavy chain circulating in low amounts in the serum as a free unassembled subunit is a major feature of HCDD. The CH1 deletion is most likely responsible for the premature secretion in blood of the heavy chain by a clone of plasma cells.

The influence of geometric mismatch between the native aortic, native pulmonary and homograft pulmonary valve on the results of the pulmonary autograft operation.

BACKGROUND: The pulmonary autograft operation has achieved broad acceptance and may be the ideal aortic valve substitute. Both the pulmonary autograft and the aortic homograft are more complicated procedures than prosthetic valve replacement. The trend to insert the pulmonary autograft as a root replacement rather than in the subcoronary position has achieved greater uniformity in the results, but there is still confusing diversity in opinions on technical details and anatomical dimensions. The importance of both size and shape mismatches between the three valves involved has received little attention. The valves often differ in diameter and in the shape of the recipient aortic annulus. This uncertainty and the diversity of opinions on essential technical details was disconcerting when we proceeded from aortic homograft-to-pulmonary autograft operations, this was compounded by only a single homograft being available for every operation as we have no homograft bank. METHODS: We compared the hemodynamic results regarding various geometric mismatches. All operative details were the same and patients were studied at regular intervals. Comparisons were made in patients with mismatch between recipient aortic annulus and pulmonary autograft. Patients with a normal tricuspid aortic annulus were compared to those with either a circular redo prosthetic valve annulus or a bicuspid recipient annulus. Thirdly we compared the patients with plication of the aortic annulus to those with remodeling of the distal aorta. Lastly we compared mismatch between donor homograft and pulmonary autograft. RESULTS: No influence of geometric mismatch between the three valves could be found on the results of the pulmonary autograft operation. CONCLUSIONS: Good results are obtainable without a painful learning curve if one keeps to certain surgical principles. It need not be a complicated operation and geometric mismatches between the three valves involved may be compensated for adequately.