Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy.

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Association of painful and painless diabetic polyneuropathy with different patterns of nerve fiber degeneration and regeneration.

We evaluated neuropathological abnormalities in sural nerve biopsies from 6 nondiabetic control subjects and 16 age-matched diabetic patients with different syndromes of sensory polyneuropathy (6 with chronic painful neuropathy [CPN], 4 with newly presenting painful neuropathy [NPN], and 6 with painless neuropathy associated with recurrent neurotrophic foot ulcers [RFU]). Although all but one of the evaluated features of myelinated and unmyelinated fiber pathology could be found in every diabetic patient, certain myelinated fiber abnormalities were associated with the clinical characteristics of the neuropathy. Thus, myelinated fiber density was severely reduced, "empty" Schwann tubes (an index of myelinated fiber degeneration) were increased, and early regeneration (bands of Büngner [BB], nonmyelinated axons) was pronounced in the RFU group. Progression from BB to regenerating myelinated fiber cluster (myelination and maturation) was more successful in patients with CPN and NPN than in those with RFU, and the finding of fibers with disproportionately large Schwann cells (cytoplasm and myelin) relative to axon caliber was exclusive to patients with neuropathic pain. We concluded that 1) unequal rates of successful fiber regeneration may underlie the apparent difference in the extent of myelinated fiber loss between painful and painless diabetic polyneuropathy; 2) myelinated and unmyelinated fiber degeneration and regeneration per se are probably not the cause of neuropathic pain in diabetic polyneuropathy, because each occurred in patients with RFU; and 3) axonal atrophy may be involved in neuropathic pain generation.

Relationship of endoneurial capillary abnormalities to type and severity of diabetic polyneuropathy.

Endoneurial capillary abnormalities have been assessed quantitatively in sural nerve biopsies from diabetic patients with different syndromes of sensory polyneuropathy: chronic painful neuropathy, newly presenting painful neuropathy, and painless neuropathy associated with neurotrophic foot ulceration. Comparisons were made with age-matched nondiabetic control subjects. The diabetic groups showed no abnormality in capillary density or mean endoneurial area per fascicle. Compared with control subjects, all diabetic patients had an increase in mean capillary diameter, capillary wall thickness, and outer tunic (basement membrane and pericytes) thickness. The increase in wall thickness was most pronounced in patients with painless neuropathy (200%) and less marked in similar patients with painful neuropathy (100%). The pericyte volume fraction of the outer tunic was reduced in all diabetic patients, implying that basement membrane hypertrophy and reduplication were responsible for outer tunic thickening. There was evidence of endothelial cell hyperplasia rather than hypertrophy. There was a correlation between the degree of basement membrane thickening and the severity of myelinated fiber abnormality assessed neurophysiologically and morphologically. This study shows a link between the degree of endoneurial capillary basement membrane thickening, the type of neuropathology, and the clinical expression of neuropathy in diabetes mellitus.

The effect of pancreatic islet transplantation on experimental diabetic neuropathy.

Quantitative light and electronmicroscopical morphometric techniques were used to determine the effect of pancreatic islet transplantation on experimental diabetic neuropathy. Groups of STZ-diabetic rats were given islet transplants at 3 weeks after diabetes onset (prevention) and at 6 months after diabetes onset (reversal). Comparisons were made with onset controls, age-matched non-diabetic controls and untreated diabetic controls 6 months later (n = 8 for all groups). Euglycaemia and normal levels of glycosylated haemoglobin were achieved in both groups of diabetics after islet transplantation. Loss of body weight in diabetic animals was prevented by early islet transplantation, but was only partially reversed following delayed islet transplantation. Normal growth of myelinated fibres and axons during development was retarded in untreated diabetics, but was normal in rats given islet transplants soon after the onset of diabetes (cross-sectional perimeter and area). Diabetics transplanted with islets after a delay had myelinated fibres and axons with diminished calibre. Teased fibre preparations of nerves from diabetics which had received islet transplants showed no excess of abnormalities. This study has shown that the development of certain structural abnormalities of peripheral nerve fibres is prevented in diabetic rats which receive transplants of islets of Langerhans soon after the onset of diabetes. However, once established abnormal fibre morphology can not be completely ameliorated merely by achieving and sustaining euglycaemia through delayed islet transplantation.

Bioassay development: the implications of cardiac myocyte motility in vitro.

Cardiac myocytes cultured over microfabricated extracellular recording devices can be used to assay bioactive compounds. However, electrophysiological signals recorded from these devices vary in amplitude with time. Theoretically, changes in signal amplitude arise from myocytes being moved over recording sites by cocultured fibroblasts. To test this, neonatal rat cardiac myocytes were cultured at high densities and low densities on fibronectin-coated glass. After 36.5 h, myocytes were identified by their rhythmic contractions and then time-lapse-recorded for 3.5 h. Length, width, and angle of orientation was then determined every 30 min for five cells in low density and five cells in high-density culture. Low-density cells had mean lengths of 65.3 microm and widths of 35.1 microm, whereas cells in high-density culture had greater mean lengths of 74.2 microm and lower mean widths of 24.3 microm. Length, width, and angle of orientation of cells in low- and high-density culture changed by 4.1%, 11.8%, and 2.7 degrees, and 6.4%, 10%, and 4.6 degrees, respectively, every half hour. We found no evidence of myocyte-fibroblast interactions influencing cell position or shape in low density, but in high density, we found evidence that fibroblast-myocyte interactions could transiently influence cell shape. We conclude that fibroblast-independent changes in cell shape are largely responsible for the changes in signal amplitude recorded from cardiac myocytes cultured on microfabricated extracellular recording devices. However, there is some evidence that myocyte-fibroblast interactions may augment this process in high-density culture. The implications of these findings for bioassay development are discussed.

Preliminary study on the suitability of a pharmacological bio-assay based on cardiac myocytes cultured over microfabricated microelectrode arrays.

There are a range of techniques that can be used to assay bioactive compound. One potentially promising technique is a system consisting of microfabricated extracellular recording devices over which electrogenic cells can be grown. To date, research in this area has concentrated on the use of neurons as an electrogenic cell type. However, these cells have limitations. Only small extracellular potentials have been recorded from mammalian neurons cultured over microfabricated electrode arrays. Although such potentials may be of use in assays examining the effects of bio-active compound analogues on firing frequency, they are of little use for more detailed pharmacological studies involving analyses of signal shape. What is required is a system from which much larger extracellular potentials can be recorded. This preliminary study reports on a system based on cardiac myocytes cultured over microfabricated metal microelectrode arrays, from which potentials with a mean amplitude of 16.9 microV can be reliably recorded, which can be reversibly blocked with mumoll-1 concentrations of the sodium ion channel blocker lidocaine. Less common potentials with amplitudes of up to 3.5 mV were also recorded. It is demonstrated that cardiac myocytes cultured over microfabricated micro-electrode arrays can be used in assays of cardioactive compound analogues.

Cell reactions to dielectrophoretic manipulation.

The phenomenon of dielectrophoretic particle manipulation holds promise for many biotechnology applications, including cell sorting. In our system cell manipulation normally involves transient exposure (15 minutes) to radio-frequency AC electric fields generated using planar microelectrodes. The present study was designed to investigate the range of acute effects of dielectrophoretic manipulation on the normal physiology of isolated cells. Cells were suspended in isoosmotic Mannitol and exposed to a 5 MHz, 21 V (peak to peak) electric field with 100 micrometer gap electrodes. Cells were assigned to three experimental groups; non-exposed controls, exposed cells processed immediately after cessation of the field, and exposed cells processed after a time delay. SEM observations of spread cells cultured on the devices showed no apparent acute effects of field exposure on cell morphology. Cell-doubling rates in exposed cells subsequent to field-exposure or transient incubation in mannitol were no different from control cells. An MTT 'mitochondrial stress' assay indicated no alteration in the rate of oxidative respiration in exposed cells 0.5 hour after exposure to the field. Western blot analysis indicated upregulation of fos protein in cells 0.5 hour after field-exposure, which was confirmed using densitometry. Reverse transcription of cellular mRNA followed by PCR amplification, polyacrylamide gel electrophoresis and autoradiography of cDNA banding revealed differential gene expression between controls and exposed cells processed immediately after cessation of the field. Differential gene expression persisted in exposed cells at least 0.5 hours after removal from the field. Observations indicated that temperature fluctuation in the mannitol solution was minimal, suggesting that upregulated mRNA may not have been related to thermally-induced heat shock protein. The present study has indicated that exposure to AC fields during dielectrophoretic cell manipulation is associated with upregulation of the intermediate-early gene cfos and also transcription of other as yet unidentified genes. These transcriptional events were not manifest as gross changes in cell morphology or cell-cycle dynamics.

Ultrastructural observations on myelinated fibres in the tibial nerve of streptozotocin-diabetic rats: effect of insulin treatment.

Four groups of male Sprague-Dawley rats aged 11 weeks and weighing approximately 450 g were studied over 16 weeks: onset and end controls, untreated diabetics and diabetics treated with a daily subcutaneous injection of Ultralente insulin. Good metabolic control was achieved in the insulin-treated group as judged by daily blood glucose estimations, glycosylated haemoglobin levels (HbA1c) and body weight. Cross-sectional myelinated fibre area significantly increased between onset and end controls; growth thus occurred. In the untreated diabetic rats the values were significantly less when compared with age matched controls and not different to onset controls. The values for the insulin-treated diabetic group did not show a significant increase when compared with untreated diabetics and onset controls and were intermediate between end controls and untreated diabetics without any significant difference when compared with either group. Cross-sectional axonal area was significantly less in the untreated diabetic group as compared with age matched controls and this was corrected by insulin therapy, as there was no difference between end controls and insulin-treated diabetic group. Insulin treatment corrects the reduction in axon size but total myelinated fibre size is not normalised. It seems that in addition to the axon, the myelin sheath and Schwann cells are also affected and these may be less influenced by insulin therapy.

Vagus nerve morphology in diabetic gastropathy.

Two patients were treated with gastroenterostomy and vagotomy for intractable vomiting due to diabetic gastropathy. A morphometric examination of nerve fibres and capillaries in their resected abdominal vagi was performed and the results were compared with findings from two diabetic and two non-diabetic patients undergoing gastroenterostomy and vagotomy for duodenal ulceration. All four diabetic patients had pathological changes of a similar character: reduced myelinated fibre density, degeneration and regeneration of unmyelinated fibres, and capillary basement membrane thickening. Abnormalities were more pronounced in the two diabetic patients with gastropathy but intact and regenerating nerve fibres were still present. The findings support the view that vagal neuropathy could be a causal factor in diabetic gastropathy but imply that severe gastropathy with vomiting is not simply a consequence of autovagotomy. The morphological observations indicated that structural changes can occur in the autonomic nerves of diabetic patients who do not develop autonomic symptoms or have easily detectable abnormal autonomic physiology.