Heart rate variability and plasma nephrines in the evaluation of heat acclimatisation status.

PURPOSE: Heat adaptation (HA) is critical to performance and health in a hot environment. Transition from short-term heat acclimatisation (STHA) to long-term heat acclimatisation (LTHA) is characterised by decreased autonomic disturbance and increased protection from thermal injury. A standard heat tolerance test (HTT) is recommended for validating exercise performance status, but any role in distinguishing STHA from LTHA is unreported. The aims of this study were to (1) define performance status by serial HTT during structured natural HA, (2) evaluate surrogate markers of autonomic activation, including heart rate variability (HRV), in relation to HA status. METHODS: Participants (n = 13) were assessed by HTT (60-min block-stepping, 50% VO2peak) during STHA (Day 2, 6 and 9) and LTHA (Day 23). Core temperature (Tc) and heart rate (HR) were measured every 5 min. Sampling for HRV indices (RMSSD, LF:HF) and sympathoadrenal blood measures (cortisol, nephrines) was undertaken before and after (POST) each HTT. RESULTS: Significant (P < 0.05) interactions existed for Tc, logLF:HF, cortisol and nephrines (two-way ANOVA; HTT by Day). Relative to LTHA, POST results differed significantly for Tc (Day 2, 6 and 9), HR (Day 2), logRMSSD (Day 2 and Day 6), logLF:HF (Day 2 and Day 6), cortisol (Day 2) and nephrines (Day 2 and Day 9). POST differences in HRV (Day 6 vs. 23) were + 9.9% (logRMSSD) and - 18.6% (logLF:HF). CONCLUSIONS: Early reductions in HR and cortisol characterised STHA, whereas LTHA showed diminished excitability by Tc, HRV and nephrine measures. Measurement of HRV may have potential to aid real-time assessment of readiness for activity in the heat.

The involvement of the serotonergic transmission system in neonatal and adult rat ileum contractility varies with age.

The relevance of age on serotonergic involvement in the control of alimentary contractility has not been pharmacologically described. Experiments were performed to investigate the effects of acetylcholine, atropine, 5-hydroxytryptamine (5-HT) and its related drugs on intestinal segments taken from the neonatal and adult ileum. 5-HT induced concentration-dependent contractions of ileum irrespective of age; however, these contractions were diminished by pretreatment with atropine only in neonatal tissues. In tissues taken from both the neonatal and adult ileum, methysergide (5-HT(1/2/5-7) receptor antagonist), ritanserin (5-HT(2) receptor antagonist), and RS23597-190/SB204070 (5-HT(4) receptor antagonists) all differentially reduced 5-HT-induced contractions at a concentration <100 µmol/l. At higher concentrations, the contractions were comparable to those in control tissues. Granisetron and ondansetron (5-HT(3) receptor antagonists) significantly reduced contractions induced by 5-HT at concentrations >30 µmol/l in both neonatal and adult ileum. Combined treatments with ritanserin, granisetron, plus RS23597-190 reduced or abolished contraction responses induced in neonatal ileum by 5-HT. SB269970A (5-HT(7) receptor antagonist) and WAY100635 (5-HT(1A) receptor antagonist) failed to influence contractile responses induced by 5-HT or 5-HT receptor agonists. Pretreatments with WAY100635 and SB267790A also had no influence on the contractile responses induced by 5-HT(1A/7) receptor agonist, 5-CT, and 5-HT(1A) receptor agonist, 8-OH-DPAT, which itself failed to induce a measurable response. It is concluded that the 5-HT-induced contractions in segments taken from both the neonatal and adult rat ileum were mediated via 5-HT(2) receptors, 5-HT(3) receptors and 5-HT(4) receptors. However, the effect of atropine on the neonatal rat intestine indicates that the mechanism of serotonergic involvement in ileal contractility is influenced by age.

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.

Micropatterning proteins and synthetic peptides on solid supports: a novel application for microelectronics fabrication technology.

In this paper, we describe a method for immobilizing proteins and synthesizing peptides in micrometer-dimension patterns on solid supports. Microelectronics fabrication technology was adapted and used to lithographically direct the location of immobilization of proteins on appropriately derivatized surfaces. As examples, we micropatterned the protein bovine serum albumin (BSA) and the enzyme horseradish peroxidase (HRP). The catalytic activity of HRP was shown to be retained after being cross-linked to the support. When coupled with solid-phase peptide synthesis, the technique allowed synthetic peptides to be constructed in patterns again having micrometer dimensions. Synthetic polypeptides, polylysine, were constructed in patterns with dimensions that approached the practical limit of resolution for optical lithography at 1-2 microns. The patterns of immobilized molecules and synthetic peptides were visualized using histochemical methods together with light and fluorescence microscopy. The protein and peptide patterning technique described here is an advance in the field of bioelectronics. In particular, it should now be possible to devise novel methods for interfacing with biological systems and constructing new devices for incorporation into miniaturized biosensors.

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.

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones.

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 degrees C) in 0.25% trypsin for periods of 30-90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 microg mL(-1)) and anti-mitotic cytosine arabinoside (6 microm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle alpha-actin, alpha-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to alpha-smooth muscle actin and alpha-actinin. Conversely, ISMC were stained with antibodies to alpha-smooth muscle actin and alpha-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types.

A comparison of the effects of conventional insulin treatment and continuous insulin infusion therapy on established morphological and biochemical abnormalities of peripheral nerve in experimental diabetes.

This study compared the relative efficacy of conventional insulin therapy (CIT) and continuous subcutaneous insulin infusion therapy (CSII) in correcting a range of morphological and biochemical abnormalities of peripheral nerve in rodents with experimental diabetes mellitus. Both treatments were applied for two months and were commenced two months after the induction of diabetes in 11 week old rats. Better diurnal glycaemic control was achieved with CSII therapy than with CIT. An established deficit in body weight in the diabetic animals was partially, but not fully corrected by both treatments. Retardation of long bone growth in the diabetic animals was corrected by CSII therapy but not CIT. Elevated levels of sciatic nerve glucose, sorbitol and fructose found in diabetic animals were normalized by both treatments. However, depleted levels of nerve myo-inositol which were also found in diabetic animals were restored to normal only by CSII therapy. Abnormal myelinated nerve fibre morphology, indexed by fibre and axon cross-sectional area measurements, was again corrected more effectively by CSII therapy. This study has demonstrated that often partial and sometimes complete amelioration of established biochemical and morphological abnormalities of peripheral nerve in diabetic animals can be achieved by the application of either CSII therapy or CIT. However, under the controlled conditions of this experimental study better overall performance was gained from CSII therapy.

A new perspective on myelinated nerve fibre pathology in experimental diabetes.

Quantitative electron-microscopical techniques were used to investigate the effects of experimental streptozotocin-diabetes on the longitudinal morphology of myelinated fibres in rat sural nerve. The morphology of myelinated fibres was symmetrical at proximal and distal levels in the nerves of control animals aged three months. Further development of myelinated fibres in control animals between three and seven months of age involved proximal and distal fibre growth with the simultaneous formation of a proximo-distal taper. The same pattern of fibre growth was not observed in animals with experimental diabetes. Although both fibre and axon size increased with age in both proximal and distal regions of sural nerve in the diabetic animals, a taper was not established. This impairment of fibre maturation by experimental diabetes was manifest more strongly in the proximal part of the nerve. There was no evidence favouring selective involvement of the axon or Schwann cell in the fibre pathology found in the diabetic animals. The results of this study do not support previous suggestions that experimental diabetic neuropathy is mainly a dying-back axonopathy. This report reaffirms that normal maturation of myelinated fibres is prevented by experimental diabetes mellitus.

Embryonic Xenopus neurites integrate and respond to simultaneous electrical and adhesive guidance cues.

Nerve cells detect and respond to multiple extrinsic guidance cues during development and regeneration using a motile growth cone. Navigational decisions may be required of the growth cone when different guidance cues are encountered simultaneously. We have tested the relative potencies of two opposing cues by presenting Xenopus spinal cord nerve cells growing on a micropatterned laminin culture substratum with an orthogonal DC electric field. Substrata composed of repeating 25-micron laminin tracks and spaces failed to influence the position of neuritogenesis from nerve cell soma. Once established, however, growth cone movement was constrained by laminin tracks such that neurites of 65% of cells were aligned after 5 h in vitro. Two hours after the application of a 100-140 mV/mm DC field the majority of cells remained aligned with the laminin tracks. Around 70% of Xenopus neurites normally orient cathodally on homogenous laminin substrata; therefore the galvanotropic response was impeded by prior exposure to a patterned laminin substrate. However, a proportion of aligned neurites did orient cathodally and evidence of a response to both directional cues was even found within the same cell. Video-enhanced contrast, differential interference contrast (VEC-DIC) microscopy was used to examine the detailed behavior of growth cones on micropatterned laminin substrata. The present study has demonstrated that growth cones can detect and integrate at least two morphogenetic guidance cues simultaneously. The strength of the galvanotropic response in Xenopus growth cones, however, was often insufficient to override established adhesive guidance in this model system.

Growth cone guidance and neuron morphology on micropatterned laminin surfaces.

Neurite growth cones detect and respond to guidance cues in their local environment that determine stereotyped pathways during development and regeneration. Micropatterns of laminin (which was found to adsorb preferentially to photolithographically defined hydrophobic areas of micropatterns) were here used to model adhesive pathways that might influence neurite extension. The responses of growth cones were determined by the degree of guidance of neurite extension and also by examining growth cone morphology. These parameters were found to be strongly dependent on the geometry of the patterned laminin, and on neuron type. Decreasing the spacing of multiple parallel tracks of laminin alternating with non-adhesive tracks, resulted in decreased guidance of chick embryo brain neurons. Single isolated 2 microns tracks strongly guided neurite extension whereas 2 microns tracks forming a 4 microns period multiple parallel pattern did not. Growth cones appear to be capable of bridging the narrow non-adhesive tracks, rendering them insensitive to the smaller period multiple parallel adhesive patterns. These observations suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by oriented extracellular matrix or certain axon fascicle structures, but could be guided by isolated adhesive tracks. Growth cone morphology became progressively simpler on progressively narrower single tracks. On narrow period multiple parallel tracks (which did not guide neurite extension) growth cones spanned a number of adhesive/non-adhesive tracks, and their morphology suggests that lamellipodial advance may be independent of the substratum by using filopodia as a scaffold. In addition to acting as guidance cues, laminin micropatterns also appeared to influence the production of primary neurites and their subsequent branching. On planar substrata, dorsal root ganglion neurons were multipolar, with highly branched neurite outgrowth whereas, on 25 microns tracks, neurite branching was reduced or absent, and neuron morphology was typically bipolar. These observations indicate the precision with which growth cone advance may be controlled by substrata and suggest a role for patterned adhesiveness in neuronal morphological differentiation, but also highlight some of the limitations of growth cone sensitivity to substratum cues.

Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type.

We used an in vitro system that eliminates competing guidance cues found in embryos to determine whether substratum topography alone provides important neurite guidance information. Dissociated embryonic Xenopus spinal cord neurons and rat hippocampal neurons were grown on quartz etched with a series of parallel grooves. Xenopus neurites grew parallel to grooves as shallow as 14 nm and as narrow as 1 microm. Hippocampal neurites grew parallel to deep, wide grooves but perpendicular to shallow, narrow ones. Grooved substrata determined the sites at which neurites emerged from somas: Xenopus neurites sprouted from regions parallel to grooves but presumptive axons on rat hippocampal neurons emerged perpendicular to grooves and presumptive dendrites emerged parallel to them. Neurites grew faster in the favored direction of orientation and turned through large angles to align on grooves. The frequency of perpendicular alignment of hippocampal neurites depended on the age of the embryos from which neurons were isolated, suggesting that contact guidance is regulated in development. Collectively, the data indicate that substratum topography is a potent morphogenetic factor for developing CNS neurons and suggest that in addition to a role in pathfinding the geometry of the embryo assists in establishing neuronal polarity. In the companion paper (A. M. Rajnicek and C. D. McCaig (1997) J. Cell Sci. 110, 2915-2924) we explore the cellular mechanism for contact guidance of growth cones.

Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior.

It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable.

Differential response of fetal and neonatal myoblasts to topographical guidance cues in vitro.

Fusion of mononucleated myoblasts into parallel arrays of mutinucleated myotubes is an essential step in skeletal myogenesis. The formation of such a highly ordered structure requires myoblasts to come together, orient and align in the correct location prior to fusion. We report here that fetal and neonatal myoblasts can use topographical features as strong guidance cues in vitro. Myoblasts were cultured on multiple grooved substrata of varying dimensions, and the axial orientations of individual cells were recorded. Both fetal and neonatal myoblasts aligned parallel with the direction of deep grooves (2.3-6.0 micron), which is correlated well with the location of myoblasts in similar sized grooves during secondary myogenesis. Fetal myoblasts also responded to shallower grooves (0. 04-0.14 micron) by aligning parallel or perpendicular to the direction of the grooves, indicating the ability of these cells to respond to fine elements normally encountered within the developing muscle architecture. In contrast, neonatal myoblasts failed to respond to shallow grooves, adding to the suggestion that fetal and neonatal myoblasts may represent separate populations of myoblasts. Overall, the results demonstrate that myoblasts respond to large and small features of the physical topography in vitro and indicate that structural elements in the microenvironment of the muscle may play a critical role in myoblast spatial organization during myogenesis.

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.

Synergistic and hierarchical adhesive and topographic guidance of BHK cells.

Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 microm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 microm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-microm-wide adhesive tracks and to 5-microm-wide, 6-microm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-microm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.

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.