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Biomedical subjects

L R Mills

Publications and source records attributed to L R Mills.

At least 19 recordsLinked to original sources
PubMed2003

A lipoxygenase product, hepoxilin A(3), enhances nerve growth factor-dependent neurite regeneration post-axotomy in rat superior cervical ganglion neurons in vitro.

Hepoxilins are 12-lipoxygenase metabolites of arachidonic acid found in the CNS. They can modulate neuronal signaling but their functions are not known. We examined the effects of hepoxilin A(3) on neurite outgrowth post-axotomy in an in vitro model of spinal cord transection using superior cervical ganglion neurons. In the absence of nerve growth factor, hepoxilin A(3) did not support neuronal survival, or regeneration post-axotomy but did significantly enhance neurite regeneration in the presence of nerve growth factor. As early as 1 h post-injury hepoxilin A(3)-treated cultures (+nerve growth factor) had significantly more neurites than controls (nerve growth factor alone). Average hourly rates of outgrowth in hepoxilin A(3)-treated cultures were significantly higher than in controls for at least 12 h post-injury, suggesting that the effect of hepoxilin A(3) is maintained in vitro for several hours post-injury. In uninjured neurons hepoxilin A(3) caused a rapid but transient increase in intracellular calcium in the somata; by 2 min post-addition, calcium levels decreased to a new stable plateau significantly higher than pre treatment levels. In injured neurons, hepoxilin A(3) addition immediately post-transection caused a rapid transient increase in intracellular calcium in cell bodies; however, peak calcium levels were significantly lower than in uninjured neurons and the new baseline lower than in uninjured cells. In uninjured cells hepoxilin A(3) addition in zero calcium produced the same pattern, a transient elevation and subsequent decline to a new stable baseline significantly above rest but in injured cells levels fell rapidly to pretreatment values. Taken overall, these findings demonstrate a novel role for hepoxilins as a potentiator of neurite regeneration. They also provide the first evidence that this lipoxygenase metabolite can alter intracellular calcium in neurons by causing release of calcium from intracellular stores and modulating calcium influx mechanisms.

8,11,14-Eicosatrienoic Acid↗
PubMed2000

Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy.

Reactive oxygen species have been implicated in the development of seizures under pathological conditions and linked to seizure-induced neurodegeneration. There has been little direct evidence, however, of free radical production resulting from seizures. Using amygdala-kindled rats, we have examined the generation of reactive oxygen species following seizures, and their possible contribution to seizure development and seizure-induced neuronal loss. The concentrations of two products of free radical-induced lipid peroxidation, malonaldehyde and 4-hydroxy-2(E)-nonenal, were measured using colorimetric assays. Lipid peroxidation was increased in both hemispheres of kindled rats as compared to sham-operated controls. Cell death was also significantly increased in all hippocampal areas. Antioxidants (vitamin E and glutathione) prevented the rise in lipid peroxides and hippocampal neuronal death during kindling, but did not arrest the development of seizures.Thus, epileptiform activity can result in free radical production which may be one of the factors leading to cell death.

Animals↗
PubMed1999

F-actin at newly invaginated membrane in neurons: implications for surface area regulation.

Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization of F-actin. At earliest detection (1-2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e. , at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics.

Actins↗
PubMed1999

Group I and II metabotropic glutamate receptor expression in cultured rat spinal cord astrocytes.

We have examined the development of expression of group I and II metabotropic glutamate receptors (mGluRs) in pure rat spinal cord astrocyte cultures, using immunocytological and calcium imaging techniques. mGluR1alpha and mGluR2/3 antibodies were found to label roughly 10% of the total astrocyte population at all time points examined, whereas mGluR5 was poorly expressed in our culture system. Results from intracellular Ca2+ imaging experiments, measured using fura-2 ratio imaging, suggest that 20% of these cultured astrocytes express functional group I mGluRs (mGluR1 and/or 5). Our results contrast with previously published work in cultured cortical astrocytes where mGluR5 and not mGluR1 is expressed, suggesting that cultured astrocytes from different parts of the CNS exhibit different patterns of mGluR expression.

2-Amino-5-phosphonovalerate↗
PubMed1999

Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores.

Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+ from voltage-dependent Ca2+ channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+ released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12-14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+ stores using two conventional drugs, dantrolene, which blocks the Ca2+ release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+ stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16. 5 +/- 4.4, 33.7 +/- 7.1, 11.6 +/- 2.1, respectively; means +/- SE; n = 7) compared with the appropriate time-matched control (4.1 +/- 2.2, 7.3 +/- 2.0, 2.8 +/- 0.9, respectively; n = 6). Dantrolene (30 microM; n = 5) and thapsigargin (1 microM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+ entering a cell from the outside, in addition to the Ca2+ contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+ release), may be necessary for seizure-induced cell death.

Animals↗
PubMed1998

Neuronal plasma membrane dynamics evoked by osmomechanical perturbations.

When neurons swell and shrink they extensively reorganize their plasma membrane. A striking aspect of these membrane dynamics is the transient appearance of vacuole-like dilations (VLDs) which, counterintuitively, expand as the neurons shrink. Here, confocal microscopy of cultured molluscan (Lymnaea) neurons was used in conjunction with aqueous phase and membrane dyes to examine changing VLD membrane topology as VLDs form, reverse or recover. We show that VLDs start as discrete invaginations at the adherent surface, so VLD and plasma membranes are initially contiguous. Over the next few minutes VLDs expand and penetrate the cytoplasm. At the substratum, the mouths of VLDs develop into irregular annuli of motile adherent processes whereas deeper in the cytoplasm, VLD membrane profiles are smooth. Subsequently VLDs spontaneously shrink; as this recovery proceeds, constriction of the motile VLD mouth leads to the internalization of plasma membrane. Washout experiments with aqueous phase dyes demonstrated that VLD constriction yields bona fide vacuoles, i.e., membrane-bound compartments isolated from the external medium. VLDs can also be experimentally eliminated by returning cells to swelling conditions; this reversal process drives membrane back to the surface. VLD formation and reinternalization of VLD membrane can be seen as aspects of plasma membrane surface area regulation. We postulate that area adjustments, driven by regional membrane tension differences, become noticeable when excessive perturbations overload normal membrane reprocessing steps. Both the changes in VLD membrane topology, and previously established capacitance changes accompanying cell shrinking and swelling, argue that osmomechanically perturbed neurons regulate their surface area as their volume changes.

Animals↗
PubMed1998

A novel and rapid method for culturing pure rat spinal cord astrocytes on untreated glass.

Astrocytes are the major population of glial cells, and are key players in the development, maintenance, and functioning of the central nervous system (CNS). Their potential as targets of therapeutic intervention following CNS injury makes the elucidation of their cellular and subcellular physiology a primary research goal. Well defined and pure cell culture systems are required to examine astrocytic physiology, biochemical pathways and underlying responses to pathophysiologically altered conditions. Previously published protocols for establishing primary astrocyte cultures are time- and resource-consuming or suffer high contamination from other undesired cell types. Here we describe a new and simple procedure for producing highly pure ( > 99%) rat primary astrocyte cultures. The method involves a simple mechanical dissociation of harvested spinal cord tissue through a porous membrane and the subsequent plating of the cells on plain, untreated glass coverslips. Astrocytes adhere very well to the untreated glass while other cell types require a substrate such as poly-L-lysine. The method described here is, therefore, ideal for experiments which require highly pure astrocyte cultures.

Animals↗
PubMed1997

N-Type calcium channels in the developing rat hippocampus: subunit, complex, and regional expression.

The expression of multiple classes of voltage-dependent calcium channels (VDCCs) allows neurons to tailor calcium signaling to functionally discrete cellular regions. In the developing hippocampus a central issue is whether the expression of VDCC subtypes plays a role in key phases such as migration and synaptogenesis. Using radioligand binding and immunoblotting, we show that some N-type VDCCs exist before birth, consistent with a role in migration; however, most N-VDCC subunit expression is postnatal, coinciding with synaptogenesis. Immunoprecipitation studies indicate that the increased expression of N-VDCCs in early development occurs without subunit switching because there is no change in the fraction of beta3 subunits in the N-VDCC alpha1B-beta3 heteromers. Fluorescence imaging of cell surface N-VDCCs during this period reveals that N-VDCCs are expressed on somata before dendrites and that this expression is asynchronous between different subfields of the hippocampus (CA3-CA4 before CA1-CA2 and dentate gyrus). Our data argue that N-VDCC expression is an important cue in the genesis of synaptic transmission in discrete hippocampal subfields.

Animals↗
PubMed1997

Increased immunoreactivity for Rab11, a small GTP-binding protein, in low-grade dysplastic Barrett's epithelia.

Adenocarcinoma of the esophagus develops from metaplastic Barrett's columnar epithelia through the evolution of dysplastic epithelial intermediates. Although the role of dysplasia leading to adenocarcinoma is well established, far less is known regarding the cellular changes involved in this process. Because the development of dysplasia is characterized by the loss of apical secretory specializations, we hypothesized that changes in apical trafficking might be involved in the dysplastic process. We have sought to evaluate the expression of an important candidate regulator of apical trafficking, the small GTP-binding protein, Rab11, in resection and biopsy tissue from patients with Barrett's esophagus. Sections from esophageal resection specimens from 4 patients and endoscopic biopsies from 60 patients were stained with antibodies against Rab11 and Rab25 as well as protein markers of the Golgi apparatus and p53 protein. Rab11 staining in low-grade dysplastic regions was similar to that observed with monoclonal antibodies against Rab25 and gamma-adaptin and colocalized with staining for the Golgi marker, the mannose-6-phosphate receptor. In the esophageal adenocarcinoma resections, prominent Rab11 immunostaining was observed in the supranuclear region of low-grade dysplastic cells. In contrast, regions of high-grade dysplasia demonstrating strong nuclear p53 staining showed only diffuse or absent Rab11 staining. In endoscopic biopsies, 91% of biopsies that were read unanimously as low-grade dysplasia demonstrated supranuclear Rab11 staining. Fourteen percent of biopsies unanimously graded as being without dysplasia demonstrated perinuclear Rab11 staining. No p53 immunostaining was observed in any of the low-grade dysplasia biopsy specimens. An increase in Rab11 immunoreactivity seems to correlate with low-grade dysplasia, whereas p53 immunostaining correlates with high-grade dysplasia. The colocalization of Rab11 staining with increased immunoreactivity for markers of the trans-Golgi system is consistent with a defect in apical trafficking due to an expansion of either the trans-Golgi compartment or the apical recycling vesicle system.

Barrett Esophagus↗
PubMed1996

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43.

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

Animals↗
PubMed1996

Three distinct neuronal phenotypes exist in embryonic rat hippocampal neurons cultured in basic fibroblast growth factor.

The possibility that neurons cultured in basic fibroblast growth factor (bFGF) are heterogeneous raises concerns about their subsequent use in gene transfection and transplantation studies. We have examined the fate of embryonic hippocampal neurons in bFGF culture, and now conclude that these cells are not only heterogeneous, but possess neurons of various stages of development. Morphological and immunocytochemical analysis reveal three distinct developmental classes of neurons are present in extended bFGF culture. This tripartite classification is supported by electrophysiological analysis, which reveals that upon depolarization, neurons with precursor and juvenile neuron morphologies are unable to fire action potentials. The third class of neurons, which resemble age-matched polarized neurons in standard serum culture, fired multiple action potentials indicative of a mature phenotype. These data show neurons at multiple developmental stages co-exist in bFGF culture, and provide an experimental basis for their classification.

Action Potentials↗
PubMed1996

Distinct calcium signaling within neuronal growth cones and filopodia.

Previous findings indicate that spatial restriction of intracellular calcium levels within growth cones can regulate growth cone behavior at many levels, ranging from filopodial disposition to neurite extension. By combining techniques for focal stimulation of growth cones with those for measurement of filopodia and for capturing low intensity calcium signals, we demonstrate that filopodia on individual growth cones can respond to imposed stimuli independently from one another. Moreover, filopodia and their parent growth cones appear to represent functionally and morphologically distinct domains of calcium regulation, possessing distinct calcium sources and sinks. Both are sensitive to calcium influx; however, application of the calcium ionophore A23187 to cells in calcium-free medium demonstrated the presence of potential intracellular calcium pools in the growth cone proper, but not in isolated filopodia. Thapsigargin significantly reduced the rise in growth cone calcium levels associated with excitatory neurotransmitters, further implicating release from calcium pools as one component of growth cone calcium regulation. The relative contributions of these pools were examined in response to excitatory neurotransmitters by quantitative calcium measurements made in both growth cones and isolated filopodia. Striking differences were observed; filopodia were sensitive to a low concentration of dopamine and serotonin, while growth cones displayed an amplified rise at a higher concentration. The spatial distribution of organelles that could serve as morphological correlates to such calcium amplification was examined using confocal microscopy. While the majority of organelles were located in the central core of the growth cone proper, peripheral organelles were detected at the base of a subset of filopodia. The distinctive distribution of calcium regulation within motile growth cones suggests one mechanism by which growth cones may regulate their complex behavior.

Animals↗
PubMed1995

Long-term bFGF neuronal culture: reintroduction into serum medium yields neurons and non-neuronal cells with neuronal characteristics.

The potential use of bFGF immortalized cells as hosts for delivering foreign genes into nervous tissue led us to examine the effect of maintaining, E-18 hippocampal neurons for extended periods in bFGF culture prior to transfer into a standard, serum-containing, medium. We found: (1) many, if not most, precursors seen in bFGF, mature into glia and not into primary neurons after medium exchange; (2) the electrophysiology of the neurons which do mature after medium transfer and replating, is similar to that of neurons in standard cultures; (3) extended culture in bFGF prior to cell harvesting and replating into standard medium generates neurons from the precursors that possess proper neuronal polarization, morphology, and electrophysiology; and (4) extended bFGF also induces the expression, on transfer into standard medium, of an additional cell type with a distinct non-neuronal morphology that stains with the neuronal marker MAP-2. These results illustrate the need for additional characterization of long-term growth factor effects on maintained progenitor cells prior to their use in gene therapy and transplantation.

Action Potentials↗
PubMed1995

Discrete and reversible vacuole-like dilations induced by osmomechanical perturbation of neurons.

In cultured Lymnaea stagnalis neurons, osmolarity increases (upshocks) rapidly elicited large membranous dilations that could be dislodged and pushed around inside the cell with a microprobe. Subsequent osmolarity decreases (downshocks) caused these vacuole-like dilations (VLDs) to disappear. Additional upshock/downshock perturbations resulted in repeated appearance/disappearance (formation/reversal) of VLDs at discrete sites. Confocal microscopy indicated that VLDs formed as invaginations of the substrate-adherent surface of the neuron: extracellular rhodamine-dextran entered VLDs as they formed and was expelled during reversal. Our standard VLD-inducing perturbation was: 2-4 min downshock to distilled water, upshock to normal saline. However, a wide range of other osmotic perturbations (involving osmolarities up to 3.5 x normal, perturbations with or without Ca2+, replacement of ions by sucrose) were also used. We concluded that mechanical, not chemical, aspects of the osmo-mechanical shocks drove the VLD formation and reversal dynamics and that extracellular Ca2+ was not required. Following a standard perturbation, VLDs grew from invisible to their full diameter (> 10 microns) in just over a minute. Over the next 0.5-3 hr in normal saline, neurons recovered. Recovery eliminated any visible VLDs and was accompanied by cytoplasmic turmoil around the VLDs. Recovery was prevented by cytochalasin B, brefeldin A and N-ethylmaleimide but not by nocodazole. In striking contrast, these drugs did not prevent repeated VLD formation and reversal in response to standard osmo-mechanical perturbations; VLD disappearance during reversal and during recovery are different. The osmo-mechanical changes that elicited VLDs may, in an exaggerated fashion, mimic tension changes in extending and retracting neurites. In this context we postulate: (a) the trafficking or disposition of membrane between internal stores and plasma membrane is mechanosensitive, (b) normally, this mechanosensitivity provides an "on demand" system by which neurons can accommodate stretch/release perturbations and control cell shape but, (c) given sudden extreme mechanical stimuli, it yields VLDs.

Animals↗
PubMed1995

Merkel cells are not the mechanosensory transducers in the touch dome of the rat.

The identity of the mechanosensory transducing elements in the vertebrate touch receptors that contain Merkel cell-neurite complexes is unknown. The Merkel cells, however, have long been the favoured candidates. We have now selectively eliminated the Merkel cells from rat touch domes by first loading them with quinacrine, and then irradiating the domes with near-UV light. Mechanical stimulation of these domes revealed a range of mechanosensory function, evaluated qualitatively, that varied from non-responsive to normal. Since irradiation eliminated the quinacrine fluorescence, the status of the Merkel cells was evaluated by EM. In both responsive and unresponsive domes fixed for EM immediately following irradiation, the Merkel cells and associated nerve endings appeared to be normal. After 2 or more days, even in domes that continued to be normally responsive, there was a striking reduction in the normal complement of about 90 Merkel cells, and most of the remaining Merkel cells appeared to be degenerating. However, numerous 'isolated' (Merkel cell-free) nerve endings remained in the basal epidermis. A few of these nerve endings showed signs of damage, but in the non-responsive domes abnormal nerve endings were routinely observed. The EM studies did not exclude the possibility that a few surviving innervated Merkel cells, or even one such, had escaped detection and were responsible for a persisting mechanosensitivity. To resolve this issue a mechanical stimulating technique with a spatial resolution of 55 microns was used to map the mechanosensory profile of a single responsive dome irradiated 2.75 days earlier. This dome was then serially sectioned for EM study. Only seven Merkel cells had survived which appeared to be both viable and innervated, but almost all of the tested sites were normally responsive. When the correlation was made, seven of these sites were located 55-100 microns away from the nearest surviving Merkel cell, four were 110-165 microns away, and three were more than 165 microns away. Even when allowance is made for errors in the positioning of the stimulus, the responses at the last seven sites cannot be attributed to the presence of underlying Merkel cells. We conclude that mechanosensory transduction within touch domes is not a function of the Merkel cells, but must reside in the associated nerve endings.

Animals↗
PubMed1994

Long-term prognosis of well-differentiated adenocarcinoma in endoscopically removed colorectal adenomas.

Thirty-six malignant polyps were identified that met the following criteria: well-differentiated adenocarcinoma and complete excision endoscopically. Location, type, size, distance of the cancer to the cautery mark, and lymphovascular involvement were analyzed to determine if they affected findings at surgery or risk of recurrent cancer. There were 20 patients and 21 polyps in the nonsurgical group, and 15 patients and polyps in the surgical group. One patient from each group had residual cancer after endoscopic removal of the polyp. The only factor that had an adverse effect on outcome was the distance of the cancer to the cautery mark (< 1 mm). Although rectal location was associated with the residual cancer, poor prognosis could have been predicted by the inadequate margins. This long-term follow-up (65 months average) study supports previous observations that an adequate margin is the most important factor in predicting the prognosis of endoscopically resected colorectal adenomas containing well-differentiated adenocarcinomas.

Adenocarcinoma↗