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Andrew C Hall

Publications and source records attributed to Andrew C Hall.

9 recordsLinked to original sources
PubMed2007

Two-versus one photon excitation laser scanning microscopy: critical importance of excitation wavelength.

It is often anticipated that two-photon excitation (TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation (OPE) confocal scanning laser microscopy (CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.

Animals↗
PubMed2006

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes.

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.

Animals↗
PubMed2005

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage.

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).

Cartilage, Articular↗
PubMed2005

Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity.

OBJECTIVE: Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability. METHODS: Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10 cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of chondrocytes to a single impact. RESULTS: A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting approximately 3 min, followed by a slower progressive 'wave of cell death' away from the initial area lasting for approximately 20 min. The volume of the majority (88.1+/-5.99% (n=7) of the viable chondrocytes in this region decreased significantly (P<0.006). By monitoring LDH release, a single impact 5 min after changing the culture medium to hyper-, or hypo-osmolarity, reduced or stimulated chondrocyte injury, respectively. CONCLUSIONS: A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.

Animals↗
PubMed2005

Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B.

Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.

Actins↗
PubMed2004

Induction of calcium-activated potassium channel activity by hemin in human erythroleukemia cells.

The agent hemin has been demonstrated to be able to initiate a coordinated differentiation program in several cell types. In the present study, we examined the ability of hemin on inducing cell differentiation and Ca(2+)-activated K(+) channel activity in erythroleukemic K562 cells. Treating undifferentiated K562 cells with hemin (0.1 mM) for five days caused these cells to display differentiation-like characteristics including chromatin aggregation, nuclear degradation, pseudopod extension of the membrane and increased hemoglobin production. However, overall cell viability was not significantly changed by the presence of hemin. After hemin treatment for different periods, the Ca(2+)-activated K(+) channel was activated by the addition of ionomycin (1 microM), and was inhibited by either clotrimazole, charybdotoxin, or EGTA. Before hemin treatment there was no significant Ca(2+)-activated K(+) channel activity present in undifferentiated K562 cells. After hemin treatment for 5 days, a significant Ca(2+)-activated K(+) channel activity was detected. This increasing Ca(2+)-activated K(+) channel activity may be contributed from a subtype of Ca(2+)-activated K(+) channel, KCNN4. These results suggest that the ability of hemin to induce increasing Ca(2+)-activated K(+) channel activity may contribute to the mechanism of hemin-induced K562 cell differentiation.

Calcium↗
PubMed2002

Functional analysis of mouse hepatocytes differing in DNA content: volume, receptor expression, and effect of IFNgamma.

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule-1 (ICAM-1, CD54) or IFNgammaR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNgamma)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM-1 increases with increasing DNA content and IFNgammaR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNgamma-induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro.

Animals↗