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PubMed2004

Influence of different gases used for laparoscopy (helium, carbon dioxide, room air, and xenon) on tumor volume, histomorphology, and leukocyte-tumor-endothelium interaction in intravital microscopy.

BACKGROUND: Previous studies indicate that helium pneumoperitoneum used for laparoscopic surgery suppresses whereas carbon dioxide pneumoperitoneum increases postoperative tumor growth. The pathomechanisms of decreased tumor growth by helium are unknown. This study was designed to examine the effect of the gases helium, carbon dioxide (CO(2)), and air, and xenon, which can be used to induce pneumoperitoneum in laparoscopy on tumor volume, histomorphology, and leukocyte-endothelium interaction measured by intravital microscopy in rats with implanted liver malignoma (Morris hepatoma 3924A). METHODS: In 46 rats, Morris hepatoma 3294A cells were implanted intrahepatically. After implantation, rats were randomized into two main groups. In the first main group, 10 animals were prepared for examination of leukocyte-endothelium interaction by intravital video microscopy and were randomized into two groups. Five days after implantation they underwent laparoscopy using either helium (n = 5) or CO(2) (n = 5). Ten days after implantation the rats underwent intravital video microscopy to assess leukocyte-endothelium interaction in the tumor and liver vessels. In the second main group 36 rats were prepared for examination of tumor volume arid histomorphology. They were randomized into five groups. Five days after implantation they underwent laparoscopy using helium (n = 7), carbon dioxide (n = 7), room air (n = 7), or xenon (n = 8). The control group (n = 7) received anesthesia only. Rats were killed 10 days after tumor implantation to assess tumor volume and histomorphology. RESULTS: Compared to the control group or groups that received CO(2), room air, or xenon for pneumoperitoneum, the establishment of helium pneumoperitoneum caused a significantly smaller tumor volume (Kruskal-Wallis test, p = 0.001; median tumor-volume: control group, 44 mm(3); helium 19 mm(3)). There was no significant difference in histomorphology between the groups. There was only a statistically significant difference in the development of central tumor necrosis in accordance to tumor volume (Mann-Whitney test, p = 0.03). In the tumor samples, roller counts were statistically significantly higher in the helium group compared to the CO(2) group (p = 0.04). For sticker counts, no statistically significant effects due to liver/tumor (p = 0.13) or treatment (p = 0.48) were observed. CONCLUSIONS: There was a significant decrease in tumor volume using helium pneumoperitoneum for laparoscopy compared to the other gases. Here, we demonstrate that suppression of tumor growth is not due to variation of histomorphology. It seems that helium pneumoperitoneum effects a higher leukocyte-endothelium interaction and thereby a higher immune activation. This could be one explanation for the statistically significantly smaller tumor volume after laparoscopy with helium compared to laparoscopy with CO(2).

Air↗
PubMed2005

Novel optical and statistical methods reveal colloid-wall interactions inconsistent with DLVO and Lifshitz theories.

We present an experimental method based on video microscopy to perform nanometer scale position detection of a micrometer bead in the direction along the propagation of the detection light. Using the same bead for calibration and detection significantly improves the in depth resolution in comparison to video microscopy methods from literature. This method is used together with an optical trap to measure interaction potentials between a glass surface and colloids made of polystyrene or silica at different electrolyte concentrations. The results are confirmed by an independent method where the optical trap is used in connection with a quadrant photodiode. Also, we present a maximum likelihood analysis method which considerably improves the spatial resolution of interaction potentials by optimizing the underlying potential function to fit all observed position distributions. The measured interaction potentials agree well with DLVO theory for small electrolyte concentrations; however, for larger electrolyte concentrations the potentials differ qualitatively from both DLVO and Lifshitz theory.

Journal Article↗
PubMed1999

Structural skin capillary rarefaction in essential hypertension.

A reduction in the density of capillaries (rarefaction) is known to occur in many tissues in patients with essential hypertension. This rarefaction may play a role in increasing peripheral resistance. However, the mechanism underlying this capillary rarefaction is not understood. The aim of this study was to assess the extent of structural versus functional capillary rarefaction in the skin of dorsum of fingers in essential hypertension. The capillary microcirculation was examined with video microscopy before and after maximizing the number of perfused capillaries by venous congestion. The study group comprised 17 patients with essential hypertension (mean supine blood pressure, 155/96 mm Hg) and 17 closely matched normotensive controls (mean blood pressure, 127/77 mm Hg). We used intravital video microscopy with an epi-illuminated microscope to examine the skin of the dorsum of left middle phalanx before and after venous congestion at 60 mm Hg for 2 minutes. A significantly lower mean capillary density occurred at baseline in hypertensive subjects versus normotensive subjects. With venous occlusion, capillary density increased significantly in both groups; however, maximal capillary density remained significantly lower in the hypertensive subjects than in the normotensive subjects. The study strongly suggests that much of the reduction in capillary density in the hypertensive subjects is caused by structural (anatomic) absence of capillaries rather than functional nonperfusion.

Adult↗
PubMed2006

Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.

The prevailing view is that the beta2-integrins Mac-1 (alphaMbeta2, CD11b/CD18) and LFA-1 (alphaLbeta2, CD11a/CD18) serve similar biological functions, namely adhesion, in the leukocyte recruitment cascade. Using real-time and time-lapse intravital video-microscopy and confocal microscopy within inflamed microvessels, we systematically evaluated the function of Mac-1 and LFA-1 in the recruitment paradigm. The chemokine macrophage inflammatory protein-2 induced equivalent amounts of adhesion in wild-type and Mac-1-/- mice but very little adhesion in LFA-1-/- mice. Time-lapse video-microscopy within the postcapillary venules revealed that immediately upon adhesion, there is significant intraluminal crawling of all neutrophils to distant emigration sites in wild-type mice. In dramatic contrast, very few Mac-1-/- neutrophils crawled with a 10-fold decrease in displacement and a 95% reduction in velocity. Therefore, Mac-1-/- neutrophils initiated transmigration closer to the initial site of adhesion, which in turn led to delayed transmigration due to movement through nonoptimal emigration sites. Interestingly, the few LFA-1-/- cells that did adhere crawled similarly to wild-type neutrophils. Intercellular adhesion molecule-1 but not intercellular adhesion molecule-2 mediated the Mac-1-dependent crawling. These in vivo results clearly delineate two fundamentally different molecular mechanisms for LFA-1 and Mac-1 in vivo, i.e., LFA-1-dependent adhesion followed by Mac-1-dependent crawling, and both steps ultimately contribute to efficient emigration out of the vasculature.

Animals↗
PubMed1996

A saporin-insulin conjugate: synthesis and biochemical characterization.

Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.

Animals↗
PubMed1995

Current issues in telepathology.

Telepathology involves rendering diagnostic opinions on specimens at remote locations using computer and telecommunications technologies. Consultations are a routine practice pattern in pathology, as there is a large amount of diagnostic and prognostic information available from the examination of biopsy material that requires an extensive knowledge of diseases and their clinical implications. Pathologists therefore frequently request opinions from those who subspecialize in various diseases. The use of video technology to examine and consult on microscopical slides has been validated, although sparsely and with a number of technical issues as yet unresolved. There are two principal paradigms for telepathology: remote dynamic screening by robotic video microscopy and remote diagnosis from selected still video microscopical images. The former is more appealing to most pathologists; however, it requires very high speed telecommunications links that are expensive and may not be available in a given location. The latter method entails a significant reduction in the data on which the diagnosis is rendered, as one is dependent on the fields selected at the remote location. In addition, there are hybrid systems that combine limited robotic capabilities with high-resolution still images. Image compression can increase the number of images that can be transmitted over an otherwise-limiting telecommunications infrastructure. JPEG is the current standard for such compression. Despite certain limitations, telepathology has been demonstrated successfully at several sites around the world. As this technology matures, systems will offer higher resolution, standardization of file formats, and better compression at lower cost.

Computer Communication Networks↗
PubMed1988

Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro.

In developing mammalian brain, many neurons migrate to their final position by moving in direct apposition to radially oriented glial cells. Glial-guided migration can be visualized in microcultures of mouse cerebellar cells by the combined use of cellular antigen markers and high resolution time-lapse video microscopy (Hatten et al., 1984; Edmondson and Hatten, 1987). Such studies have demonstrated the behavior of migrating cells and revealed a motile leading process on the migrating neuron that resembles an axonal growth cone and grows along extended glial fibers. To study the fine structural details of the migrating neuron and its neuron-glial apposition, we identified and monitored neurons in microcultures with video microscopy and examined the cytology and cellular contacts of the same cells with transmission electron microscopy. The cytology of the soma and leading process of migrating cells closely matches that described for granule cells in intact brain (Rakic, 1971). Newly observed structures include the presence of longitudinally oriented microtubules extending from a basal body in the soma into the leading process, and microfilament-rich filopodia arising from the soma and leading process. The most striking feature of actively migrating neurons is a specialized junction between the neuronal cell soma and apposing glial fibers. At this junction, here termed "interstitial density," the extracellular space is dilated to 20 nm and filamentous material in the intracellular cleft either spans the cleft or runs parallel to the cell membranes. Some interstitial fibrils are contiguous with, or are transmembranous extensions of, submembranous cytoskeletal elements that attach to microtubules. Interstitial junctions were not found between neurons that did not translocate in the observation period before fixation. Instead, stationary cells formed desmosomes (puncta and macula adhaerentia) at appositions with glial processes.

Animals↗
PubMed2000

Adenosine deaminase inhibition attenuates reperfusion low flow and improves graft survival after rat liver transplantation.

BACKGROUND: Low flow or no flow is a prefinal step after reperfusion of hepatic allografts. Adenosine is an intrinsic key regulator of physiological and pathological hepatic blood flow. METHODS: In a model of rat liver transplantation, the effect of donor pretreatment with adenosine deaminase inhibitors (0, 0.1, 1, 10 micromol erythro-9-[2-hydroxy-3-nonyl]adenine) was studied on hepatic interstitial adenosine concentrations, microcirculatory flow, leukocyte adhesion, and graft survival by means of microdialysis sampling, intravital video microscopy, and laser Doppler flowmetry. RESULTS: Donor pretreatment with 1 micromol erythro-9-[2-hydroxy-3-nonyl]adenine increased interstitial adenosine concentrations 5- to 10-fold, for more than 24 hr of cold storage. In LDF studies, mean donor blood flow was increased from 420 +/- 42 perfusion units (PU) to 832 +/- 52 PU and from 475 +/- 79 to 720 +/- 81 PU after reperfusion, and in intravital video microscopy studies from 247 +/- 24 to 281 +/- 39 pl/sec. There was no difference in the number of leukocytes sticking, but a significantly lower percentage of leukocytes rolling (26.1 +/- 1.9 vs. 36.5 +/- 7.5%) along the endothelial wall in the treatment group. Transplant survival after 44 hr cold storage in UW solution was 8/10 in the treatment group and 1/13 in the control group. CONCLUSIONS: Donor pretreatment with erythro-9-[2-hydroxy-3-nonyl]adenine increases survival of critically injured liver grafts. Donor or recipient treatment rather than addition of protectants to cold storage solutions are successful strategies to overcome preservation injury and possibly adverse donor factors.

Adenine↗
PubMed2000

Development of an on-line monitoring system of human keratinocyte growth by image analysis and its application to bioreactor culture.

Human keratinocytes were cultured in serum-free medium for the purpose of on-line cell growth monitoring by image analysis. The validity of a process using a newly developed video microscopy system with image analysis for growth-rate monitoring in real time was verified by the measurement of the degree of confluence of keratinocytes in T-flasks and Petriperm dishes. The growth rate of keratinocytes was calculated subsequently from the linear relationship between average degree of confluence and cell concentration. This technique was applied to the culture in the bioreactor "KERATOR" in which a special video microscopy system using a CCD camera was built. The cell concentration evaluated by image analysis agreed well with that evaluated by conventional direct cell counting after enzymatic digestion, and the on-line monitoring of the specific growth rate allowed identification of both lag- and exponential-growth phases of the culture.

Bioreactors↗
PubMed2002

Endothelin receptor function in mesenteric veins from deoxycorticosterone acetate salt-hypertensive rats.

OBJECTIVES: To identify the receptors by which endothelin-1 (ET-1) increases venomotor tone in hypertension. METHODS: Vascular reactivity to ET-1 and the selective endothelin receptor subtype B (ET(B)) agonist, sarafotoxin 6c (S6c), was studied in mesenteric blood vessels from deoxycorticosterone acetate (DOCA-salt) hypertensive and normotensive control rats. The diameter of small (< or = 280 microm) mesenteric arteries and veins was monitored in vitro using computer-assisted video microscopy. Contractions of mesenteric arteries (< or= 250 microm diameter) were also studied, using a myograph. ET-1 mRNA levels were measured in mesenteric arteries and veins using real-time RT-PCR techniques. RESULTS: ET-1-induced contractions were reduced in arteries of DOCA-salt hypertensive rats compared with those of normotensive control rats; S6c produced negligible contractions in arteries from both groups. ET-1 concentration-responses curves in arteries measured using video microscopy or a myograph were similar. ET-1 and S6c caused veins to contract, and there were no differences between responses to these agonists in tissues from DOCA-salt hypertensive rats or normotensive control rats. Studies using ET(A) and ET(B) receptor antagonists indicated that ET-1-induced venoconstriction was mediated by ET(A) receptors. Potassium chloride concentration-response curves were similar in arteries and veins from normotensive control rats and DOCA-salt hypertensive rats. ET-1 mRNA levels in DOCA-salt hypertensive rat arteries or veins were not different from those in normotensive control rat arteries and veins. CONCLUSIONS: These data indicate that ET-1 reactivity is maintained in mesenteric veins, but not arteries, in DOCA-salt hypertension. Therefore, the sustained increase in venomotor tone mediated by ET(A) receptors that is known to occur in vivo in DOCA-salt hypertensive rats is not caused by direct venoconstriction.

Animals↗
PubMed1996

Intrinsic optical signals in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space.

In the last decade, the measurement of activity-dependent intrinsic optical signals (IOSs) in excitable tissues has become a useful tool for collecting data about spatial patterns of information processing in mammalian brain and spread of excitation. Although the extent of the IOS correlates well with the extent of electrical excitation, its time course is much slower, suggesting that it does not directly monitor the electrical activity. The aim of this study was to investigate the mechanisms responsible for generation of IOSs. Coronal neocortical brain slices of juvenile rats were electrically stimulated at the border of layer VI and the white matter. The induced columnar-shaped IOSs were recorded using dark-field video microscopy. At corresponding locations, alterations in extracellular K+ concentration and extracellular space (ECS) volume were registered using ion-selective microelectrodes. After stimulation, a transient increase of extracellular K+ concentration up to 10 mM and a transient decrease of ECS volume by approximately 4% could be observed. The comparison of the time courses of these parameters yielded considerable differences between extracellular K+ concentration increase and IOS, but obvious similarities between alterations in ECS volume and IOS. To test the hypothesis that changes in IOS reflect changes in ECS, but not extracellular K+ concentration, we recorded under conditions that are known to prevent activity-induced changes in ECS, i.e., in low Cl- solutions and in the presence of furosemide. Both treatments similarly decreased stimulation-induced IOSs and alterations of ECS. However, the effect of these treatments on changes of extracellular K+ was different and did not correspond to the changes of IOS. We conclude that activity-dependent IOSs in rat neocortical slices measured by near-infrared video microscopy reveal changes in ECS. Furthermore, the pharmacological and ion substitutional experiments make it likely that activity-induced IOSs are attributable to cell swelling via a net KCI uptake and a concomitant water influx.

Animals↗
PubMed1992

[Cardiac function and the skin microcirculation in acromegaly].

The microcirculation was measured by nail-fold capillary video microscopy in 21 patients (12 men, 9 women; mean age 54.7 [29-74] years) with acromegaly. Levels of growth hormone (12.0-71.7 microU/ml) and of somatomedin C (2.4-10.5 IU/ml) were elevated in 10 patients, despite preceding treatment. Eleven patients had an increase in myocardial thickness and nine had impairment of left ventricular function, although only slight in most. Left ventricular hypertrophy was demonstrable even in the absence of hypertension. No patient had evidence of coronary heart disease. Nail-fold capillary video microscopy (capillary density, torque index, reactive hyperaemia, epidermal blood flow) failed to distinguish between successfully treated patients and those with persistently elevated growth hormone concentrations or disease duration of over 5 years. There was no evidence of inadequate capillary blood flow as a cause of abnormal function in hypertrophied organs.

Acromegaly↗
PubMed1987

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells.

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

p53-independent apoptosis mediated by tachpyridine, an anti-cancer iron chelator.

Iron is involved in essential biochemical reactions ranging from respiration to DNA synthesis. Consequently, iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. We recently described a novel iron chelator, tachypyridine [N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane], and demonstrated that it not only inhibited growth of cultured tumor cells, but was actively cytotoxic. Here we explore the mechanisms underlying tachpyridine cytotoxicity. Using several criteria, including time-lapse video microscopy, DNA staining and TUNEL assays, tachpyridine was shown to specifically induce apoptotic cell death. Further, unlike numerous cytotoxic chemotherapeutic drugs which induce apoptosis by activating p53-dependent pathways, tachpyridine-mediated cell death did not require p53 activation. Although immunoblotting revealed rapid accumulation of p53 following treatment with tachpyridine, p21(WAF1) was not induced. Further, neither cytotoxicity nor apoptosis required p53. p53 null human lung cancer H1299 cells transfected with an ecdysone-inducible p53 exhibited equivalent sensitivity to tachpyridine in the presence and absence of p53, demonstrating the lack of requirement for p53 in an isogenic cell system. Further, time-lapse video microscopy and TUNEL assays demonstrated that both p53 null and p53 wild-type cells underwent apoptotic cell death in response to tachpyridine. In addition, in 55 human cancer cell lines the mean GI(50) of tachpyridine in cells with mutant p53 was virtually identical to the GI(50) in cells with wild-type p53. These results demonstrate that tachpyridine initiates an apoptotic mode of cell death that does not require functional p53. Since over 50% of human tumors contain a functionally defective p53 that reduces sensitivity to commonly used chemotherapeutic agents, such as etoposide and cisplatin, the ability of tachpyridine to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.

Anticarcinogenic Agents↗
PubMed2006

Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7.

During bone development, remodeling, and repair, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells (MPCs) that enter into the osteoblastic lineage, and enhance the recruitment of MPCs and osteogenic cells. The process of migration is believed to be regulated, in part, by growth factors stored within the bone matrix, which are released by bone resorption. In this study, primary human mesenchymal stem cells (hMSCs) and MC3T3-E1 osteoblasts were examined for chemotaxis in response to recombinant human BMP-7 (rhBMP-7) produced in COS-7 cells (co-culture system). In order to produce BMP-7 transfected cells (BTCs), which serve as suppliers of rhBMP-7 under in vitro culture conditions, the encoding DNA was transferred into the pTARGET expression vector and introduced into COS-7 cells by conventional genetic engineering techniques. In cell culture studies, the rhBMP-7 produced in BTCs stimulated the specific activity of ALP, the production of cAMP in response to PTH, and the synthesis of osteocalcin. Migration assays were conducted with a computer-aided time-lapse video-microscopy system, to allow the rapid and precise analysis of cell migration and for the dynamic measurement of cell position and morphology. The migration distance and speed of the MC3T3-E1 cells, or hMSCs, co-cultured with BTCs, using a band-type seeding method, were significantly increased (p < 0.001), compared to those of the MC3T3-E1 cells (or hMSCs) only. In conclusion, these studies revealed that rhBMP-7 plays a role in the migration of bone-forming cells, and that the co-culture model (co-culture of bone-forming cells with BMP-7-producing cells) using a computer-aided, time-lapse video-microscopy system, is useful for the chemotactic migration assay of other chemotactic growth factors.

3T3 Cells↗
PubMed1992

Intravital video-capillaroscopy for the study of the microcirculation in psoriasis.

Intravital capillaroscopy using a video-microscopy system permits real-time imaging of the skin microvasculature with retrospective analysis of capillary dynamics. The addition of fluorescein angiography improves contrast and detects aspects of blood vessel behaviour, such as perfusion homogeneity and transcapillary solute diffusion, not detectable under native conditions. This study was performed to evaluate whether the method can be applied to the investigation of a skin disease and in particular the understanding of the role of the blood vessel in the pathogenesis of psoriasis. Results demonstrated clear differences between normal and psoriatic skin. More capillaries were red-cell perfused in both plaque and uninvolved skin compared to normal skin (P less than 0.001) and 0.01 less than P less than 0.02, respectively). The capillaries in psoriatic plaque skin were much larger than those in normal skin (P less than 0.001). The density of capillaries was not increased in plaque or uninvolved psoriatic skin, indicating expansion of existing vessels and not new vessel formation. The area of fluorescence seen around each capillary at 60 s was greater in plaque (P less than 0.001) and in uninvolved psoriatic skin (P less than 0.001) than in normal skin, indicating greater vessel transcapillary diffusion. This study confirms the value of video-microscopy as a non-invasive technique for the examination of the cutaneous microcirculation in vivo.

Adolescent↗
PubMed1992

Cerebellar target neurons provide a stop signal for afferent neurite extension in vitro.

The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.

Afferent Pathways↗
PubMed1997

Development of polarity in human erythroleukemia cells: roles of membrane ruffling and the centrosome.

Cultured human erythroleukemia (HEL) cells were used to study the genesis of polarity in single cells. HEL cells grow in suspension in culture medium, but attach and spread on fibronectin when treated with 10 nM phorbol myristate acetate. If the spread cells are treated with dibutyryl cyclic adenosine monophosphate, about 50% of the cells polarize and form very striking elongated processes. Time-lapse video microscopy showed that elongation develops in these cells because the anterior pole of the cell, which bears a small ruffled membrane, moves slowly (approximately 0.16 microgram/min) forward on the substratum elongating the posterior pole or tail behind it. Using indirect immunofluorescence we found that elongation of the tail correlates with the development of long microtubule bundles emanating from the centrosome, which is located posterior to the nucleus on the trailing side of the cell. Incubation with nocodazole, which inhibited development of the long microtubules and the elongation, resulted in a centrosome positioned over the nucleus in 45% of the cells and extension of the membrane ruffling to many points around the cell's periphery. Unexpectedly, time-lapse video microscopy demonstrated that the treated cultures also contained some smaller cells with very marked anterior ruffles and short tails. These cells moved rapidly about the culture dish (maximum 0.8 microgram/min; average 0.5 microgram/min). In these fast moving cells the centrosome was also located posterior to the nucleus. Several recent reports have stressed the importance of relocation of the centrosome to an anterior position in cells developing polarity after experimental wounding. Our results show that both striking polarization and rapid motility can occur without such a relocation. The polarity induced in the HEL cells correlates most clearly with the limitation of membrane ruffling to one region; this limitation is removed by microtubule disassembly. We therefore propose that localized ruffling is the critical first step in polarized motility generally, and that centrosomal position is related to other factors.

Bucladesine↗