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PubMedDate not supplied

Evaluation of absolute cerebral blood flow by laser-Doppler scanning-- comparison with hydrogen clearance.

A major limitation of laser-Doppler (LD) flowmetry, which enables noninvasive and continuous recording of tissue perfusion, is its inability to evaluate the absolute cerebral blood flow (CBF). Using a computer-controlled micromanipulator, the LD scanning technique provides information on the brain microcirculation in many different locations, information which is not available from a single stationary probe. The purpose of the current study was to examine whether LD scanning estimates can be calibrated for the absolute CBF by comparing LD scanning with the hydrogen clearance (HC) method. In Wistar rats (n = 31) including old rats (122-123 weeks old, n = 8), the CBF was altered using the global ischemia model by bilateral carotid artery occlusion coupled with hypobaric hypotension. The CBF was determined simultaneously by the LD scanning technique and HC at each mean arterial blood pressure step, and the correlation of CBF between the two techniques was analyzed. CBF measured by LD scanning was expressed as LD units. Absolute CBF values obtained by methods were correlated (r = 0.87), and the formula to calibrate absolute CBF values from LD units was y = 1.8x - 0.6. On the other hand, in old rats the formula to calibrate the absolute values was different (y = 1.3x + 8.3, r = 0.85). The results suggest that CBF data obtained by LD scanning could be calibrated into absolute blood flow values in particular circumstances, and that LD scanning could compensate in part for the weakness of LD flowmetry.

Animals↗
PubMed2000

Primary cutaneous T-cell-rich B-cell lymphoma and Hodgkin's disease in a patient with Gardner's syndrome.

A 50-year-old patient, suffering from familial polyposis (Gardner's syndrome), initially presented with several nodules on his left arm. Histological examination revealed primary cutaneous T-cell-rich B-cell lymphoma (TCRBCL). Staging procedures failed to detect any systemic involvement. Three years after total excision of the tumours, the patient presented with a non-specific dermatitis, enlarged axillary lymph nodes and splenomegaly. Histological and immunohistochemical examination of lymph node and spleen biopsy specimens resulted in the diagnosis of Hodgkin's disease (HD) of the nodular sclerosis type. Sequence analysis of single cells micromanipulated from skin and from lymph node lesions indicated that both lymphoma infiltrates were derived from the same precursor germinal centre B-cell clone. This is a case showing a clonal relationship between TCRBCL and HD, providing support to the B-cell origin of Hodgkin and Reed-Sternberg cells.

Gardner Syndrome↗
PubMed2007

Intraocular fluid-air exchange reduces retinal ganglion cell activity.

AIM: To demonstrate the influence of intraocular fluid-air exchange on retinal ganglion cell activity. METHODS: In two cats lensectomy and vitrectomy were performed in a two-step approach 2 weeks prior to recording experiments. To measure retinal activity, up to two fiber microelectrodes were inserted via a scleral port. Precise micromanipulator positioning of the electrodes' tips onto the inner retinal surface were verified by ophthalmoscopy and simultaneous electrical recording. Neuronal activity of the retina was continuously recorded with or without visual stimulation. We maintained the intraocular pressure constant at 29 mm Hg. Fluid-air exchange and fluid refill were performed with an infusion tube integrated in the manipulator and slowly to avoid direct mechanical/stream-based damage of the retina. RESULTS: We found that fluid-air exchange leads to a rapid reduction of spontaneous as well as visually evoked retinal ganglion cell activity. The activity started to diminish within few minutes, and then it halved approximately every 3 min. Even 1 h after eye fluid refill, the retinal activity recovered only partially. CONCLUSION: Decrease in retinal activity after fluid-air exchange can be explained by mechanical stress-induced drying of the inner retinal surface and by potential changes of extracellular ion concentration. Since even moderate airstream-based fluid-air exchanges can lead to retinal damage, the surgeon should minimize the quantity of air passing open sclerotomy sites until a better understanding of the retinal effects of this essential technique in modern vitrectomy is available.

Action Potentials↗
PubMed1986

Characterization of endothelial cell transport in the developing mouse blood-brain barrier.

The cerebromicrovasculature of immature mice ranging in age from 1-24 days after birth was studied by electron microscopy. A micromanipulator apparatus enabled us to facilitate direction of a needle cannula and injection into leg veins or hearts with horseradish peroxidase (HRP) or ferritin (anionic and cationic) tracers and subsequent perfusion fixatives. Numerous HRP-filled vesiculocanalicular transport structures appearing in endothelial cells (ECs) of brain microblood vessels (MBVs) were observed in time periods ranging from 1-14 days. In addition to HRP transport across the ECs by tubulovesicular profiles, some of these structures appeared to become connected to multivesicular bodies. Between 14 and 24 days after birth, limited HRP was transported across the ECs to the basement membrane in only a few short segments of subpial arterioles. The decoration pattern with cationized ferritin (CF) on the luminal surface of the ECs depends upon whether the surface was exposed to the ligand before or after fixation. Quenching of aldehyde groups in fixed brain tissue has critical importance for the decoration pattern of CF on the luminal plasmalemmal surface. The absence of CF labeling on the delimiting membranes of plasmalemmal vesicles and tubular structures suggests that these structures represent differentiated microdomains engaged in macromolecular transport in the ECs of the developing mouse brain.

Animals↗
PubMed1980

Cytofluorometric analysis of anaphylactic secretion of 5-hydroxytryptamine and heparin from rat mast cells.

Actively sensitized rat mast cells were stimulated to secretion in vitro by means of an anaphylactic IgE antigen reaction or with polymyxin B. Release of 5-hydroxytryptamine (5-HT) and heparin from the cells and from individual granules, extruded or displaced from stimulated cells with micromanipulation, was quantitated cytofluorometrically. Several similarities in action between the two secretagogues were found. All extruded and some intracellular granules from stimulated cells lacked 5-HT, and no heparin was released from the granules. Heparin secretion from mast cells was due to granule extrusion alone. Further, the proportions between 5-HT and heparin released from mast cells indicated that amine release, too, is mainly, but not exclusively, associated with overt granule exocytosis. The results suggested that the first extruded granules are the richest in both 5-HT and heparin. Antigen-induced secretion was not as vigorous as that induced by polymyxin and, unlike the latter, followed after a short lag of about 20 sec.

Anaphylaxis↗
PubMedDate not supplied

Local modulation of intracellular calcium levels near a single-cell wound in human endothelial monolayers.

An endothelial cell monolayer with a single mechanically lysed cell was used as a model to examine the extent, kinetics, and nature of local calcium mobilization in the neighborhood of a wound. Individual endothelial cells from confluent monolayers were mechanically lysed with a minutien needle coupled to a micromanipulator while producing no observable mechanical trauma to the neighboring cells. Changes in calcium levels in individual cells surrounding the wound site were monitored by epifluorescence microphotometry with the calcium-sensitive fluorophore indo-1. Individual cells adjacent to the wound site showed a substantial increase in their intracellular calcium levels, almost as high as the calcium levels attained by ionophore controls. The magnitude of intracellular calcium mobilization in confluent monolayers decreased with distance from the wound site, and those cells located at a radius greater than seven cells from the wound site showed no change in their calcium levels. Thus, lysis of a single cell resulted in calcium mobilization in approximately 200 neighboring cells. The time necessary for intracellular calcium to reach maximum levels also increased with distance from the wound site. Calcium mobilization was partly intracellular and was inhibited by disrupting cell-cell coupling or by increasing gap junction resistance by heptanol. This mobilization was greatly attenuated in subconfluent endothelial monolayers, and it was not observed in fibroblasts or smooth muscle cells; furthermore, the effect was defective in monolayers intentionally contaminated with smooth muscle cells. This study examines the extent and possible mechanisms of local endothelial activation near a microscopic endothelial wound.

Calcium↗
PubMed1997

Artificial evolution.

Most of us know about specific biotechnologies but may be less aware of the underlying process. This essay analyzes that process and speculates on its meaning. It introduces the term artificial evolution, here defined as the controlled micromanipulation of genetic information from one generation to the next, where the first variational step is engineered and the second selection step is insured by humankind. This is qualitatively different from natural evolution. The characteristics of this artificial mode of evolution are immediacy, as opposed to Darwin's law of gradualism, transclass descent, unlike Darwin's common descent, identity, as opposed to variety, and an artificial rate of mutational change, as opposed to a natural one. It constitutes evolution out of evolution, and redoubles our ethical responsibility for the future.

Animals↗
PubMed2004

Experimental retinal vein occlusion: effect of acetazolamide and carbogen (95% O2/5% CO2) on preretinal PO2.

PURPOSE: To evaluate the variations of preretinal oxygen partial pressure (Po(2)) in normal and in ischemic postexperimental branch retinal vein occlusion (BRVO) areas, during normoxia, hyperoxia (100% O(2)), and carbogen (95% O(2), 5% CO(2)) breathing before and after intravenous injection of acetazolamide. METHODS: Preretinal Po(2) measurements were obtained in intervascular retinal areas, distant from the retinal vessels of 13 anesthetized mini-pigs with oxygen-sensitive microelectrodes (10 microm tip diameter) introduced through the vitreous cavity by a micromanipulator. The microelectrode tip was placed <50 microm from the vitreoretinal interface in the preretinal vitreous. Po(2) was measured continuously for 10 minutes under systemic normoxia, hyperoxia, and carbogen breathing. A BRVO was induced with an argon green laser, and oxygen measurements were repeated under normoxia, hyperoxia, and carbogen breathing, before and after intravenous injection of acetazolamide (500 mg bolus). RESULTS: In hyperoxia, a moderate nonsignificant preretinal Po(2) increase in both normal (DeltaPo(2) = 2.20 +/- 4.16 mm Hg; n = 25) and ischemic retinas (DeltaPo(2) = 4.30 +/- 3.57 mm Hg; n = 16) was measured in spite of a substantial increase in systemic Pao(2). Carbogen breathing induced a significant increase in systemic Paco(2) and a higher systemic Pao(2) than hyperoxia. Furthermore, it significantly increased the preretinal Po(2) in normal areas (DeltaPo(2) = 19.37 +/- 16.41 mm Hg; n = 26), and in ischemic areas (DeltaPo(2) = 14.94 +/- 8.53 mm Hg; n = 14). Intravenous acetazolamide did not affect the preretinal Po(2). Acetazolamide induced an increase of the preretinal Po(2) to a greater extent when it was associated with carbogen breathing (DeltaPo(2) = 15.15 +/- 9.15 mm Hg; n = 7) than when it was combined with hyperoxia (DeltaPo(2) = 6.96 +/- 4.49 mm Hg; n = 7). CONCLUSIONS: Carbogen breathing significantly increased preretinal Po(2) in normal and in ischemic postexperimental BRVO areas of mini-pigs. The concomitant use of acetazolamide injection and carbogen breathing or hyperoxia could restore an appropriate oxygenation of BRVO areas.

Acetazolamide↗
PubMed2004

Corneal fibroblasts respond rapidly to changes in local mechanical stress.

PURPOSE: To investigate the response of corneal fibroblasts to local changes in extracellular matrix (ECM) tension. METHODS: Rabbit and human corneal fibroblasts were plated inside fibrillar collagen matrices. After 18 to 72 hours, a glass microneedle was inserted into the ECM and either pushed toward a cell to reduce local tension, or pulled away to increase tension. Time-lapse differential interference contrast (DIC) imaging was performed both before and after needle micromanipulation. ECM displacements were quantified, and strain maps were generated by finite element modeling. In some experiments, cells were treated with the Rho-kinase inhibitor Y-27632 either 30 minutes before, or 1 hour after they were pushed with the microneedle. Changes in focal adhesion organization were also evaluated in a subset of cells expressing green fluorescent protein (GFP)-zyxin, by simultaneous fluorescent and DIC imaging. RESULTS: Pulling on the ECM resulted in initial cell elongation, followed by disengagement and retraction of pseudopodia. In contrast, pushing the ECM toward a cell induced rapid shortening (contraction), presumably since existing cellular forces were no longer counterbalanced by ECM tension. Pseudopodial extension (spreading) was then observed at both ends of the cell. The ECM was pulled inward during this secondary spreading, and rapid turnover of focal adhesions was observed along extending pseudopodia. Preincubation with Y-27632 or cytochalasin D blocked both the initial contractile and secondary spreading responses. CONCLUSIONS: Overall, the data suggest that corneal fibroblasts actively respond to increases or decreases in local matrix stress in an attempt to maintain tensional homeostasis (constant tension), and that this response may be mediated by Rho and/or Rac.

Amides↗
PubMed1984

Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes.

In this paper, we report analysis of differentiation in human hemopoietic colonies derived from a single cell. Cord blood mononulear cells and panned My-10 antigen-positive bone marrow and cord blood cells were plated in methylcellulose medium containing erythropoietin and conditioned medium. Initially, we performed mapping studies to identify candidate colony-forming cells. Subsequently, using a micromanipulator, we transferred single cells individually to 35-mm dishes for analysis of colony formation. Cellular composition of the colony was determined by identifying all of the cells in the May-Grunwald-Giemsa stained preparation. Of 150 single candidate cells replated, 63 produced colonies. The incidences of single lineage colonies included 19 erythroid, 17 monocyte-macrophage, and 9 eosinophil colonies. There were 18 mixed hemopoietic colonies consisting of cells in two, three, four, and five lineages in varying combinations. In some instances, we noted the predominance of one lineage and the presence of very small populations of cells in a second or third lineage. These results provide evidence for the single-cell origin of human multilineage hemopoietic colonies, and are consistent with the stochastic model of stem cell differentiation in man. They also indicate that restriction of the proliferative potential of committed progenitors is a stochastic process.

Bone Marrow Cells↗
PubMed2000

Microsurgery of sulcus vergeture with carbon dioxide laser and injectable collagen.

Between January 1989 and June 1998, we operated on 45 patients for sulcus vergeture. The studied population encompassed 38 women (84%) and 7 men (16%). The median age was 36 (range 12 to 71 years). The surgical technique is based on a concept of Cornut and Bouchayer according to which the dissection of the epithelium adherent to the deep subepithelial plane improves the vocal fold vibration. Dissection is performed with a single-pulsed carbon dioxide laser at 2 to 3 W with a pulse duration of 0.1 second. We use the Super-pulse microwave. The Acuspot micromanipulator provides a spot size of 250 microm at 350-mm focal length. When the vocal fold is atrophic, surgery is completed with a bovine or autologous collagen injection; the median injected quantity is 0.3 mL (range 0.1 to 0.4 mL). The epithelial microflap is redraped with fibrin glue. Voice therapy is indispensable for correcting the associated hyperkinetic dysphonia. The median postoperative follow-up period is 5 months (range 1 to 18 months). In terms of median values, the maximum phonation time improved from 9 to 13 seconds, the phonation quotient improved from 296.5 to 228.5 mL/s, and the spectral analysis distribution improved by 1 class. Stroboscopic examination reveals an improvement of the vibratory symmetry, amplitude, and wave. Subjectively, the patients describe an improved ability for vocal effort and the regression or disappearance of vocal fatigue. Although the timbre is improved, the voice often remains breathy and hoarse.

Adult↗
PubMed2005

Anatomic study of laser-assisted endoscopic cricopharyngeus myotomy.

OBJECTIVES: Laser-assisted endoscopic cricopharyngeus muscle (CPM) myotomy has been used to correct dysphagia caused by CPM dysfunction. The aim of this study was to anatomically characterize this procedure in hopes of demonstrating its safety and efficacy. METHODS: A Dohlman endoscope was used to isolate the CPM in 5 lightly preserved, thawed cadavers. A carbon dioxide laser at 10 W continuous power was used to section through the CPM in conjunction with a micromanipulator connected to an operating microscope. The specimens were then carefully dissected and photographed to demonstrate the anatomy of the pharyngoesophageal segment, including the location of the incision and the condition of the tissue planes. The CPM was harvested for histologic studies, sectioned, and prepared with modified Gomori trichrome stain. RESULTS: Gross examination of the retropharyngeal region revealed the presence of intact buccopharyngeal fascia between the lasered region and the retropharyngeal space. Histologic analysis demonstrated sectioning of the CPM with preservation of this fascia layer. Placement of the endoscope was difficult in 1 cadaver, in which we were unable to properly identify the CPM. CONCLUSIONS: The carbon dioxide laser-assisted endoscopic CPM myotomy is a potentially anatomically safe and viable procedure when properly performed. However, the potential for violation of the retropharyngeal space is real.

Cadaver↗
PubMed1999

Carbon dioxide laser microsurgery of benign vocal fold lesions: indications, techniques, and results in 251 patients.

Two hundred fifty-one carbon dioxide laser-assisted cases of microphonosurgery are reported. Our series includes 167 women (66.5%) and 84 men (33.5%), with a mean age of 41 (+/-11) years. Single lesions represent 67.8% (n = 170) of the cases, with 20% (n = 50) being nodules, 18% (n = 44) Reinke's edema, 9% (n = 23) polyps, 8% (n = 19) sulci and related lesions, 6% (n = 16) mucosal cysts, 4% (n = 10) scars, 2% (n = 4) granulomas, and 2% (n = 4) vascular corditis. The cases with 2 or 3 lesions represented 32% (n = 81). Carbon dioxide laser-assisted microphonosurgery is efficient, provided the working parameters are strictly adhered to: micromanipulator micropoint providing a 250-microm laser beam for a 400-mm working distance; 0.1-second single pulses; and maximum power of 3 W with the superpulse wave. Glutaraldehyde-cross-linked collagen remains our filling material of choice in cases of vocal fold atrophy. Fibrin glue is useful for covering the resection area and for setting the microflaps. Microphonosurgery cannot be dissociated from speech therapy, the planning and duration of which, in relation to the procedure, depend on the nature of the initial lesion. Twenty to 30 sessions are usually adequate, but 6 months may be necessary in the case of sulcus vergetures. Our operating technique is derived from the microphonosurgery procedures with cold instruments. In addition to the classic advantage with regard to hemostasis, the carbon dioxide laser micropoint seems to make the dissection of microflaps easier.

Adult↗
PubMed1993

Effects of CO2 laser irradiation in vivo on rat alveolar bone and incisor enamel, dentin, and pulp.

Previous studies have shown that a surgical 'window' can be drilled in alveolar bone for experimental manipulation of the underlying enamel organ and enamel. To determine whether similar and/or improved access could be obtained by use of the surgical capabilities of laser optics, and to note the effects of laser irradiation in vivo on the extracellular matrices and cells of bone, enamel, and dentin, tissue responses to laser-created lesions were examined histologically. Briefly, samples were prepared in which the alveolar bone along the inferior mandibular border of Wistar rats was exposed, and a continuous-wave CO2 laser equipped with a custom-made micromanipulator was used to penetrate the bone and to create lesions within the lower incisor. Animals were perfusion-fixed at either 10 min or 10 days post-treatment, and affected tissues were processed for light and transmission electron microscopy. At 10 min, all lesions consisted of a void of ablated tissue containing some organic debris. Tissues immediately surrounding the lesion were generally intact, but showed some damage, presumably resulting from elevated temperature effects. At 10 days, lesions in the bone, dentin, odontoblast layer, or pulp showed morphological evidence of tissue repair represented by the presence of cell infiltrates, new bone, or reparative dentin. In lesions that were created during the secretory stage of amelogenesis that had moved into the maturation stage, there was evidence of delayed or incomplete maturation of enamel (i.e., retention of organic matrix normally lost during maturation) related to the enamel organ affected by the laser treatment. In the bone lesion at 10 days, new bone formation was observed, while bone fragments originally created at the time of lasing were surrounded by mononuclear and large multinucleated giant cells. It is thus concluded that the application of this laser system is an alternative method for exposing unerupted dental tissues for experimental manipulation, and that laser irradiation may also be useful for the study of mineralized tissue repair.

Alveolar Process↗
PubMed1990

Argon laser stapedectomy using an endo-otoprobe system.

In summary, we believe that the endo-otoprobe system offers several advantages to the otologist. The system is safe and relatively inexpensive. The probes are designed to provide tactile feedback similar to that of standard otologic instruments and avoid the use of a cumbersome micromanipulator. There are currently shapes that are similar to a Rosen needle, Shea pick, and bayonet. We hope most otologists will find that one of the endo-otoprobes fits their particular style of surgery.

Humans↗
PubMed1991

Advanced microspot microslad for the CO2 laser.

New advances in instrumentation have facilitated the development of a second generation carbon dioxide (CO2) laser microspot micromanipulator. The 710 Acuspot has unique advantages over the previous generation of microspots. The compact design is easier to handle and has attachment points for sterile draping. The unit produces a spot size of 250 microns at a 400-mm focal length and 160 microns at 250 mm; the maximum de-focus is 3.2 mm at all focal lengths. An innovative dichroic mirror allows use of the laser unit's own HeNe laser as the aiming beam, eliminating possible aiming error introduced with a virtual image-aiming system. The dichroic mirror also allows better light transmission, resulting in a brighter field of view. As with first generation microspots, the laser beam path is coincident with the microscope optical path, eliminating parallax; this feature has been especially advantageous in pediatric and otologic cases. Having used this unit for 6 months on more than 50 patients, we now consider the use of a microspot to be our delivery system of choice for most microlaryngeal laser surgical applications.

Adult↗
PubMed1994

Early effects of cerebellopontine angle compression on rabbit distortion-product otoacoustic emissions: a model for monitoring cochlear function during acoustic neuroma surgery.

A rabbit model was developed to simulate the effects of ischemia that may occur during surgical removal of tumors involving the cerebellopontine angle or internal auditory canal. Specifically, the internal auditory artery was visualized through a posterior craniotomy and mechanically compressed for repetitive 1-minute intervals with a micromanipulator-controlled glass pipet terminating in a smooth bead. The 2f1-f2 distortion-product otoacoustic emissions were used to monitor the susceptibility of cochlear function to compressive effects. Distortion-product otoacoustic emissions were measured during discrete preblock, block, and postblock periods to determine the time course of distortion-product otoacoustic emission reduction and its return to baseline levels after rapid obstruction and resumption, respectively, of the cochlear vascular supply. Comparisons during these times indicated that preblock distortion-product otoacoustic emission levels were very stable, often varying by less than 1 dB. Additionally, distortion-product otoacoustic emissions were very sensitive to brief vascular occlusions in that, within approximately 25 seconds of blockage onset, emission levels at all frequencies decreased at rates of about -1.5 dB/second. On alleviation of the occlusion, distortion-product otoacoustic emissions rapidly and completely returned to preblock levels with a delay of about 4 seconds and recovery slopes of about 10.5 dB/second. A notable finding in some animals was that early and reproducible variations in distortion-product otoacoustic emission levels occurred within 5 to 8 seconds of internal auditory artery compression. When present, these transitory changes in distortion-product otoacoustic emission levels acted as early warning signs for vascular compromise of cochlear function.

Animals↗
PubMed2004

Microinjection of living adherent cells by using a semi-automatic microinjection system.

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.

Animal Testing Alternatives↗