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Capillary reperfusion after L-arginine, L-NMMA, and L-NNA treatment in cheek pouch microvasculature.

The effects of arginine (L-arg), promoter of nitric oxide (NO) production and NO synthesis inhibitors, NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine (L-NNA), on arteriolar responses and capillary perfusion after 30 min ischemia were studied in the cheek pouch preparation under pentobarbital anesthesia and intravenous drug infusion. Capillary density, venular leukocyte sticking, and vessel diameters were investigated by fluorescence microscopy. Damage due to photoactivation of intravascular dyes was investigated by injecting fluorescent dextran 150,000 MW prior to and after ischemia reperfusion. No difference was found indicating that effects were independent from exposure time to photoactivated dyes. Capillary perfusion reduction was always present after reperfusion in untreated, L-NMMA-treated, and L-NNA-treated animals, with increased venular leukocytes adhesion. Arteriolar vasomotion was induced by L-NMMA treatment. Capillary perfusion recovered in L-arg-treated hamsters, where capillary blood flow velocity was lower than in L-NMMA group and the number of adhering leukocytes was lower than in untreated controls, L-NMMA, and L-NNA groups. It is concluded that L-arg determines perfusion with increased blood flow heterogeneity while inhibition of NO preserves capillary perfusion causing appearance of vasomotion in the arterial network.

Animals↗

Novel neuropeptide Y receptor antagonists block vasoconstriction in the hamster cheek pouch microcirculation.

We investigated the efficacy of novel neuropeptide Y (NPY) antagonists to inhibit the microcirculatory dynamics of NPY in the hamster cheek pouch microcirculation using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter served as an index of vasomotor alterations. Fluorescein isothiocyanate-labeled Dextran 150 served as a tracer for measurements of macromolecular transport. GW 383 and GW 1229, two novel NPY receptor antagonists, were applied topically in separate experiments. Pretreatment with 10(-5), 10(-6) and 10(-7) M GW 383 and with 10(-6) and 10(-8) M GW 1229 attenuated the vasoconstriction induced by 10(-7) M NPY in a dose-dependent manner. Furthermore, pretreatment with 10(-7) and 10(-8) M GW 1229 significantly inhibited the 10(-9) M NPY-induced vasoconstriction. At these doses, the NPY antagonists did not alter microvascular permeability. Our results demonstrate that the novel NPY antagonists inhibit the vasoconstriction induced by NPY in the hamster check pouch microcirculation. We suggest that the inhibition is due to binding of antagonists to Y1-type NPY receptors.

Animals↗

Red blood cell velocity and volumetric flow assessment by enhanced high-resolution laser Doppler imaging in separate vessels of the hamster cheek pouch microcirculation.

An enhanced high-resolution laser Doppler imager (EHR-LDI), configured to fit the demands of a measurement area containing separate microvessels, was evaluated for perfusion measurements in hamster cheek pouch preparations during ischemia, reperfusion, and pharmacologically induced vasodilation and vasoconstriction. Measurements in separate microvessels where the laser beam was smaller than the vessel diameter were referred to as red blood cell (RBC) velocity estimates, as previously validated in vitro, whereas a relative flow index, RFI (mean RBC velocity/tissue area), was introduced as a volumetric flow measure. Microvessel diameter and RBC velocity changes during ischemia, reperfusion, as well as during vasoconstriction and vasodilation correlated to the data obtained from the microscope. Correspondingly, during the described provocations anticipated volumetric flow changes were registered as changes in the RFI. When data on intravessel RBC velocity profiles are presented they reflect a parabolic flow profile usually seen in this size microvessel. The EHR-LDI appears a promising tool for investigation of the microvasculature, as it almost simultaneously provides information on relative changes of both in vivo RBC velocity and volumetric flow (RFI), although the latter estimate needs to be further refined.

Adenosine↗

Platelet-activating factor modulates microvascular dynamics through phospholipase C in the hamster cheek pouch.

We studied the interactions between platelet-activating factor (PAF) and phospholipase C (PLC) in the modulation of microvascular responses in the hamster cheek pouch using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter and in integrated optical intensity (IOI, an index of vascular permeability) were measured. Fluorescein-isothiocyanate-labeled dextran 150 (FITC-Dx 150) served as a tracer for macromolecular transport. 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5,-dione (U-73122), two PLC inhibitors, were applied topically in separate experiments. PAF at 10(-7) M elevated IOI from baseline to a mean +/- SEM value of 70. 7 +/- 8.9 units. Pretreatment with 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated the maximal increment in mean IOI (+/-SEM) induced by PAF at 10(-7) M to mean +/- SEM values of 30.6 +/- 6.5, 39.3 +/- 6.0, 12.1 +/- 4.8, and 41.5 +/- 6.0, respectively. The simultaneous vasoconstrictor action of 10(-7) M PAF was expressed as the experimental-to-baseline ratio, with the baseline diameter adjusted to a value of 1. PAF constricted the arterioles to a mean +/- SEM ratio of 0.30 +/- 0.07. Pretreatment with the PLC inhibitors NCDC at 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated 10(-7) M PAF-induced vasoconstriction to mean +/- SEM diameter ratios of 0.55 +/- 0.05, 0. 48 +/- 0.06, 0.55 +/- 0.08, and 0.58 +/- 0.06, respectively. Our results demonstrate that PLC is an element of the biochemical pathway involved in PAF modulation of microvascular permeability and in PAF modulation of arteriolar diameter.

Administration, Topical↗

Increased viscosity is protective for arteriolar endothelium and microvascular perfusion during severe hemodilution in hamster cheek pouch.

We hypothesized that during severe hemodilution (SH), i.e., hemodilution beyond 50%, the reduced conditions of shear stress result in endothelium dysfunction and subsequent vasoconstriction. To evaluate the endothelial responses associated with the reduction of hematocrit we tested the responsiveness of arterioles to acetylcholine. Infusion with high-molecular-weight dextran 500,000 following SH was characterized by laser Doppler flowmetry (LDF), total perfused capillary length (TPCL), vessel diameter, and red blood cell (RBC) velocity as visualized by fluorescence microscopy in the hamster cheek pouch. Hemodilution was performed by blood removal and simultaneous infusion of 6% dextran 70. LDF increased significantly during hemodilution in arterioles and in venules (100 +/- 20 vs 37 +/- 11 and 34.2 +/- 3.5 vs 28.6 +/- 4.0 perfusion units, PU, respectively). During the final step of hemodilution LDF decreased significantly to 12 +/- 4.5 PU in arterioles and 6.2 +/- 1.5 PU in venules, which correlated with the decrease of arteriolar RBC velocity. Arterioles constricted and TPCL decreased significantly (-35 +/- 5.3%). Hyperviscosity infusion significantly raised arteriolar and venular LDF to 184 +/- 15 and 40.2 +/- 3.5 PU, arterioles dilated, RBC velocity, TPCL, and mean blood pressure. There was an impairment of endothelial-dependent dilation that was not present in the group with dextran 500, which suggests that viscosity was involved in the development of vasoconstriction during SH. In conclusion, an increase in plasma viscosity has beneficial effects on the microcirculation during SH that might preserve arteriolar endothelium and capillary perfusion.

Animals↗

Effects of L-NA and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in hamster cheek pouch venules.

Our objective was to study how the topical application of a nitric oxide synthase inhibitor (l-NA, Nomega-nitro-L-arginine) and a nitric oxide donor, sodium nitroprusside (SNP), could modulate leukocyte adhesion (sticking) and microvascular permeability as altered by ischemia/reperfusion (I/R) and topically applied histamine after I/R. Golden hamsters were prepared for intravital microscopy. Ischemia was induced by an inflatable silicon rubber cuff mounted around the neck of the cheek pouch prepared for intravital microscopy. Saline, L-NA, sodium nitroprusside, and histamine were applied in the superfusion solution. FITC-dextran was injected iv 30 min before initiation of ischemia as a marker of microvascular permeability. L-NA 10(-5) M inhibited both the increase in number of sticking leukocytes and the increase in vascular permeability after I/R compared with the untreated control group of hamsters. SNP neutralized this effect of L-NA on leukocytes and vascular permeability and caused arteriolar dilation at the concentration used, 10(-6) M. Both SNP and L-NA + SNP enhanced the I/R-induced macromolecular leakage. The topical application of SNP and SNP + L-NA did not modify the response to histamine after I/R compared with the untreated control group. In hamsters not subjected to I/R, histamine-induced macromolecular leakage was inhibited by L-NA and L-NA + SNP but was unchanged by SNP. It is concluded that inhibition of nitric oxide formation by L-NA reduced both leukocyte adhesion in postcapillary venules and the increase in macromolecular leakage and that a NO donor such as SNP could enhance the macromolecular leakage response to I/R.

Animals↗

A quantitative ultrastructural analysis of changes in hamster cheek-pouch epithelium treated with vitamin A.

The ultrastructural changes induced by the topical application of retinol acetate on hamster cheek pouch epithelium were evaluated using stereological analysis. Electron micrographs were prepared of the basal and superficial regions of the nucleated cell layer of the epithelium obtained from 3 treated and 3 control animals and examined at two levels of magnification. A total of 528 micrographs were analyzed using a coherent double lattice test system. Although the mean thickness of the nucleated cell layer did not change significantly after 10 days of treatment with retinol acetate the formation of keratinized squames was completely inhibited. This was paralleled by significant changes in the volume density of a number of organelles in both the basal and superficial strata. Rough endoplasmic reticulum increased significantly whereas filaments, which maintained a constant diameter of approximately 9 nm, keratohyalin granules and membrane-coating granules decreased in both strata. Desmosomes also showed a significant decrease in numerical area density in the treated tissues. In contrast, no changes were observed in the volume density of the Golgi apparatus, free ribosomes or mitochondria in the treated epithelium. It is concluded that this treatment provides an epithelium lacking all features of keratinization and may be a useful model for examining metabolic activities specifically associated with keratinization.

Animals↗

Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy.

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01 x 10(-9) cm2/sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4 x 10(-9) cm2/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.

Cell Membrane↗

Cellular and nuclear volumetric alterations during differentiation of normal hamster cheek pouch epithelium.

The aim of this investigation was to determine whether volumetric changes occurred in differentiating layers of stratified squamous epithelium. Samples of cheek pouch mucosa from 5 hamsters were obtained, prepared for electron microscopy using carefully controlled methods, and electron micrographs obtained from defined basal, spinous and granular layers of the epithelium. Stereological point counting procedures were used to determine the ratio of nucleus to cytoplasm for each of the defined cell layers. From direct measurement of nuclear profiles, major and minor axes were transformed to diameters of circles of equivalent area and it was thus possible to obtain an estimate of nuclear volume. Using the previously determined nuclear-cytoplasmic ratio, the volume of cytoplasm and hence cell volume, could be estimated for the cell layers. Between basal and granular layers, nuclear-cytoplasmic ratios decreased from 0.42 to 0.08, whereas cytoplasmic and cellular volumes increased progressively from 248 to 1052 microns 3 and from 352 to 1144 microns 3, respectively. Nuclear axial ratios were highest in the granular layers. These methods can be used in a variety of comparative ultrastructural studies of epithelia, and will also prove valuable in generating additional biological information from more conventionally presented stereological data.

Animals↗

The trifurcated SMAS flap: three-part segmentation of the conventional flap for improved results in the midface, cheek, and neck.

While SMAS surgery revolutionized facelift procedures, the single flap created by conventional dissections suffers the drawback that it can only be advanced in one direction and sutured in place under uniform tension. Division of the flap into three segments overcomes this problem and allows independent pull to be applied in different directions to the upper midface, cheek, and neck.

Adult↗

Polycations induce microvascular leakage of macromolecules in hamster cheek pouch.

The microvascular response to two polycationic proteins, poly-L-lysine (mol wt 104,000) and leukocyte elastase, was studied in the hamster cheek pouch microcirculation model. A 2-min topical application of polylysine (100 micrograms/ml) induced vigorous macromolecular leakage from venules only that declined within 30 min. A second application induced significantly less leakage. The leakage was inhibited by admixing polylysine with dextran sulfate prior to application or by giving hamsters an intravenous injection of dextran sulfate. The histamine antagonist pyrilamine did not interfere with the leakage, and only a few degranulated mast cells were found after polylysine application. No intravascular adhesion of leukocytes could be detected. Elastase (100 micrograms/ml) was deposited adjacent to venules with micropipets. The resulting leakage response was not inhibited by L658,758, an inhibitor of elastase enzymatic activity, but by dextran sulfate. These results may prove significant in light of the numerous polycationic proteins present within neutrophil granules.

Animals↗

Activation and migration of leukocytes and vascular leakage induced by serum-opsonized zymosan particles in hamster cheek pouch.

The effect of topically applied serum-opsonized zymosan (SOZ) and zymosan-activated serum (ZAS) was studied in the hamster cheek pouch. Our data suggest that early vascular leakage, evaluated by intravital fluorescence microscopy after injection of FITC-dextran, and accumulation of polymorphonuclear granulocytes (PMNGs), quantitated in fixed whole tissue specimens, induced by ZAS and SOZ were caused by complement activation, whereas the late leakages (greater than 20 min after SOZ application) was PMNG-dependent. Inhibition of prostaglandin and leukotriene synthesis by indomethacin, nordihydroguaiaretic acid (NDGA), and BW755C influence early and late vascular leakage as well as accumulation of PMNGs. These drugs also inhibited the activation of hamster PMNGs in vitro, as evaluated by chemiluminescence, in a dose-dependent manner. (-)-Terbutaline, an adrenergic beta agonist, decreased vascular leakage but had no effect on PMNG activation and accumulation.

Animals↗

Experimental paracoccidioidomycosis of hamster inoculated in the cheek pouch.

We compared the granuloma morphology and immune response of hamsters inoculated with Paracoccidioides brasiliensis (Pb) into the cheek pouch, which lacks lymphatic drainage, and into the footpad, which is rich in lymphatics. Our objective was to better understand the modulation of Pb granuloma in an immunocompetent animal inoculated in an immunologically privileged site. The humoral immune response (ELISA) and cell mediated immunity (footpad test) became positive on days 7 and 14, respectively in animals inoculated into footpad and on days 35 and 60 in animals inoculated into the pouch. Typical epithelioid granulomas were observed at both sites on day 14. The number of fungi gradually decreased from the beginning of the experiment in footpad lesions, but only after day 35 in pouch granulomas, when cell mediated immunity was detectable. The results indicate that typical epithelioid paracoccidioidomycotic granulomas may develop in the absence of a detectable immune response; however, they are incapable of controlling fungal reproduction. Lack of lymphatic drainage delays the appearance of a detectable immune response, but with time fungi escape from the pouch, elicit an immune response and reach other organs. Our results further indicate the importance of the lymphatics in the pathogenesis of paracoccidioidomycosis.

Animals↗

Anatomic and magnetic resonance imaging bases for the naso-maxillo-cheek flap technique.

A transfacial approach to the deep cranio-maxillo-facial areas by the naso-maxillo-cheek flap technique (NMCF) is indicated for the treatment of some bulky tumors of the naso-pharynx. The procedure requires precise preoperative imaging. This study presents the morphologic bases of this surgical access and the reasonable limits of the excision preoperatively determined by magnetic resonance imaging (MRI). 18 facial and skull specimens were submitted to surgical facial dismantling by the NMCF technique according to Curioni's method. The clinical application in a 66-year-old patient suffering from a neuroblastoma of the olfactory nerve extended into the naso-pharynx is presented. Pre- and postoperative MRI correlations were made in transverse, sagittal and frontal acquisitions. Several structures were preserved in the procedure: facial reliefs, inferior orbital rim and orbital floor, posterior wall of the maxillary sinus covering the pterygopalatine fossa, lateral and medial pterygoid plates and pterygopalatine ganglion with its branches, lateral facial neurovascular pedicle, teeth and soft palate. Other structures were sacrificed: arteries and nerves located at the sites of skin and mucosal incision, and at the sites of osteotomies, ie the infraorbital nerve, the distal part of the greater palatine nerve, the nerves supplying the naso-pharynx, the nasal septum and the nasal conchae, nasolacrimal groove and lacrimal canal. The NMCF technique gives wide access to the deep nasal and nasopharyngeal areas. It is essential to preserve the lateral facial neurovascular pedicle to prevent necrosis of the midface structures. Preservation of the bony architecture surrounding the osteotomy sites is of great importance to allow precise final bone reassembly. Preoperative MRI appears of paramount importance to determine the borders of the lesion and the possibility of block resection.

Aged↗

Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A.

We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells. The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy. The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release. It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release. The lectins at high concentrations quench the histamine release. This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Animals↗

Large liponecrotic pseudocyst formation following cheek augmentation by fat injection.

A case in which a growing cystic mass developed in the submandibular area, 6 months following fat injection to the cheek and mandibular area, is described. Histological examination revealed a liponecrotic pseudocyst. It is suggested that subcutaneous masses appearing in an area, which was previously injected with fat, should be excised. Diagnostic aspiration procedures may cause leakage of its oily content and possible further granulomatotic reactions and cysts.

Adipose Tissue↗

Ultrastructural and autoradiographic observations of hamster cheek pouch epithelium grown in vitro.

Epithelial outgrowths from hamster cheek pouch explants were cultured for varying periods of time up to 22 days. Growth of the epithelial sheets was monitored, employing colcemid for demonstrating mitotic activity and tritiated thymidine for DNA synthesis. Mitoses and thymidine uptake were observed among epithelial outgrowths at a considerable distance form the original explant. The epithelial nature of the growing cell sheets was confirmed, employing electron microscopic techniques. The cells exhibited the presence of tonofilaments, desmosomes, ribosomes, Golgi, mitochondria, and rough endoplasmic reticulum. The cultured explants were treated with cyclic nucleotides in order to investigate their modulatory effects on epithelial cell differentiation. Dibutyryl cAMP induced marked mitotic inhibition (46.3%) in our assay, which was increased to 57% with the addition of theophylline. Dibutyryl cGMP showed only a mild (5%) stimulatory effect on mitotic activity. Dibutyryl cAMP enhanced keratinization in the epithelial cell outgrowths with the biogenesis of keratohyalin granules, whereas dibutyryl cGMP did not produce any observable alterations.

Animals↗

Functional lower lip reconstruction with bilateral cheek advancement flaps: revisitation of Webster method with a minor modification in the technique.

Major defects of the lower lip have been repaired in many ways. Of these, some employed flaps from the chin, cheek or upper lip. Some of these procedures employed flaps without regard for the facial grooves or landmarks. Some methods required incisions through nerves supplying the orbicularis oris and the flaps used for the lower lip reconstruction. Of the many methods of reconstruction that have been reported, we believe that the Webster technique should be considered as the first choice for lower lip defects larger than 80% of the total. Because this technique interferes minimally with the sensation of the lower lip and a satisfactory cosmetic outcome with a functional lower lip is achieved, the sensation and muscle function of the upper lip are unaffected. In addition to discussing the advantages of the Webster method, in this paper we present a minor modification in the dissection of the orbicularis oris muscle in an attempt to improve the motor innervation and to provide adequate oral competence and labial functioning in expression and speaking. Motor function and innervation of the lips after reconstruction was documented by clinical findings as well as electrophysiological methods.

Aged↗