Skinfold Chamber Model Research Articles

Dynamic Assessment of Angiogenesis in Renal Cell Carcinoma Spheroids by Intravital Microscopy

Renal cell cancer represents a suitable tumor model for in vivo observation of neo-angiogenesis. We used intravital microscopy and the well established dorsal skin fold chamber model to characterize neo-angiogenesis in freely implanted renal cell cancer spheroids. Tumor spheroids were implanted into dorsal skin fold chambers of 8 nude mice. At days 3, 6, 10 and 14 after implantation the newly vascularized spheroid area, density of perfused microvessels in the spheroid versus the periphery, capillary center erythrocyte velocity and capillary diameter were recorded by intravital microscopy. Video images were analyzed by a computer assisted image analysis device. After the experiments the chambers were analyzed morphologically. The model enabled quantitative analysis of microcirculation and angiogenesis in the renal cell cancer spheroids during 14 days of observation. Mean spheroid center perfused microvessel density +/- SEM increased from 3 +/- 2 to 269 +/- 21 cm.-1 on days 3 to 10 and subsequently decreased to 189 +/- 38 cm.-1 on day 14. Spheroid periphery perfused microvessel density was significantly higher throughout the experiments, attaining a mean maximum of 522 +/- 34 cm. on day 14. Mean capillary diameter decreased continuously from 14.2 +/- 0.9 to 8.4 +/- 0.4 microm. on days 3 to 14. In contrast, mean capillary center erythrocyte velocity significantly increased during 14 days of observation from 0.09 + 0.02 mm. per second on day 3 to 0.24 +/- 0.08 mm. per second on day 14. Histological analysis after 14 days revealed the spheroids as cell clusters in the upper layers of the dorsal skin fold chamber. The model is suitable for the analysis of renal cell cancer angiogenesis. Although it is heterotopic, angiogenesis in renal cell cancer spheroids mimics important characteristics of human renal cell cancer.

Microvascular response of striated muscle to metal debris

Wear products of metal implants are known to induce biological events which may have profound consequences for the microcirculation of skeletal muscle. Using the skinfold chamber model and intravital microscopy we assessed microcirculatory parameters in skeletal muscle after confrontation with titanium and stainless-steel wear debris, comparing the results with those of bulk materials. Implantation of stainless-steel bulk and debris led to a distinct activation of leukocytes combined with a disruption of the microvascular endothelial integrity and massive leukocyte extravasation. While animals with bulk stainless steel showed a tendency to recuperation, stainless-steel wear debris induced such severe inflammation and massive oedema that the microcirculation broke down within 24 hours after implantation. Titanium bulk caused only a transient increase in leukocyte-endothelial cell interaction within the first 120 minutes and no significant change in macromolecular leakage, leukocyte extravasation or venular diameter. Titanium wear debris produced a markedly lower inflammatory reaction than stainless-steel bulk, indicating that a general benefit of bulk versus debris could not be claimed. Depending on its constituents, wear debris is capable of eliciting acute inflammation which may result in endothelial damage and subsequent failure of microperfusion. Our results indicate that not only the bulk properties of orthopaedic implants but also the microcirculatory implications of inevitable wear debris play a pivotal role in determining the biocompatibility of an implant.

Effects of Ultra-Purified Polymerized Bovine Hemoglobin on Local Tissue Oxygen Tension in Striated Skin Muscle – An Efficacy Study in the Hamster

The development of hemoglobin-based oxygen carriers has been propagated for replacement of the oxygen carrying properties of red blood cells for almost one century. Using a Clark-type multi-wire oxygen surface electrode and the dorsal skin fold chamber model of the awake Syrian golden hamster, local tissue pO₂ was analyzed in the thin striated skin muscle before and after administration of an ultrapurified polymerized bovine hemoglobin solution (U-PBHb^®, Biopure Corp., Boston, Mass., USA) under the following experimental conditions: (a) hypervolemic infusion with U-PBHb at ∼10% of calculated blood volume, and (b) isovolemic exchange transfusion with U-PBHb by replacing ∼50% of calculated blood volume. Control animals of group a received equivalent treatment with either isotonic saline or dextran 60, control animals of group b received dextran 60. Local tissue pO₂ was found slightly decreased after both hypervolemic infusion and isovolemic exchange transfusion with U-PBHb, while frequency distribution curves of local tissue pO₂ were found more narrow (less values <10 mm Hg and >25 mm Hg), suggesting a more homogeneous tissue pO₂ distribution. The data thus indicate that U-PBHb slightly decreases mean tissue pO₂ after both hypervolemic infusion and isovolemic exchange transfusion which is accompanied by an effective homogenization of local tissue pO₂ distribution as compared to dextran 60.

Hirudin protects from leukocyte/endothelial cell interaction induced by extracorporeal circulation.

The clinical complications of Extracorporeal Circulation (ECC) have been linked to disturbances in the microcirculation. In order to prevent these deleterious effects, a biodegradeable agent to coat the extracorporeal circuit was tested. Intravital fluorescence microscopy was used on the hamster skinfold chamber model in permanently instrumented, awake animals. ECC was introduced via a micro-roller-pump and a silicon tube shunted between the carotid artery and the jugular vein. The ECC-tube system was coated with PEG-Hirudin-Iloprost, two additional groups received either Iloprost i.v. (0.8 mg/kg/h) or Hirudin i.v. (1 mg/kg b.w.). ECC for 20 minutes resulted in an increase in rolling and adherent leukocytes in postcapillary venules (Roller 9 to 36 [%]; Sticker 24 to 330 [n/mm2]). Use of the coated tube system reduced L/E cell interaction (Roller 9 to 24* [%], Sticker 28 to 194* [n/mm2]; *p<0.05), whereas Hirudin i.v. nearly abolished it. The protective effects of the coating and of Hirudin i.v are probably a result of an attenuated activation of the coagulation-fibrinolytic system.

Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies.

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

Microcirculatory Dysfunction Induced by Cigarette Smoking

This review deals with the deleterious effects of cigarette smroking on the microcirculation, both in terms of morphological (i.e., vessel wall injury, capillary loss) and functional aspects. The latter concerns predominantly changes in tissue perfusion and its regullatory mechanisms (i.e., reactive hyperemia, sequestration of blood cells in the microcirculation). The mechanisms of action of cigarette smoking on the microcirculation include compromised endothelial-dependent voasorelaxation, platelet aggregation, emdothelial cell dysfunction and the activation of circulating leukocytes. Through these mechanisms, cigarette smoking elicits the aggregation and adhesion of leukocytes and/or platelets to the microvascular endothelium in venules and arterioles, as assessed by intravital fluorescence microscopy in the hamster skinfold chamber model. This model has allowed us to learn more about the participation of reactive oxygen species, inflammatory mediators, and adhesion molecules in the orchestration of microcirculatory dysfunction after cigarette smoking. In the final part of this review, the clinical consequences of microcirculatory dysfunction are discussed and an outlook is offered on potential prophylactic interventions (i.e., antioxidant vitamins) aimed at abrogating the deleterious action of cigarette smoking on the microcirculation.

Microcirculatory Dysfunction Induced by Cigarette Smoking

This review deals with the deleterious effects of cigarette smoking on the microcirculation, both in terms of morphological (i.e., vessel wall injury, capillary loss) and functional aspects. The latter concerns predominantly changes in tissue perfusion and its regulatory mechanisms (i.e., reactive hyperemia, sequestration of blood cells in the microcirculation). The mechanisms of action of cigarette smoking on the microcirculation include compromised endothelial-dependent vasorelaxation, platelet aggregation, endothelial cell dysfunction, and the activation of circulating leukocytes. Through these mechanisms, cigarette smoking elicits the aggregation and adhesion of leukocytes and/or platelets to the microvascular endothelium in venules and arterioles, as assessed by intravital fluorescence microscopy in the hamster skinfold chamber model. This model has allowed us to learn more about the participation of reactive oxygen species, inflammatory mediators, and adhesion molecules in the orchestration of microcirculatory dysfunction after cigarette smoking. In the final part of this review, the clinical consequences of microcirculatory dysfunction are discussed and an outlook is offered on potential prophylactic interventions (i.e., antioxidant vitamins) aimed at abrogating the deleterious action of cigarette smoking on the microcirculation. Microcirculation (2000) 7, 367–384.

Compatibility of different colloid plasma expanders with perflubron emulsion: an intravital microscopic study in the hamster.

Perfluorocarbon-based oxygen carriers have been proposed as an adjunct to autologous blood conservation techniques during elective surgery. To date, the effects of perfluorocarbon emulsions at the microcirculatory level have not been studied extensively. In this study the effects of perflubron emulsion on the microcirculation after acute normovolemic hemodilution (ANH) were investigated using different colloid plasma expanders. The dorsal skin fold chamber model and intravital fluorescence microscopy were used for analysis of the microcirculation in the thin striated skin muscle of conscious hamsters (body weight, 40-60 g). Measurements of microvascular perfusion and leukocyte adhesion (n = 6 animals per experimental group) were made before and at 10, 30, and 60 min after ANH (to hematocrit 0.3) with either 6% hydroxyethyl starch 200/0.6 (HES), 3.5% gelatin, 5% human serum albumin (HSA), or 6% dextran 60 (DX-60) followed by intravenous injection of 3 ml/kg body weight of a 60% weight/volume perfluorocarbon emulsion based on perflubron (perfluorooctyl bromide) emulsified with egg yolk lecithin. Acute normovolemic hemodilution with HES, gelatin, or HSA followed by injection of perflubron emulsion elicited no alterations of local microvascular perfusion or leukocyte-endothelium interaction as assessed in arterioles and postcapillary venules. However, ANH with DX-60 followed by injection of perflubron emulsion led to a significant reduction of erythrocyte velocity in postcapillary venules and an increase in venular leukocyte sticking that was never observed with DX-60 alone. Hydroxyethyl starch, gelatin, and HSA are compatible with perflubron emulsion in the setting of ANH. Only DX-60 appeared to be incompatible with perflubron emulsion, as evidenced by impairment of capillary perfusion.

Morphine Attenuates Leukocyte/Endothelial Interactions

Gram-negative sepsis and subsequent endotoxic shock after surgery remain problematic in the United States and throughout the world. While morphine is widely prescribed for postoperative trauma pain management, there are reports that morphine may compromise the immune system and contribute to postoperative sepsis. The current study tested the hypothesis that morphine attenuates leukocyte rolling and sticking in both arterioles and venules via nitric oxide production. Nude mice implanted with slow-release morphine pellets were used in this study. The dorsal skinfold chamber model for intravital fluorescence microscopy on awake mice was used. Leukocyte/endothelial interactions were evaluated after bolus injection of oxidized low density lipoprotein. Morphine was found to significantly attenuate leukocyte rolling and sticking in both the arterial and venular side of the microcirculation. This attenuation was reversed by simultaneous implantation of naloxone pellets. The mechanisms of this attenuation were further investigated by administration of the nitric oxide synthase inhibitors NG-nitro-l-arginine (NOLA) and aminoguanidine (AG) in drinking water. NOLA was found to significantly reverse this morphine-induced attenuation of leukocyte rolling and sticking in both arterioles and venules. However, AG did not have the same effect. The results indicate that morphine interferes with leukocyte/endothelial cell interactions via stimulation of nitric oxide production.

The antiangiogenesis effect of interleukin 12 during early growth of human pancreatic cancer in SCID mice.

Interleukin 12 (IL-12) is a heterodimeric cytokine that exerts a potent antitumor effect through its pleiotropic actions. It was recently reported that IL-12 has also a potent antiangiogenic effect through the induction of IFN-gamma, which triggers the production of chemokines such as IP-10 that has been shown to have antiangiogenesis properties. In this study we transfected the IL-12 gene into a human pancreatic adenocarcinoma cell line (PK-1). PK-1 cells transfected with the green fluorescence protein (gfp) gene were used as positive controls. The in vitro growth curve and in vivo tumor growth of transfectants (IL-12/PK-1 and gfp/PK-1) were compared with those of parental cells. The SCID mice used in this study were administered antiasialo GM-1 Ab (100 microg, i.p., twice weekly) to deplete the remaining immunoeffector cells, NK cells. Using a skinfold chamber model, we observed and recorded tumor angiogenesis by intravital microscopy. In vitro growth of IL-12/PK-1 and gfp/PK-1 cells was not different from that of wild-type PK-1 cells (wt/PK-1). However, IL-12 transfected PK-1 cells did not develop into tumors as did the wt/PK-1 cells after subcutaneous inoculation in antiasialo GM-1 Ab administered SCID mice. The growth of IL-12/PK-1 tumors was restored in mice treated with anti-IL-12 antibody. We found that IL-12/PK-1, in contrast to gfp/PK-1 and wt/PK-1, failed to initiate an angiogenic response, as observed in the skinfold chamber model. These results indicate that the antiangiogenesis effect of IL-12 alone, without immune system involvement, is sufficient to block the growth of human pancreatic cancer.

Quantification of video-taped images in microcirculation research using inexpensive imaging software (Adobe Photoshop).

Study end-points in microcirculation research are usually video-taped images rather than numeric computer print-outs. Analysis of these video-taped images for the quantification of microcirculatory parameters usually requires computer-based image analysis systems. Most software programs for image analysis are custom-made, expensive, and limited in their applicability to selected parameters and study end-points. We demonstrate herein that an inexpensive, commercially available computer software (Adobe Photoshop), run on a Macintosh G3 computer with inbuilt graphic capture board provides versatile, easy to use tools for the quantification of digitized video images. Using images obtained by intravital fluorescence microscopy from the pre- and postischemic muscle microcirculation in the skinfold chamber model in hamsters, Photoshop allows simple and rapid quantification (i) of microvessel diameters, (ii) of the functional capillary density and (iii) of postischemic leakage of FITC-labeled high molecular weight dextran from postcapillary venules. We present evidence of the technical accuracy of the software tools and of a high degree of interobserver reliability. Inexpensive commercially available imaging programs (i.e., Adobe Photoshop) provide versatile tools for image analysis with a wide range of potential applications in microcirculation research.

Microcirculatory Dysfunction Induced by Cigarette Smoking

This review deals with the deleterious effects of cigarette smoking on the microcirculation, both in terms of morphological (i.e., vessel wall injury, capillary loss) and functional aspects. The latter concerns predominantly changes in tissue perfusion and its regulatory mechanisms (i.e., reactive hyperemia, sequestration of blood cells in the microcirculation). The mechanisms of action of cigarette smoking on the microcirculation include compromised endothelial-dependent vasorelaxation, platelet aggregation, endothelial cell dysfunction, and the activation of circulating leukocytes. Through these mechanisms, cigarette smoking elicits the aggregation and adhesion of leukocytes and/or platelets to the microvascular endothelium in venules and arterioles, as assessed by intravital fluorescence microscopy in the hamster skinfold chamber model. This model has allowed us to learn more about the participation of reactive oxygen species, inflammatory mediators, and adhesion molecules in the orchestration of microcirculatory dysfunction after cigarette smoking. In the final part of this review, the clinical consequences of microcirculatory dysfunction are discussed and an outlook is offered on potential prophylactic interventions (i.e., antioxidant vitamins) aimed at abrogating the deleterious action of cigarette smoking on the microcirculation. Microcirculation (2000) 7, 367–384.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension.

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na(+) and K(+) and urinary Na(+) and K(+) excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO(2)(-)/NO(3)(-) (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-D,L-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60-70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to approximately 70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Effects of the phlebotropic drug Daflon 500 mg on postischemic microvascular disturbances in striated skin muscle: An intravital microscopic study in the hamster

The objective of this study was to investigate the effects of the micronized purified flavonoid fraction Daflon 500 mg (90% diosmin and 10% hesperidin) on IR-induced microvascular leukocyte-endothelium interaction and leakage of the high molecular weight plasma tracer FITC-dextran (relative molecular mass, 150 kd) as assessed in the striated skin muscle of the dorsal skin fold chamber model in the hamster. Intravital fluorescence microscopy was used for analysis of microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage of FITC-dextran 150 kd in the striated skin muscle of the hamster. A toumiquet ischemia of 4 hours' duration was induced followed by reperfusion. Animals were treated with an oral administration of Daflon 500 mg (n = six) or its vehicle (5% Arabic gum solution, n = six) for 8 days at a daily dose of 30 mg/kg body weight. Measurements in the microcirculation were made before the 8-day feeding profocol before induction of ischemia and 0.5, 2, and 24 hours of reperfusion. In the absence of IR, no differences in microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage were found in Daflon 500 mg and vehicle-treated control animals before and after administration of the drugs. Induction of ischemia and reperfusion, however, elicited a significant increase in venular leukocyte rolling and sticking in vehicle-treated animals, which was accompanied by enhancement of leakage of FITC-dextran 150 kd into the perivascular tissue. Treatment with Daflon 500 mg had no effect on postischemic leukocyte rolling and sticking, and macromolecular leakage of FITC-dextran 150 kd from arterioles and postcapillary venules was significantly reduced. These data indicate that Daflon 500 mg preserves the endothelial barrier function of striated skin muscle arterioles and venules after IR, which appears to be independent of an action on postischemic intravascular leukocyte rolling and sticking.

Polynitroxylated hemoglobin-based oxygen carrier: inhibition of free radical-induced microcirculatory dysfunction

Reactive oxygen species have been identified as key mediators of leukocyte/endothelial cell interaction under various pathological conditions and diseases such as ischemia/reperfusion injury, inflammation, and after exposure to cigarette smoke. Consequently, antioxidants have been shown to successfully prevent the sequelae of these conditions, ranging from tissue infarction to atherogenesis. In this study we investigated whether, via its established superoxide dismutase-like activity, a novel polynitroxyl hemoglobin-based oxygen carrier (PNH), could affect the stimulation of leukocyte rolling and adhesion to endothelial cells in response to cigarette smoke. Using the dorsal skin fold chamber model for intravital microscopic observation of leukocyte/endothelium and −/platelet interactions in hamsters, we could demonstrate that cigarette smoke exposure elicited in control animals the rolling and adhesion of leukocytes along the endothelium of postcapillary venules and also of arterioles, as well as the formation of leukocyte/platelet aggregates. In contrast to the hemoglobin based oxygen carrier (HBOC) alone, that showed no therapeutic benefit, PNH significantly inhibited these proadhesive processes secondary to cigarette smoke. Also, PNH significantly reduced the formation of leukocyte/platelet aggregates in the blood stream of the cigarette smoke-exposed animals. These effects are not due to changes in microhemodynamic conditions, because wall shear rates remained unchanged in all three groups of animals.

Deposition of complement C3 and factor H in tissue traumatized by burn injury

Activation of complement is known to accompany burn injury. To study deposition of complement proteins within tissue traumatized by burn we employed the technique of intravital microscopy using a murine dorsal skinfold chamber model. C3, factor H, factor B, HSA, and transferrin were labeled fluorescently and injected into the tail vein of mice which had been subjected to a small third degree burn within the skin fold. Only C3 and factor H deposited within blood vessels of the traumatized tissue. Binding was specific because it occurred only in and proximal to burn sites, and neither C3 nor factor H was observed to accumulate in blood vessels of healthy tissue. Furthermore, fluorescently labelled HSA, factor B, and transferrin all failed to deposit at or around burn loci. The deposition of C3 and factor H occurred within 10 min of injury and was intravascular occurring in major blood vessels, capillaries, and post-capillary venules, with little evidence of accumulation in the interstitium. Since both C3 fragments and factor H are recognized as adhesion molecules by granulocyte receptors, these deposited proteins could promote leukocyte accumulation, thereby contributing to an initiation of an inflammatory cascade at a site of burn injury.

Microvascular response to compartment syndrome-like external pressure elevation: an in vivo fluorescence microscopic study in the hamster striated muscle.

To quantitatively assess the nature and the magnitude of the microvascular response of striated muscle tissue upon elevation of external pressure, as in compartment syndrome. Using the skinfold chamber model in Syrian golden hamsters and intravital fluorescence microscopy, we studied the individual response of the different segments of the microcirculation, i.e., the arterioles, capillaries, and postcapillary venules, in terms of vasomotor control (change of vessel diameter) and cessation of blood flow upon defined changes in external tissue pressure. The unique findings of our study are that (1) arteriolar flow ceased at mean external pressures of 25.6+/-2.4, 28.3+/-2.8, 34.5+/-4.6, and 44.4+/-6.8 mm Hg in vessels with diameters of less than 20, 20 to 40, 40 to 60, and greater than 60 microm, respectively, without signs of spasm or collapse even at a pressure maximum of 70 mm Hg, whereas (2) in venules the increase of external pressure was associated with a diameter reduction ranging from 5 to 25% with cessation of blood flow at mean external pressures between 27 and 33 mm Hg. Blood flow ceased in 50% of the muscle capillaries already at an external pressure of 12 mm Hg. Thus, at distinct external pressure levels venous and capillary blood flow ceased, but arterioles were still capable of carrying flow, which was directed along arteriolo-arteriolar "thoroughfare" channels. To restart blood flow, external pressure had to be decreased by 9, 11, 15, and 17 mm Hg in arterioles with diameters of less than 20, 20 to 40, 40 to 60, and greater than 60 microm, and by approximately 9 mm Hg in venules regardless of vessel diameter. Capillary blood flow was found to be restored at a mean reduction of external tissue pressure of approximately 4 mm Hg. Our study disproves the critical closing theory but complies-in particular because of the supposed constriction-induced increase of venular resistance-with the hypothesis of reduced arteriovenous pressure gradients as the cause of flow cessation in compartment syndrome. The necessity of a substantially increased perfusion pressure gradient to restart blood flow in arterioles, capillaries, and venules confirms the existence of yield stress in these microvessels. The high susceptibility of capillaries to elevated external pressure indicates the necessity of early fasciotomy to restore impaired nutritive circulation in cases of compartment syndrome.

Effects of cyclosporine A on the process of vascularization of freely transplanted islets of Langerhans.

We studied in vivo the effect of cyclosporine A (CsA) on both pancreatic islet vascularization and microvascular perfusion using intravital fluorescence microscopy and the dorsal skinfold chamber model in Syrian golden hamsters. Syngeneic transplantation was performed in order to exclude allograft- or xenograft-induced microvascular alterations. To study the effect of CsA on islet angiogenesis and vascularization, animals received 20 mg/kg CsA daily from day 0 until day 14 after transplantation (group A). To study toxic effects of CsA on islet microcirculation, the grafts were allowed to vascularize without immunosuppression, and 20 mg/kg CsA was given daily from day 10 until day 20 after transplantation (group B). Quantitative analysis of the process of islet vascularization in group A revealed a functional capillary density (FCD) of 515.6+/-72.7 cm(-1) at day 6 after transplantation without further increase until day 14 (504.3+/-16.7 cm(-1)). Islet transplants which were not treated with CsA during the process of angiogenesis/vascularization (group B) demonstrated a slightly but significantly (P<0.05) higher FCD (604.7+/-42.5 cm(-1)) at day 14 after transplantation, indicating slightly improved vascularization when compared to transplants of group A. Additional CsA treatment of these islet grafts until day 20 did not induce derangements of microvascular perfusion (601.2+/-67.0 cm(-1)), indicating that the immunosuppressive, drug has no toxic/detrimental effects on the transplants nutritional blood supply. We conclude that CsA only slightly alters the process of final vascularization of freely transplanted islets, and does not deteriorate nutritive perfusion of completely vascularized grafts.

Attenuation of postischemic reperfusion injury in striated skin muscle by diaspirin-cross-linked Hb.

Hemoglobin-based oxygen carriers have been suggested to enhance the formation of oxygen free radicals, especially under conditions of ischemia-reperfusion (I/R), in which activation and adhesion of leukocytes play a pivotal role for propagation of reperfusion injury. This study investigates the effects of the hemoglobin-based oxygen carrier diaspirin-cross-linked hemoglobin (DCLHb) in an I/R model of hamster striated skin muscle. The dorsal skinfold chamber model in the awake Syrian golden hamster was used for analysis of the microcirculation and local tissue PO2 in striated skin muscle utilizing the technique of intravital fluorescence microscopy and a multiwire platinum surface (Clark type) electrode. Measurements were made before 4 h of pressure-induced ischemia and at 0.5, 2, and 24 h of reperfusion. Animals were treated with 5 ml/kg body wt of either 10% DCLHb (n = 8), 6% Dextran 60 (Dx-60; 60 kDa, n = 8), or 0.9% NaCl (n = 7), which was given intravenously 15 min before reperfusion. In animals treated with DCLHb or Dx-60, a significant decrease of leukocytes rolling along and sticking in postcapillary venules, associated with a recovery of functional capillary density and red blood cell velocity, was observed compared with saline-treated controls. In the early reperfusion period (0.5 h), DCLHb and Dx-60 efficiently restored local tissue PO2, whereas tissue PO2 decreased from 18.3 +/- 1.9 to 15.3 +/- 5.3 mmHg in 0.9% NaCl-treated animals. Electron microscopic analysis of the postischemic tissue at 24 h of reperfusion revealed markedly reduced tissue damage in animals treated with DCLHb compared with Dx-60 or isotonic saline. These results indicate that DCLHb attenuates postischemic reperfusion injury of striated skin muscle, presumably through alterations of leukocyte-endothelial cell interactions.

Striated muscle microvascular response to zymosan-induced generalized inflammation in awake hamsters.

The effect of zymosan-induced generalized inflammation on the microcirculation of distant striated skin muscle was studied for a 12 day period in awake Syrian golden hamsters (n = 18) using the dorsal skinfold chamber model and intravital fluorescence microscopy. Intraperitoneal zymosan exposure (125 mg/100 g body weight) induced significant nutritive perfusion failure in the distant striated muscle tissue at Day 1 without complete recovery over the 12 day observation period, as indicated by the marked reduction of functional capillary density when compared with both baseline values and values of sham-treated control animals. Moreover, intraperitoneal zymosan exposure induced endothelial disintegration, as demonstrated by the continuous increase of macromolecular leakage throughout the 12 days of observation. Strikingly, zymosan did not induce significant leukocyte adherence to the endothelial lining of postcapillary and collecting venules of the striated muscle tissue. Thus, we conclude that in this model of generalized inflammation nutritive perfusion failure and loss of endothelial integrity in distant striated muscle is not mediated by activated leukocytes, but must rather be attributed to direct toxic effects of mediators, elicited by the local (intraperitoneal) zymosan challenge and systemically released.