Window Chamber Research Articles

CCR2+ and CX3CR1+ Bone Marrow‐Derived Cells Differentially Regulate Microvascular Remodeling in an Inflammation/Injury Model Without Transdifferentiating into Smooth Muscle

Monocytes are generally known to regulate vascular remodeling. However, monocyte subpopulations with distinct functional roles exist, and their relative contributions to remodeling are poorly understood. Here, the relative expression of CX3CR1 and CCR2, which mark inflammatory (CCR2high/CX3CR1low) and resident (CCR2low/CX3CR1high) monocytes, was used to study the roles of these cells in regulating microvascular remodeling during injury/inflammation. Dorsal skin‐fold window chambers were implanted into wild‐type (WT) c57bl/6 mice engrafted with bone marrow‐derived cells (BMCs) from WT (control), CCR2−/−, CX3CR1−/−, or EGFP+ mice. WT control chimeras exhibited significant increases in maximum arteriolar (68%) and venular (40%) diameters from day 7 to 14. In contrast, arteriolar remodeling (‐0.8%) was completely blocked in CCR2−/− chimeras, but no change in venular remodeling was observed compared to control (37%). Interestingly, CX3CR1−/− chimeras exhibited only a partial attenuation of arteriolar (32%) and venular (14%) remodeling. EGFP+ chimeras revealed no evidence that recruited EGFP+ BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. We conclude that CCR2+ and CX3CR1+ BMCs differentially regulate microvascular remodeling during the injury/inflammation response. Because these cells do not transdifferentiate into vascular cells, it is likely that both subpopulations serve as paracrine growth factor sources. Supported by NIH HL74082.

Microvascular response to cell free Hb infusion in transgenic mouse model of sickle cell anemia

Targeting O2 delivery to anoxic regions may reduce the incidence of complete deoxygenation of the RBC and thus be an effective therapy in sickle cell disease. Knockout transgenic mice (n = 6) were treated with an infusion of MP4, a blood substitute that targets oxygen delivery to hypoxic areas (malemide polyethylene glycol‐conjugated Hb, Hemospan®, Sangart Inc., San Diego, CA) and then subjected to a hypoxic challenge (FiO2 = 0.08). Results were compared to a control group which received saline (n = 5). A 10% blood volume (BV) topload was infused (BV estimated as 6% body weight). Changes of microvessel diameter and functional capillary density (number of capillaries perfused per area of tissue) were studied in vivo using the dorsal skin fold window chamber model. Mean arterial pressure was higher after treatment during normoxia and hypoxia challenge as compared to the control group. Arterioles dilated and venules remained unchanged from baseline during hypoxia in both study groups. FCD was statistically reduced from baseline during hypoxia; however MP4 infused animals maintained FCD at a level that was statistically higher than observed with saline infusion; 60 ± 15% and 27 ± 24%, respectively. Thus MP4 treatment reduces the severity of hypoxia‐mediated decline in FCD. Mechanistically the higher FCD could be due to the higher perfusion pressure in the absence of vasoconstriction. Reduced sickling and inflammatory responses (i.e., lesser leukocyte‐adhesion) are likely concomitant mechanisms which support and enhance capillary perfusion.

Improved small volume resuscitation from hemorrhagic shock with acellular hemoglobin

Systemic and microvascular hemodynamic responses to hemorrhagic shock volume resuscitation with hypertonic saline (HTS) followed by infusion of polymerized bovine Hb (PBH) at different concentrations were studied in the hamster window chamber model, to determine role of plasma oxygen carrying capacity and vasoactivity during resuscitation. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of blood volume (BV), and a hypovolemic state was maintained for one hour. Volume was restituted by infusion of HTS (7.5% NaCl) 3.5% of BV of followed by 10% of BV of PBH at two different concentrations. Resuscitation was followed for 90 minutes and was carried out using 13 gPBH/dl [PBH13], PBH diluted to 4 gPBH/dl in albumin solution at matching colloidal osmotic pressure (COP) [PBH4], and albumin only solution at matching COP [PBH0]. Systemic parameters, microvascular hemodynamics and functional capillary density (FCD) were determined during hemorrhage, hypovolemic shock and resuscitation. PBH13 caused higher arterial pressure without reverting vasoconstriction and hypoperfusion. PBH4 and PBH0 had lower MAP and partially reverted vasoconstriction. Only treatment with PBH4 restored perfusion and FCD when compared to PBH13 and PBH0. Blood gas parameters and acid‐base balance recovered proportionally to microvascular perfusion. Tissue pO2 was significantly improved in the PBH4 group showing that limited restoration of oxygen carrying capacity is beneficial and compensates for the effects of vasoactivity characteristic of molecular hemoglobin solutions proposed as blood substitutes.

Oxygen‐independent degradation of HIF‐α via bioengineered VHL tumour suppressor complex

Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-α subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-α is carried out by the von Hippel–Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-α for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL–HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-α or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-α while preserving its E3 enzymatic activity. Using speckle variance–optical coherence tomography (sv–OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of ‘bio-tailored’ enzymes in the treatment of HIF-overexpressing tumours.

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO(3) selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by (31)P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO(3) therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO(3) therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease.

DURABILITY OF RESPONSE: THE ACHILLES HEEL OF SALVAGE COMBINATION IMMUNOTHERAPY WITH INTRAVESICAL BACILLUS CALMETTE-GUERIN AND INTERFERON-ALPHA 2B IN BLADDER CANCER

complex in cancer cell. Combination of Geldanamycin and HER2 monoclonal antibody enhanced the anticancer effects to breast cancer (Cancer Res2004), we hypothesized combination of Geldanamycin and IF7 has potential to improve the anticancer effects. Here we report that the cancer targeting chemotherapy using IF7 and GA to prostate tumor and other cancers. METHODS: Anxa1 binding peptide (IF7) was identified from peptide displaying phage library. In vivo phage targeting was determined by recovery of IF7 displaying phage. To confirm peptide binding in vitro, affinity of Anxa1 and Alexa 488 conjugated with IF7 (IF7-A488) were measured. For in vivo binding assay, IF7-A488 was visualized by dorsal skinfold window chamber and its signal was inhibited by anti-Anxa1 Antibody. Reverse sequence peptide (RQ7-A488) was used for control. For cancer treatment, IF7 was conjugated with Geldanamycin analogue; 17GMB-APA-GA (GA) and product (IF7-GA) was purified by silica gel chromatography. Drug effects were evaluated by PC3, MDA-MB-231, LLC and B16F1. Mice were divided 4 groups, Control, IF7, GA, and IF7GA. Drug toxicity study was performed by C57BL/6 mice. RESULTS: IF7 peptide showed strong specificity and affinity to Anxa1 in vitro and in vivo. Tumor sizes of IF7-GA group were significant small (p<0.05) and tumor apoptosis were increased compare to other groups. There was no major side effect in IF7-GA group. CONCLUSIONS: Our results show that intravenously injected IF7GA suppresses tumor growth in the mouse by inducing apoptosis. These results suggest the potential of annexin binding peptide for therapies

Multimodal optical imaging of microvessel network convective oxygen transport dynamics

Convective oxygen transport by microvessels depends on several parameters, including red blood cell flux and oxygen saturation. We demonstrate the use of intravital microscopy techniques to measure hemoglobin saturations, red blood cell fluxes and velocities, and microvessel cross-sectional areas in regions of microvascular networks containing multiple vessels. With these methods, data can be obtained at high spatial and temporal resolution and correlations between oxygen transport and hemodynamic parameters can be assessed. In vivo data are presented for a mouse mammary adenocarcinoma grown in a dorsal skinfold window chamber model.

POLYMERIZED BOVINE HEMOGLOBIN CAN IMPROVE SMALL-VOLUME RESUSCITATION FROM HEMORRHAGIC SHOCK IN HAMSTERS

Systemic and microvascular hemodynamic responses to hemorrhagic shock volume resuscitation with hypertonic saline followed by infusion of polymerized bovine hemoglobin (PBH) at different concentrations were studied in the hamster window chamber model to determine the role of plasma oxygen-carrying capacity and vasoactivity during resuscitation. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of blood volume (BV), and a hypovolemic state was maintained for 1 h. Volume was restituted by infusion of hypertonic saline (7.5% NaCl), 3.5% of BV, followed by 10% of BV of PBH at 2 different concentrations. Resuscitation was followed for 90 min and was carried out using 13 gPBH/dL (PBH13), PBH diluted to 4 gPBH/dL in albumin solution at matching colloidal osmotic pressure (PBH4), and an albumin-only solution at matching colloidal osmotic pressure (PBH0). Systemic parameters, microvascular hemodynamics, and functional capillary density were determined during hemorrhage, hypovolemic shock, and resuscitation. The PBH13 caused higher arterial pressure without reverting vasoconstriction and hypoperfusion. The PBH4 and PBH0 had lower MAP and partially reverted vasoconstriction. Only treatment with PBH4 restored perfusion and functional capillary density when compared with PBH13 and PBH0. Blood gas parameters and acid-base balance recovered proportionally to microvascular perfusion. Tissue PO2 was significantly improved in the PBH4 group, showing that limited restoration of oxygen-carrying capacity is beneficial and compensates for the effects of vasoactivity, a characteristic of molecular hemoglobin solutions proposed as blood substitutes.

Blood Supply in Melanoma Xenografts Is Governed by the Morphology of the Supplying Arteries

Tumor blood supply was related to the morphology of the tumor microvasculature and the supplying arteries (SAs) of A-07-GFP and D-12-GFP melanoma xenografts growing in window chamber preparations in BALB/c nu/nu mice. Blood supply and morphologic parameters were determined from first-pass imaging movies and vascular maps recorded after a bolus of 155-kDa tetramethylrhodamine isothiocyanate–labeled dextran had been administered in-travenously. Poorly supplied tumors showed microvascular networks that did not differ from those of well-supplied tumors in vessel tortuosity, diameter, and density. Conversely, the SAs of poorly supplied tumors were more tortuous and had a smaller diameter than those of well-supplied tumors, resulting in lower plasma velocities in the downstream tumor vessels. Consequently, the blood supply of A-07-GFP and D-12-GFP tumors was governed by the geometric resistance of the SAs rather than by the geometric resistance or the vessel density of the tumor microvasculature. The present study suggests that the SAs may represent an important target for physiological interventions of tumors and that it may be beneficial to focus on the tumor SAs rather than the tumor microvasculature when searching for novel therapeutic strategies for modifying tumor blood supply.

Revisiting optical clearing with dimethyl sulfoxide (DMSO)

Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering. Here we report on the ability of dimethyl sulfoxide (DMSO) alone to reduce the optical scattering of skin. We observed a threefold reduction in the scattering of skin with topical DMSO application. With an in vivo window chamber model, we observed a threefold increase in light transmittance through the preparation and enhanced visualization of subsurface microvasculature. Collectively, our data demonstrate the potential of DMSO alone to mitigate effects of scattering, which we expect will improve molecular imaging studies.

Proof-of-principle demonstration of a Mueller matrix decomposition method for polarized light tissue characterization in vivo

We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.

Microcirculatory, leukocyte/endothelium interaction and survival time effects of recombinant C-reactive protein in nonhypotensive endotoxemia in hamsters

Microcirculatory, leukocyte/endothelium interaction and 7-day survival time effects of human recombinant C-reactive protein (rCRP) associated or not with volume resuscitation were studied in the hamster window chamber model during resuscitation from nonhypotensive endotoxemia [1,2].

Drug and light dose responses to focal photodynamic therapy of single blood vessels in vivo

As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.

Microcirculatory, leucocyte/endothelium interaction and survival time effects of dobutamine in nonhypotensive endotoxemia

Microcirculatory, leucocyte/endothelium interaction and survival time effects of dobutamine in the dose of 5 μg/kg/minute associated or not with volume resuscitation were studied in the hamster window chamber model during resuscitation from nonhypotensive endotoxemia [1,2].

Analysis of dose perturbation factors of a NACP-02 ionization chamber in clinical electron beams

For well-guarded plane-parallel ionization chambers, international dosimetry protocols recommend a value of unity for electron perturbation factors in water. However, recent data published by various groups have challenged this. Specifically for the NACP-02 chamber, non-unity electron perturbation factors have already been published by Verhaegen et al (2006 Phys. Med. Biol. 51 1221–35) and Buckley and Rogers (2006 Med. Phys. 33 1788–96). Recently it was found that the mass thickness of the front chamber window can be 35% greater than is listed in the IAEA's TRS-398 absorbed dose protocol (Chin et al 2008 Phys. Med. Biol. 53 N119–26). This study therefore recalculated NACP-02 electron perturbation correction factors for energies 4–18 MeV at depths zref and R50 to determine the effect of the chamber model change. Results showed that perturbation factors at zref are fairly stable for similar chamber models but become highly sensitive to small changes at deeper depths. The results also showed some dependence on using 1 keV versus 10 keV for the transport cut-off. Additional investigations revealed that the wall perturbation factor, pwall, is strongly influenced by the chamber back wall at zref and at larger depths small changes in the positioning of the effective point of measurement cause large fluctuations in the final value. Finally, the cavity perturbation factor, pcav, was found to be primarily influenced by electron backscatter.

DNA loaded carrier preferential extravasation from tumor blood vessel

Non-viral gene delivery carriers were prepared by using DNA/polyethylenimine/polymethacrylic acid (DPP) polyplexes and its extravasation from tumor blood vessel was evaluated with mouse dorsal skin fold window chamber model. The DNA/PEI (DP) complex with a ratio of N to P (10/1) was coated with polymethacrylic acid, and the ratio of PMA to DNA complex in DNA/PEI/PMA (DPP) polyplex was fixed 0.03 (w/w). The surface charges of the DP and DPP polyplex were positive 26 and 15, respectively. The size of DP and DPP polyplex were 161 nm and 195 nm. The transfection efficiencies in HepG2 cells were about 30-fold and 20-fold higher than that in HeLa and L/C cells in the presence of 50% serum, respectively. The DPP polyplex showed a reduced erythrocyte aggregation activity and a decreased cytotoxicity in cancer cells. After being incubated 30 min, Fluorescently labelled DPP polyplex uptaken by cancer cells decreased, compared with DP by measuring flowcytometry. DPP polyplex penetrating through tumor blood vessel appeared fast and stayed longer in tumour interstitial, this fact was observed from mouse dorsal skin fold window chamber model.

Influence of poly(D,L-lactic-co-glycolic acid) microsphere degradation on arteriolar remodeling in the mouse dorsal skinfold window chamber

Poly(D,L-lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is widely used for drug delivery. However, the degradation of PLGA alters the local microenvironment and may influence tissue structure and/or function. Here, we studied whether PLGA degradation affects the structure of the arteriolar microcirculation through arteriogenic expansion of maximum lumenal diameters and/or the formation of new smooth muscle-coated vessels. Single microspheres comprised of 50:50 PLGA (521 +/- 52.7 microm diameter), 50:50 PLGA with bovine serum albumin (BSA) (547 +/- 62.2 microm), 85:15 PLGA (474 +/- 52.6 microm), or 85:15 PLGA with BSA (469 +/- 57.2 microm) were implanted into mouse dorsal skinfold window chambers, and longitudinal arteriolar diameter measurements were made in the presence of a vasodilator (10(-4)M adenosine) over 7 days. At the end of the 7-day period, the length density of all smooth muscle-coated microvessels was also determined. Implantation of the window chamber alone elicited a 22% increase in maximum arteriolar diameter. However, the addition of 85:15 and 50:50 PLGA microspheres, bearing either BSA or no protein, elicited a significant enhancement of this arteriogenic response, with final maximum arteriolar diameters ranging from 36 to 46% more than their original size. Interestingly, the influence of PLGA degradation on microvascular structure was limited to lumenal arteriolar expansion, as we observed no significant differences in length density of smooth muscle-coated microvessels. We conclude that the degradation of PLGA microspheres may elicit an arteriogenic response in subcutaneous tissue in the dorsal skinfold window chamber; however, it has no apparent effect on the total length of smooth muscle-coated microvasculature.

Percutaneous Window Chamber Method for Chronic Intravital Microscopy of Sensor—Tissue Interactions

A dorsal, two-sided skin-fold window chamber model was employed previously by Gough in glucose sensor research to characterize poorly understood physiological factors affecting sensor performance. We have extended this work by developing a percutaneous one-sided window chamber model for the rodent dorsum that offers both a larger subcutaneous area and a less restrictive tissue space than previous animal models. A surgical procedure for implanting a sensor into the subcutis beneath an acrylic window (15 mm diameter) is presented. Methods to quantify changes in the microvascular network and red blood cell perfusion around the sensors using noninvasive intravital microscopy and laser Doppler flowmetry are described. The feasibility of combining interstitial glucose monitoring from an implanted sensor with intravital fluorescence microscopy was explored using a bolus injection of fluorescein and dextrose to observe real-time mass transport of a small molecule at the sensor-tissue interface. The percutaneous window chamber provides an excellent model for assessing the influence of different sensor modifications, such as surface morphologies, on neovascularization using real-time monitoring of the microvascular network and tissue perfusion. However, the tissue response to an implanted sensor was variable, and some sensors migrated entirely out of the field of view and could not be observed adequately. A percutaneous optical window provides direct, real-time images of the development and dynamics of microvascular networks, microvessel patency, and fibrotic encapsulation at the tissue-sensor interface. Additionally, observing microvessels following combined bolus injections of a fluorescent dye and glucose in the local sensor environment demonstrated a valuable technique to visualize mass transport at the sensor surface.

Vascular effects of photodynamic and pulsed dye laser therapy protocols

Pulsed dye laser (PDL) treatment of cutaneous vascular lesions is associated with variable and unpredictable efficacy. Thus, alternative treatment modalities are needed. Previous in vitro and in vivo studies have demonstrated enhanced selective vascular destruction with benzoporphyrin derivative (BPD) monoacid ring A photodynamic therapy (PDT) followed immediately by PDL irradiation (PDT+PDL). Here, we evaluate PDT alone, PDL alone, and PDT+PDL protocols using an optimized in vivo rodent dorsal window chamber model. A dorsal window chamber was surgically installed on male Golden Syrian hamsters. BPD solution was administered intravenously via a jugular venous catheter. Evaluated interventions included: (1) Control (no BPD, no light); (2) Control (BPD, no light); (3) PDT alone (lambda = 576 nm; 25, 50, 75, or 96 J/cm2 radiant exposure; 15 minutes post-BPD injection); (4) PDL alone at 7 J/cm2 (585 nm, 1.5 milliseconds pulse duration, 7 mm spot); and (5) PDT (25 or 75 J/cm2)+PDL (7 J/cm2). Laser speckle imaging was used to monitor blood flow dynamics before, immediately after, and 1, 3, and 5 days post-intervention. Perfusion reduction on day 1 post-intervention was achieved with PDT>50 J/cm2, PDL alone, and PDT+PDL. However, by day 5 post-intervention, recovery of flow was observed with PDT alone at 50 J/cm2 (-15.1%) and PDL alone (+215%). PDT (75 J/cm2)+PDL resulted in the greatest prolonged reduction in vascular perfusion (-99.8%). Our in vivo data suggest that the PDT+PDL therapeutic protocol can result in enhanced and persistent vascular shutdown compared to PDT or PDL alone. The PDT+PDL approach has potential for considerable superficial vascular destruction and should be considered as a treatment modality for cutaneous vascular lesions. Monitoring of blood flow changes for as long as possible is crucial for accurate assessment of light-based vascular interventions.

Isovolemic exchange transfusion with increasing concentrations of low oxygen affinity hemoglobin solution limits oxygen delivery due to vasoconstriction

O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.