Hemodynamics and tissue oxygenation after hemodilution with ultrahigh molecular weight polymerized albumin

Abstract

Background.

Compared to blood transfusion, plasma expanders (PEs) are more cost effective, have a longer shelf-life, and elicit a milder immune response. High molecular weight (MW) dextrans preserve microvascular function during extreme hemodilution. Dextrans, however, evokes negative hemostatic effects, including red blood cell (RBC) aggregation and reduce platelet adhesion, that limit their clinical use. Therefore, polymerization of human serum albumin (HSA) presents a simple strategy to increase HSA’s molecular size.

Methods.

This study was designed to test the hypothesis that polymerized HSA (PolyHSA) biophysical properties improves systemic and microvascular hemodynamics when used as a PE under anemic conditions. The study was implemented using the hamster window chamber model. Animals were first hemodiluted to 18% hematocrit (Hct) using 6% dextran 70 kDa and then to 11% Hct using either 10% PolyHSA, 10% unpolymerized HSA, or 6% dextran 70 kDa. Systemic and microvascular hemodynamics, including cardiac output (CO), mean arterial blood pressure (MAP), functional capillary density (FCD), microvascular perfusion, and oxygen tension were measured.

Results.

Posthemodilution, PolyHSA improved MAP, CO, and oxygen delivery compared to HSA and dextran. Additionally, PolyHSA improved microvascular function in terms of blood flow and FCD. Although oxygen carrying capacity is limited at 11% Hct, tissue pO₂ and oxygen delivery were higher for PolyHSA compared to HSA and dextran.

Conclusion.

PolyHSA during extreme anemia supported systemic and microvascular hemodynamics by increasing plasma viscosity without increasing vascular resistance. These findings can aid to design of studies to understand the role of the PE biophysical properties in clinical scenarios.

Plasma expanders (PEs) are used to increase, maintain, and/or restore blood volume in hemorrhage treatment or during major surgery. The use of PEs is advantageous to red blood cell (RBC) transfusion due to the absence of immunologic reactions, longer shelf-life, better cost-effectiveness, and reduced risk of infection. PEs in clinical use include crystalloids and colloids such as gelatin, dextran, hydroxyethyl starch (HES), and human serum albumin (HSA).¹ Colloids such as gelatin exhibit poor volume expansion and are frequently associated with allergic reactions and edema.² On the other hand, dextran and HES restore circulatory volume and microvascular perfusion.³ Unfortunately, both have been shown to affect blood coagulation, RBC aggregation, and renal function with prolonged usage.^4–7 Clinical studies have suggested that HES can be harmful, since more patients resuscitated with HES were treated with renal-replacement therapy due to renal failure compared to patients treated with saline.⁵ Albumin or HSA is considered a near optimal PE, since it occurs naturally in blood. It has a unique molecular size, shape and electrical charge that ensures its vascular retention.⁸ Albumin also accounts for most of the plasma colloid osmotic pressure (COP).⁹ Unfortunately, meta analysis of clinical studies of HSA indicate increased mortality in patients with compromised endothelia.¹⁰ Therefore, preventing extravasation of HSA by increasing the molecular size of HSA should reduce the HSA toxicity.

Therefore, glutaraldehyde cross-linking represents a simple cost-effective strategy to synthesize large molecular radii protein aggregates with high molecular weight (MW) and viscosity. Glu-taraldehyde can react with lysine, histidine, tyrosine, arginine, and primary amine residues, thus forming both intra- and intermolecular cross-links.¹¹, ¹² HSA has several of these residues, especially lysines, which are solvent exposed, facilitating glutaraldehyde polymerization.¹³ Therefore, increasing the cross-link density (i.e. molar ratio of glutaraldehyde to HSA) during the polymerization reaction yields high MW fractions with high solution viscosity. Payne et al. used this approach to synthesize polymers of bovine serum albumin; however, their chemistry did not yield a stable product, leaving the polymer susceptible to hydrolysis.¹² We have synthesized a chemically stabilized polymerized HSA (PolyHSA) that preserves HSAs secondary structure.¹⁴

Preoperative hemodilution is strongly recommended to reduce the risk of thromboembolic complications and to avoid homologous transfusions.¹⁵ Low MW dextrans and other PEs can be used as diluents, but they fail to preserve microvascular perfusion.¹⁶, ¹⁷ PolyHSA is a PE designed to preserve endothelial shear stress in anemic states and sustain microvascular perfusion and oxygenation.¹⁸, ¹⁹ The aim of this study was to evaluate PolyHSA PE properties during anemic conditions by analyzing the hemodynamics (systemic and microvascular) and oxygenation using the hamster window chamber model.

Materials and methods

PolyHSA Synthesis

The synthesis of PolyHSA was previously described in the literature.¹⁴ Briefly, Albuminar® (ABO Pharmaceuticals, San Diego, CA) was diluted to 25 mg/mL with phosphate buffered saline. Glutaraldehyde at 70% (Sigma Aldrich, Atlanta, GA) was then added to the HSA solution at a molar ratio of glutaraldehyde to HSA of 60:1. The polymerization reaction was incubated at 37 °C for 3 hours, then quenched with 25 ml of 1 M sodium borohydride and incubated for 30 minutes. The PolyHSA solution was subjected to diafiltration against a modified lactated Ringer’s buffer on a 100 kDa hollow fiber filter (Spectrum Labs, Rancho Dominguez, CA). The PolyHSA solution was then sterile filtered (0.2 mm). The endotoxin level of PolyHSA was below 0.5 EU/ml (Pyrogent Plus, Lonza, Walkersville, MD), and aliquots for experiments were stored at −80 ^oC.

Viscosity and COP

Viscosity was measured in a cone and plate viscometer DV-II+ (Brookfield Engineering Laboratories, Middleboro, MA). COP was measured using a 4420 membrane colloid osmometer (Wescor, Logan, UT). Viscosity is reported at a shear rate of 160 s⁻¹.

Animal preparation

Animal handling and care followed the NIH Guide for the Care and Use of Laboratory Animals and approved by UCSD Institutional Animal Care and Use Committee. Studies were performed in 55 to 65 g male Golden Syrian Hamsters (Charles River Laboratories, Boston, MA) fitted with a dorsal skinfold window chamber. The hamster window chamber model is widely used for microvascular studies in unanesthetized animals. The complete surgical technique is described in detail elsewhere.²⁰, ²¹ Briefly, the animal was anesthetized for chamber implantation with a 50 mg/kg IP injection of pentobarbital sodium. After hair removal, sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal’s back. The window chamber consisted of two identical titanium frames with a 15-mm diameter circular window (12 mm circular visible field). With the aid of backlighting and a stereom-icroscope, one side of the skinfold was removed following the outline of the window until only a thin monolayer of retractor muscle and subcutaneous skin of the opposing side remained. A cover glass was placed on the exposed skin and held in place by the other frame of the chamber. The other side of the skin remained intact. The animal was allowed at least 2 days for recovery; then its chamber was assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium and arterial (carotid) and venous (jugular) catheters (PE-50) were implanted. The catheters were filled with a heparinized saline solution (30 IU/mL) to ensure their patency at the time of experimentation. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck where they were attached to the chamber frame. An image of the dorsal skinfold window chamber preparation is presented in Figure 1A. The experiment was performed at least 24 h after catheter implantation.

Figure 1.—

A) Window chamber model. The hamster window chamber model facilitates simultaneous observation of systemic and microhemodynamic parameters over prolonged periods (>7 days) of time. The implanted titanium frames on the dorsal skinfold for microvascular studies (12 mm window) are used to study microhemodynamics in the absence of surgical trauma or anesthesia. B) Hemodilution protocol. Hemodilution was attained by means of a progressive, stepwise, isovolemic hemodilution protocol in which the RBC volume (dark bar) was continuously decreased, and the plasma volume was increased (clear bar), while maintaining total blood volume constant (represented by the dotted line). The extreme anemic state was achieved by two hemodilution exchanges (first 40% of the blood volume was exchanged followed by 35% of the blood volume) and a final exchange hemodilution step (35% of the blood volume) using either PolyHSA, HSA or Dextran 70.

Inclusion criteria

Animals were considered suitable for experiments if systemic parameters were within the normal ranges, namely: heart rate (HR) >340 beats/min, mean arterial blood pressure (MAP) >80 mmHg, systemic hematocrit (Hct) >45%, and arterial O₂ partial pressure (P_AO₂) >50 mmHg. Additionally, only animals without signs of low perfusion, inflammation, edema or bleeding in their microvasculature were included in the study.

Systemic parameters

MAP and HR were monitored continuously (MP150, Biopac System Inc., Santa Barbara, CA). Cardiac output (CO) was measured by a modified thermodilution technique.²² Blood chemistry was measured using a blood gas analyzer 248 (Bayer, Norwood, MA).

Experimental setup

The unanesthetized animal was placed in a restraining tube where it had access to wet feed during the experiment. The well ventilated restraining tube mimics hamsters’ natural burrow environment; it has a longitudinal slit from which the window chamber protrudes. The conscious animal in the tube was then affixed to the stage of a transillumination intravital microscope (BX51WI, Olympus, New Hyde Park, NY). The animal was given 20 min to adjust to the tube environment before systemic or micro-vascular parameters were measured. Images were visualized using a CCD camera (4815, COHU, San Diego, CA). Measurements were carried out using a 40′ (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective. The animal was repositioned on the microscope after each exchange to follow changes in microvascular parameters. Fields of observations and vessels were chosen for study at locations in the tissue where the vessels were in sharp focus.

Microhemodynamics

Arteriolar and venular blood flow velocities were measured online using the photodiode cross-correlation method (Photo-Diode/Velocity, Vista Electronics, San Diego, CA).²³ The measured centerline velocity (V) was corrected according to blood vessel size to obtain the mean RBC velocity.²⁴ A video image-shearing method was used to measure blood vessel diameter (D).²⁵ Blood flow (Q) was calculated from the measured values as Q=π×V (D/2).² Wall shear stress (WSS) was defined as WSS=WSR×η, where WSR is the wall shear rate given by 8VD⁻¹, and n is the plasma viscosity.

Functional capillary density

Functional capillaries are defined as capillaries with transit of at least a single RBC within a period of 45 s. Functional capillaries measurements include 10 successive microscopic fields with a total area of 0.46 mm². FCD is defined as the total length of RBC perfused capillaries divided by the area of the microscopic fields (cm⁻¹).

Microvascular pO₂

pO₂ measurements were made using phosphorescence quenching microscopy (PQM).²⁶ PQM is based on the oxygen-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complexes after pulsed light excitation. Tissue pO₂ was measured in tissue regions in between functional capillaries. PQM allows for precise localization of pO₂ measurements without subjecting the tissue to injury.

Oxygen delivery and extraction

The microvascular methodology used in our studies allows a detailed analysis of O₂ supply in the tissue. Calculations are made using equations 1 and 2:

$$O_{\mathit{2}\mathit{\text{delivery}}}=\left[\left(RB{C}_{Hb}\times \gamma \times S_A\right)\right]\times Q$$ (1)

$$O_{2extraction}=\left[\left(RB{C}_{Hb}\times \gamma \times S_{A-V}\right)\right]\times Q$$ (2)

RBC_Hb is the total hemoglobin (Hb) in the RBCs [gHb/dL_blood], γ is the O₂ carrying capacity of saturated Hb [1.34 mLO₂/g_Hb], S_A is the arteriolar blood O₂ saturation, _A-V indicates the arteriolar/venular differences in O₂ saturation and Q is the average microvascular blood flow (arterioles and venules).

Isovolemic hemodilution protocol and experimental groups

Acute anemia was induced by three isovolemic hemodilution steps. This protocol has been described in detail in previous publications and it is schematically shown in Figure 1B.²⁷ Briefly, 40% of the animal’s blood volume (BV), estimated as 7% of body weight (BW), was withdrawn and immediately restored with 6% dextran 70 kDa in two consecutive steps to achieve a systemic Hct of 18% (moderate hemodilution). After moderate hemodilution, similarities between animals were statistically verified before they were divided into three experimental groups. The hemodilution protocol was then continued by exchanging 35% of the BV with the test PE solution (extreme hemodilution). Experimental groups were defined based on the test PE solution used: i) Dextran 70, hemodiluted with 6% dextran 70 kDa (N.=6); ii) HSA, hemodiluted with 10% HSA (N.=6); or iii) PolyHSA, hemodiluted with 10% PolyHSA (N.=6). Systemic and microhemodynamic parameters were measured after a stabilization period of 15 min. All parameters were measured at three time points, namely: baseline (before any hemodilution), at moderate hemodilution (18% Hct) and at extreme hemodilution (11% Hct). Results are presented at the extreme hemodilution time point,since they reflect the effects of the test PEs. Base-line data is a combination of data from all experimental groups. All animals were euthanized at the end of the experiment.

Statistical analysis

Results are presented as the mean ± standard deviation. Values are presented relative to baseline. As the data was collected, interim analysis were implemented, and following animal care regulation at our institution, no more animals were included as statistical significance was reached. Data changes between interested time points in the same group were analyzed using analysis of variance for repeated measures (ANOVA, Kruskal-Wallis test) and followed by post hoc analyses with the Dunn’s Multiple Comparison test when appropriate. Statistics were calculated using Prism 4.01 (GraphPad, San Diego, CA). Results were considered statistically significant if P<0.05.

Results

Eighteen animals were entered into the study. All animals tolerated the protocol without any visible signs of discomfort. Systemic baseline data was obtained by combining all experimental groups

PE and blood biophysical properties

Dextran 70 and HSA have approximately equal MW (0.07 MDa) and similar COP (49 and 42 mmHg, respectively), although their viscosities are different due to the nature of the materials (2.8 and 1.4 cP, respectively). Poly-HSA was the most viscous PE: it’s four and eight times more viscous than Dextran 70 and HSA, respectively. Baseline blood and plasma viscosity (4.2±0.4 and 1.2±0.2 cP, respectively) was obtained from animals that did not enter the hemodilution protocol. Animals in the Poly-HSA group showed higher blood and plasma viscosity compared to the HSA and Dextran 70 groups (blood: 3.6±0.5, 1.7±0.2 and 2.1±0.3 cP; plasma: 2.5±0.3, 1.1±0.2 and 1.4±0.2 cP for the three groups respectively) after hemodilution. Plasma COP was not different between groups and compared to baseline (baseline COP: 17 mmHg).

Systemic parameters

Blood gas parameters are presented in Table I. After extreme hemodilution with Dextran 70, HSA and PolyHSA, the Hct and Hb concentration decreased as expected and the arterial pO₂ significantly increased compared to base-line but showed no changes between groups. Arterial pCO₂ for Dextran 70 was significantly lower compared to baseline but not to the other groups. Arterial blood pH was not different after hemodilution for any of the groups compared to baseline. Blood base excess (BE) was significantly decreased after hemodilution with Dextran 70 compared to baseline and the groups hemodiluted with HSA and PolyHSA. The MAP after hemodilution with PolyHSA was not different to baseline, while it was significantly lower for Dextran 70 and HSA compared to baseline. There were no significant changes in the HR between test solutions after extreme hemodilution. The CO was significantly higher for PolyHSA compared to baseline, and to Dextran 70 and HSA and it was significantly lower for Dextran 70 compared to baseline. Cardiac function after hemodilution restricted cardiac hydraulic power (MAPxCO) to 47% and 64% of baseline for hemodilution with Dextran 70 and HSA, respectively, whereas cardiac hydraulic power of animals’ hemodiluted with PolyHSA increased by 20% from baseline. Peripheral vascular resistance (PVR=MAP/CO) was 78% (Dextran 70: 0.50±0.12 dynes.s/cm⁵) and 72% (HSA: 0.39±0.08 dynes.s/cm⁵) of baseline for conventional PEs and 62% (PolyHSA: 0.31±0.07 dynes.s/cm⁵) of baseline for PolyHSA. Furthermore, vascular hindrance (PVR divided by blood viscosity), which accounts for the geometric contribution to the resistance, was 30% lower than baseline for PolyHSA (PolyHSA: 85±18 cm⁻³), whereas it was 56% and 78% increased from baseline for Dextran 70 (Dextran 70: 184±42 cm⁻³) and HSA (HSA: 210±57 cm⁻³), respectively.

Table I.—

Blood gas and systemic hemodynamic parameters. Values are means±SD. Baseline included all animals in the study. No significant differences were detected between the baseline values of each group or before the exchange with test solutions.

	Baseline	Dextran 70	HSA	PolyHSA
Hct, %	49.3 ± 1.4	11.1 ± 0.9†	11.4 ± 0.8†	11.6 ± 0.5†
Hb, g/dL	14.8 ± 0.9	3.7 ± 0.5†	3.8 ± 0.5†	3.9 ± 0.4†
MAP, mmHg	107.6 ± 5.8	64.4 ± 7.5†	73.4 ± 6.1†§	92.5 ± 6.4‡§
Heart Rate, bpm	418 ± 35	418 ± 41	454 ± 36	441 ± 33
CO, mL/min	18.2 ± 1.6	14.2 ± 1.9†	17.3 ± 1.5	25.6 ± 1.4†‡§
PO2, mmHg	57.9 ± 5.8	105.3 ± 16.7†	97.2 ± 9.8†	92.5 ± 7.2†
PCO2, mmHg	51.4 ± 5.0	39.1 ± 7.8†	42.4 ± 6.4	46.1 ± 6.9
pH	7.35 ± 0.03	7.32 ± 0.09	7.35 ± 0.04	7.35 ± 0.03
BE, mmol/L	3.3 ± 0.9	−4.6 ± 2.6†	−0.5 ± 1.4†§	0.9 ± 1.3‡§
Microvascular wall shear rate (WSR) and wall shear stress (WSS)
Arteriolar
WSR, L/s	789 ± 74	487 ± 56†	532 ± 57†	665 ± 69‡§
WSS, dyns/cm2	22 ± 6	8 ± 4†	8 ± 4†	17 ± 6‡§
Venular
WSR, L/s	435 ± 52	232 ± 47†	302 ± 46†	461 ± 56‡§
WSS, dyns/cm2	12 ± 4	4 ± 2†	5 ± 2†	10 ± 4‡§

Hct: systemic hematocrit; Hb: hemoglobin concentration; MAP: mean arterial blood pressure; CO: cardiac output; pO₂: arterial partial pressure of O₂; PCO₂: arterial partial pressure of CO₂; BE: base excess.

^†P<0.05 compared to baseline;

^§P<0.05 compared to Dextran 70;

^‡P<0.05 compared to HSA.

Microvascular hemodynamics

Changes in microvessel diameter and blood flow are presented in Figure 2. For the groups Dextran 70 and HSA, venular diameters were significantly lower after extreme hemodilution, while arteriole diameters were not different from baseline. The RBC velocity in arterioles and venules was significantly lower for the Dextran 70 and HSA groups. PolyHSA showed significant increases in arteriolar and venular diameters and an increase in venular velocity, compared to Dextran 70 and HSA. Consequently, arteriolar and venular blood flows were significantly higher for PolyHSA compared to the other groups and baseline. The FCD was significantly reduced in all experimental groups compared to baseline, though it was higher for PolyHSA (74±12 cm⁻¹) compared to Dextran 70 (38±16 cm⁻¹) and HSA (54±14 cm⁻¹).

Figure 2.—

Relative changes to baseline in arteriolar and venular hemodynamics after extreme hemodilution with PolyHSA, HAS and Dextran 70. The broken line represents the baseline level.

†P<0.05 relative to baseline; ‡P<0.05 compared to Dextran 70; §P<0.05 compared to HSA. Diameters (μm, mean±SD) at baseline in each animal group were as follows: PolyHSA (arterioles [A]: 65.7±8.4, N.=48; venules (V): 67.3±8.6, N.=56); HSA (A: 65.3±8.3, N.=46; V: 69.0±8.8, N.=52); and Dextran 70 (A: 65.7±7.2, N.=52; V: 65.6±7.8, N.=56). N.=number of small blood vessels studied. RBC velocities (mm/s, mean±SD) at baseline in each animal group were as follows: PolyHSA (A: 4.5±0.8; V: 2.0±0.6); HSA (A: 4.4±0.9; V: 1.9±0.7); Dextran 70 (A: 4.5±0.7; V: 2.0±0.6). Blood flow (nL/s, mean±SD) at baseline in each animal group were as follows: PolyHSA (A: 12.4±4.8; V: 5.8±2.5); HSA (A: 12.7±4.6; V: 6.0±2.2); Dextran 70 (A: 12.8±4.5; V:5.9±2.2).

Oxygen transport

Microvascular and tissue oxygen tensions are presented in Figure 3A. After extreme hemodilu tion with PolyHSA, arteriolar pO₂ was significantly increased compared to Dextran 70 and HSA. Additionally, PolyHSA significantly increased tissue pO₂ compared with Dextran 70 but was not statistically significantly different to the HSA group. Venular pO₂s were not different between groups or compared to baseline. Calculated oxygen delivery and extraction are presented in Figure 3B. PolyHSA provided superior systemic and microcirculatory oxygen delivery; it also maintained higher oxygen extraction in the microcirculation. Arterial (systemic) and microvascular oxygen delivery were significantly higher for PolyHSA compared to Dextran 70 and HSA groups. The microcirculation oxygen extraction was significantly increased for PolyHSA compared to Dextran 70 and HSA.

Figure 3.—

A) Intravascular and extravascular pO₂ after moderate hemodilution and extreme hemodilution with PolyHSA, HAS and Dextran 70. Baseline (non-hemodiluted) pO₂s for arterioles =51.8 mmHg, venules=32.7 mmHg, and perivascular tissue =21.7 mmHg. B. Arterial oxygen delivery and extraction after extreme hemodilution with PolyHSA, HSA and Dextran 70. Oxygen transport is not directly measurable; however, it can be calculated using the measured parameters.

†P<0.05 relative to baseline; ‡P<0.05 compared to Dextran 70; §P<0.05 compared to HSA.

Discussion

The principal finding of this study is that plasma expansion with PolyHSA sustained mi crocirculatory and systemic hemodynamics and also provided superior tissue oxygenation compared to unpolymerized HSA and dextran 70. After extreme hemodilution with PolyHSA, CO increased and facilitated improved systemic oxygen delivery without increasing vascular resistance. Microvascular hemodynamics were also superior for PolyHSA compared to the other PEs. Extreme hemodilution dramatically reduces blood viscosity; however, when PolyHSA was used to reduce the Hct, it increased plasma viscosity and prevented the decrease in blood viscosity compared to HSA or dextran 70. The lower decrease in blood viscosity with PolyHSA preserved MAP compared to the other PEs, while PolyHSA comparatively decreased vascular resistance, evidenced by changes in vascular hindrance and microvascular vasodilation. PolyH-SA also demonstrated superior recovery of blood pressure, microvascular blood flow, and FCD, and volume expansion during fluid resuscitation from hemorrhagic shock when compared to commercially available PEs.¹⁹

The acid-base balance after extreme hemodilution with PolyHSA was not different from baseline, while extreme hemodilution with conventional PEs produced a negative acid-base balance. Unfortunately, lactate could not be measured in our experimental approach, since the volume required to accurately quantify lactate is large relative to the animal’s blood volume. PolyHSA improved gas exchange and oxygenation mostly by improving hemodynamic conditions. FCD was reduced in all groups compared to baseline, which limits oxygenation and the washing out of metabolic wastes; however, PolyHSA sustained higher FCD compared to dextran 70 and HSA. Increased plasma viscosity during extreme hemodilution does not increase vascular resistance over the entire circulatory system, since vascular resistance is not uniformly distributed throughout the circulation.²⁸ During extreme hemodilution, the circulation appears to attempt to shunt blood flow to vital organs in response to the reduced oxygen-carrying capacity of blood via vasoconstriction. Vasoconstriction lowers hydraulic pressure and leads to capillary shutdown, because capillary pressure is no longer sufficient to maintain the patency of these conduits,²⁸ whereas vasodilation induced by increased mechanotransduction allows for the transmission of central blood pressure to the periphery.

In this study, the tissue pO₂ was lower in all groups relative to the normal tissue pO₂ of the window chamber (21±2 mmHg), since the oxygen carrying capacity of the blood was limited. However, PolyHSA increased tissue pO₂, microvascular oxygen delivery, and extraction compared to Dextran 70 and HSA. Differences in microcirculation oxygen extraction between HSA and Dextran 70 compared to PolyHSA suggest that they were unable to maintain tissue oxygenation, since oxygen extraction reflects oxygen consumption by the tissue. This is also confirmed by the oxygen extraction ratio (micro-circulation extraction/ arteriolar supply), as the HSA and dextran 70 extraction ratios were close to 100%, while the PolyHSA extraction ratio was 92%.

Study limitations

Extreme hemodilution is of limited clinical value, since the Hb concentration during surgery should be maintained at a minimum of 10 g/ dL to avoid the risk of circulatory problems and arrythmias such as atrial fibrillation.²⁹ However, extreme hemodilution to Hb concentrations below 4 g/dL are unavoidable when blood is unavailable or due to religious objections to blood transfusion. The hemodilution protocol implemented in this study can not be directly extrapolated to clinical practice, since all the animals included in this study were young and healthy. Cardiac dysfunction, coronary artery disease, and hepatic disease are all contraindications to severe hemodilution.²⁹ In addition, pulmonary emphysema and obstructive lung disease limit blood oxygenation. The improved rheological properties of diluted blood with PolyHSA may allow for superior perfusion in cardiovascular disease states via the increase of compensatory mechanisms necessary to tolerate hemodilution, although more experimental validation is required. Dextran provides stable hemodynamic response in hamsters; in humans, however dextran has hemostatic effects similar to those of von Willebrand disease, namely a decrease in platelet adhesion.³ An additional limitation of the study is the short observation period after hemodilution with PolyHSA; although PolyHSA and other PEs are only intended as acute therapeutic solutions at the low Hct.

Conclusions

In conclusion, analysis of microvascular function during anemic states provides great information about how PEs interact with the cardiovascular system. Engineering the biophysical properties of PEs has the potential to preserve microvascular perfusion and sustain tissue oxygen delivery to tissues. Although the techniques reported here for microvascular studies are difficult to translate to a clinical setting, they serve as an starting point for the design of clinical studies to understand the importance of the biophysical properties of PEs in clinical scenarios and their advantages over conventional PEs.

Key messages.

• —PolyHSA sustains microcirculatory and systemic hemodynamics as well as oxygen delivery to tissues during extreme anemic conditions.
• —During extreme anemic conditions, high viscosity PEs promote increased shear stress mediated endothelial mechanotransduction which promotes maintenance of vascular hindrance and vascular resistance.
• —Plasma expansion is an important component of medical intervention, thus understanding the effects of PE biophysical properties on blood flow and oxygen delivery will have significant implications in transfusion medicine.
• —Detailed analysis of the interaction between blood flow and the vascular system allows for the optimal design of PEs.