American Journal of Emergency Medicine (2012) 30, 1336-1346

Original Contribution

Small-volume resuscitation from hemorrhagic shock with polymerized human serum albumin

Catalina Messmer MD ^{a, b}, Ozlem Yalcin PhD ^a,

Andre F. Palmer PhD ^c, Pedro Cabrales PhD ^b,?

^aDepartment of Otorhinolaryngology, Head and Neck Surgery, University of Munich (LMU), Campus Grosshadern,

Munich 81377, Germany

^bDepartment of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA

^cWilliam G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA

Received 16 August 2011; revised 16 September 2011; accepted 19 September 2011

Abstract Human serum albumin (HSA) is used as a plasma expander; however, albumin is readily eliminated from the intravascular space. The objective of this study was to establish the effects of various-sized polymerized HSAs (PolyHSAs) during small-volume resuscitation from hemorrhagic shock on systemic parameters, microvascular hemodynamics, and functional capillary density in the hamster window chamber model. Polymerized HSA size was controlled by varying the cross-link density (ie, molar ratio of glutaraldehyde to HSA). Hemorrhage was induced by controlled arterial bleeding of 50% of the animal’s blood volume (BV), and hypovolemic shock was maintained for 1 hour. Resuscitation was implemented in 2 phases, first, by infusion of 3.5% of the BV of hypertonic saline (7.5% NaCl) then followed by infusion of 10% of the BV of each PolyHSA. Resuscitation provided rapid recovery of blood pressure, blood gas parameters, and microvascular perfusion. Polymerized HSA at a glutaraldehyde-to-HSA molar ratio of 60:1 (PolyHSA_60:1) provided superior recovery of blood pressure, microvascular blood flow, and functional capillary density, and acid-base balance, with sustained volume expansion in relation to the volume infused. The high molecular weight of PolyHSA_60:1 increased the hydrodynamic radius and solution viscosity. Pharmacokinetic analysis of PolyHSA_60:1 indicates reduced clearance and increased circulatory half-life compared with monomeric HSA and other PolyHSA formulations. In conclusion, HSA molecular size and solution viscosity affect central hemodynamics, microvascular blood flow, volume expansion, and circulation persistence during small-volume resuscitation from hemorrhagic shock. In addition, PolyHSA can be an alternative to HSA in pathophysiological situations with compromised vascular permeability.

(C) 2012

Introduction

* Corresponding author. Tel.: +1 858 534 5847.

E-mail address: [email protected] (P. Cabrales).

Colloidal plasma expanders (PEs) are used to increase or maintain intravascular blood volume (BV). PPE increase plasma oncotic pressure (colloid osmotic pressure [COP]) and move fluid from the interstitial space into intravascular compartment. However, they further decrease erythrocyte

0735-6757/$ - see front matter (C) 2012 doi:10.1016/j.ajem.2011.09.018

concentration and blood viscosity. Colloids such as gelatin, dextran, hydroxyethyl starch (HES), and human serum albumin (HSA) have different advantages and disadvantages [1,2]. Gelatin exhibits poor volume expansion and is associated with Allergic reactions and edema. Dextran and HES are able to effectively restore circulatory volume and microvascular perfusion. Unfortunately, dextran and HES have both been shown to inhibit coagulation, promote aggregation of red blood cells , and can even induce renal failure [3]. Conversely, HSA is a naturally produced protein and accounts for more than 50% of the total plasma proteins. Albumin is the major contributor to plasma COP; it binds toxic species and drugs and consumes free radicals [4,5]. Despite all of these beneficial properties, clinical trials and meta-analyses of HSA have provided some contradicting results, since the use of HSA has been shown to be generally safe [1,6]. However, the negative responses to HSA are likely caused by extravasation in situation with increased vascular permeability (ie, shock, sepsis, burns, etc) and associated with edema and tissues exposed to potentially toxic molecules bound to HSA [7]. Therefore, there is a need to develop a new generation of effective PEs based on HSA with increased vascular retention.

Inspired by this need, glutaraldehyde-based cross-linking represents a simple and cost-effective strategy to synthesize large protein with high molecular weight (MW). Glutaral- dehyde is able to react with lysine, histidine, tyrosine, and arginine residues on the surface of HSA, forming both intramolecular and intermolecular cross-links [8]. The abundance of these residues on the surface of HSA facilitates its polymerization with a simple and effective chemistry. Polymerized HSA (PolyHSA) MW is controlled by simply varying the molar ratio of glutaraldehyde to HSA. Payne [9] was the first to increase the size of bovine serum albumin (BSA) by reacting it with various concentrations of glutaraldehyde [10]. However, the resultant Schiff bases in the polymerized BSA molecules were not chemically stabilized, leaving the polymerized BSA susceptible to hydrolysis back into BSA and glutaraldehyde. Recently,

PolyHSA was synthesized with chemically stabilized Schiff bases, preserving HSA secondary structure, increasing solution viscosity, and decreasing COP compared with unpolymerized HSA [9,10]. Therefore, glutaraldehyde cross- linking represents an attractive option to increase HSA size and intravascular retention.

The present study was carried out to test the hypothesis that the biophysical properties of PolyHSA (size, viscosity, and COP) affect the recovery of systemic and microvascular hemodynamics, volume expansion, and plasma protein retention during small-volume resuscitation from hemor- rhagic shock model. To achieve this objective, our experi- mental hamster model was subjected to a hemorrhage of 50% of the animal’s BV, followed by 1 hour of hypovolemic shock (Fig. 1). The resuscitation was implemented in 2 steps. The initial phase consisted of the infusion of Hypertonic saline (7.5% NaCl) at 3.5% of BV, 5 minutes after a 10% of the BV infusion of the test solutions (PolyHSA) was administered. The volume resuscitation solutions evaluated in this study were adjusted to a protein concentration of 10 g/dL, namely, HSA (unpolymerized HSA), PolyHSA_24:1 (molar ratio of glutaraldehyde to HSA, 24:1), PolyHSA_60:1 (molar ratio of glutaraldehyde to HSA, 60:1), and PolyHSA_94:1 (molar ratio of glutaraldehyde to HSA, 94:1).

Methods

PolyHSA synthesis

Human serum albumin polymerization has been previ- ously published [10]. Briefly, Albuminar (ABO Pharma- ceuticals, San Diego, CA) was diluted to 25 mg/mL with phosphate-buffered saline, and 70% glutaraldehyde (Sigma Aldrich, Atlanta, GA) was then added to HSA solutions at the following molar ratios of glutaraldehyde to HSA: 24:1, 60:1, and 94:1. The polymerization reaction was incubated at 37?C for 3 hours, quenched with 25 mL of 1 mol/L

Small volume resuscitation model

Hemorrhage

50% of BV

Fluid Resuscitation HTS 3.5% of BV followed by HSA or PolyHSA of 10% of BV

Baseline

Shock Resuscitation

Time points

Time (min)

BL

Sh

R60

R90

90

60

5

Fig. 1 Hemorrhage was induced by withdrawing 50% of the animal’s BV. Hypovolemia was maintained for 1 hour. Small-volume fluid resuscitation was implemented initially by infusion of 3.5% of the BV with HTS, and 5 minutes after HTS, followed by infusion of 10% of the BV with PolyHSA resuscitation fluids. The resuscitation fluids consisted composed of 3 different formulations of PolyHSA at 10 g/dL, namely, PolyHSA_24:1 (synthesized at a 24:1 molar ratio of glutaraldehyde to HSA), PolyHSA_60:1 (synthesized at a 60:1 molar ratio of glutaraldehyde to HSA), and PolyHSA_94:1 (synthesized at a 94:1 molar ratio of glutaraldehyde to HSA). Parameters were analyzed before hemorrhage (baseline), after hemorrhage (shock, 60 minutes after hemorrhage induction), and at 60 and 90 minutes after fluid resuscitation. BL indicates baseline; Sh, shock; R60, resuscitation after 60 minutes; R90, resuscitation after 90 minutes.

resuscitation groups“>sodium borohydride, and incubated for 30 minutes. Polymerized HSA solutions were subjected to diafiltration against a modified lactated Ringer‘s buffer (115 mmol/L NaCl, 4 mmol/L KCl, 1.4 mmol/L CaCl₂, 13 mmol/L NaOH, 27 mmol/L sodium lactate, and 2 g/L N-acetyl-L- cysteine) on a 100 kDa hollow fiber filter (Spectrum Labs, Rancho Dominguez, CA) a total of 4 times. Polymerized HSA solutions were then filtered through 0.2-um filters and stored at -80?C until needed.

Light scattering

The absolute MW distribution of HSA/PolyHSA solutions was measured using a size exclusion chromatography column (Ultrahydrogel linear column, 10 um, 7.8 x 300 mm; Waters, Milford, MA) driven by a 1200 high performance liquid chromatography pump (Agilent, Santa Clara, CA), controlled by Eclipse 2 software (Wyatt Technology, Santa Barbara, CA) connected in series to a DAWN Heleos (Wyatt Technology) light-scattering photometer and an OptiLab Rex (Wyatt Technology) differential refractive index detec- tor. All data were collected and analyzed using Astra 5.3 software (Wyatt Technology).

Viscosity, COP, and protein concentration

The viscosity of HSA/PolyHSA solutions was measured in a cone/plate viscometer DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories, Middleboro, MA) at a shear rate of 160 per second, whereas the COP was measured using a Wescor 4420 Colloid Osmometer [11] (Wescor, Logan, UT). Protein concentration was determined using spectrophotometric (Lambda 20; Perkin-Elmer, Nor- walk, CT) analysis in the UV domain (280 nm).

Animal preparation

Investigations were performed in 55- to 65-g male Golden Syrian Hamsters (Charles River Laboratories, Boston, MA) fitted with a dorsal window chamber. Animal handling and care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the local animal care committee. The hamster window chamber model is widely used for microvas- cular studies without anesthesia, and the complete surgical technique is described in detail elsewhere [12,13]. Catheters were tunneled under the skin, exteriorized at the dorsal side of the neck, and securely attached to the window frame.

Inclusion criteria

Animals were suitable for the experiments if (1) systemic parameters were within reference range, namely, heart rate (HR) greater than 340 beats per minute, mean arterial blood pressure (MAP) greater than 80 mm Hg, systemic hematocrit

(Hct) greater than 45%, and PaO2 greater than 50 mm Hg and

(2) microscopic examination of the tissue in the chamber observed under a original magnification x650 did not reveal signs of edema or bleeding.

Acute hemorrhage and volume replacement protocol

Acute hemorrhage was induced by withdrawing 50% of the estimated BV via the carotid artery catheter within 5 minutes. Total BV was estimated as 7% of body weight. One hour after hemorrhage induction, animals received a single Bolus infusion of HTS comprising 3.5% of BV, and 5 minutes after HTS, 10% of the BV comprising of volume resuscitation fluids (see experimental groups) was infused within 10 minutes via the jugular vein catheter, Fig. 1. Parameters were analyzed before hemorrhage (baseline), after 45 minutes after the hemorrhage (shock), and over 90 minutes after volume infusion (resusci- tation). Animals did not receive any additional fluid during the experiment. At the end of the experiment, blood (1.5 mL) was collected in a heparinized syringe for plasma and blood viscosity, COP, and protein concentration.

Resuscitation groups

Animals were randomly divided into the following 4 experimental groups before the experiment: (1) HSA, animals were resuscitated with 10% HSA; (2) PolyHSA_24:1, animals were resuscitated with 10% PolyHSA_24:1; (3) PolyHSA_60:1, animals were resuscitated with 10% PolyHSA_60:1; and (4) PolyHSA_94:1, animals were resuscitated with 10% PolyHSA_94:1. Solution biophysical properties are presented in Table 1.

Systemic parameters

Mean arterial blood (MAP) pressure and HR were recorded continuously (MP 150; Biopac System, Santa Barbara, CA). Hematocrit was measured from centrifuged arterial blood samples taken in heparinized capillary tubes. Hemoglobin (Hb) content was determined spectrophotomet- rically (B-Hemoglobin; Hemocue, Stockholm, Sweden).

Blood chemistry and biophysical properties

Arterial blood was collected in heparinized glass capillaries (0.05 mL) and immediately analyzed for PaO2, PaCO2, base excess, and pH (Blood Chemistry Analyzer 248; Bayer, Norwood, MA).

Microvascular experimental setup

The awake animal was placed in a restraining tube with a longitudinal slit from which the window chamber protruded and then fixed to the microscopic stage of a transillumination intravital microscope (BX51WI; Olympus, New Hyde Park,

Table 1 Biophysical properties of resuscitation solutions and blood rheological properties Test solution (before infusion) and blood and plasma (after resuscitation)
	Test solutions g/dL	Viscosity ? cP		COP mm Hg	MW MDa		Blood Viscosity* cP		Plasma Viscosity* cP		COP mm Hg
HSA	10	1.5		42.0	0.07		2.4 +- 0.2		1.1 +- 0.2		16 +- 2
PolyHSA24:1	10	1.6		22.0	0.24		2.7 +- 0.3		1.2 +- 0.2		15 +- 2
PolyHSA60:1	10	11.2		4.0	2.00		3.2 +- 0.4		1.8 +- 0.3		14 +- 1
PolyHSA94:1	10	15.2		1.0	11.84		4.3 +- 0.3		2.5 +- 0.4		12 +- 2
* Shear rate of 160 s-1 at 37?C; COP at 27?C. Molecular weight (MW); megadaltons (MDa).

NY). The animals were given 20 minutes to adjust to the change in the tube environment before measurements. Measurements were carried out using a x40 (LUMPFL- WIR, numerical aperture 0.8; Olympus) water immersion objective. The same sites of study were followed throughout the experiment so that comparisons could be made directly to baseline levels.

Microhemodynamics

Arteriolar and venular blood flow velocities were measured online via the photodiode cross-correlation method (Photo-Diode/Velocity; Vista Electronics, San Diego, CA) [14]. The measured centerline velocity (V) was corrected according to blood vessel size to obtain the mean RBC blood velocity [15]. A video image-shearing method was used to measure blood vessel diameter (D) [16]. Blood flow (Q) was calculated from the measured values as Q = ? x V (D/2) ². Wall shear stress (WSS) was defined by WSS = WSR x ?, where WSR is the wall shear rate given by 8VD^-1 and ? is the microvascular blood viscosity.

Functional capillary density

Functional capillaries, defined as those capillary segments that have RBC transit of at least 1 RBC in a 60-second period in 10 successive microscopic fields, were assessed, totaling a region of 0.46 mm². Each field had between 2 and 5 capillary segments with RBC flow. Functional capillary density (FCD) (cm^-1) is the total length of RBC-perfused capillaries divided by the area of the microscopic field of view. Measurement includes length of capillaries perfused with RBCs in the field of view. The relative change in FCD from baseline levels after each intervention is indicative of the extent of capillary perfusion [17].

Volume expansion

Changes in intravascular volume were calculated as the difference in Hct before infusion (Hct₀) and the Hct at a specific time after infusion (Hct_t) as V_E = (Hct₀ - Hct_t)/Hct_t.

Pharmacokinetic analysis of PolyHSA solutions

Polymerized HSA pharmacokinetics was studied in hamsters instrumented with the dorsal window chamber (described in the animal preparation section) after a 40% of the estimated BV Exchange transfusion, to determine the role of PolyHSA molecular size on circulation persistence in normal vasculature with (uncompromised) permeability. Pharmacokinetic parameters were determined for total PolyHSA in the plasma using a noncompartmental method (NCOMP Pharmacokinetics Analysis Software; Paul B. Laub and James M. Gallo) [18]. At each time point (30 minutes and 1, 2, 4, 8, 12, 24, and 48 hours) after the exchange transfusion, a 50-uL aliquot of blood was collected, and plasma was obtained by centrifugation (6000g, 5 minutes). At 48 hours after the exchange, the experiment was terminated.

PolyHSA/HSA concentration

Polymerized HSA presented in the sampled plasma was determined by labeling it with a goat anti-HSA polyclonal antibody conjugated to fluorescein isothiocyanate (FITC) (Bethyl Laboratories, Montgomery, TX). This polyclonal antibody does not cross-react with albumin derived from hamsters or other animals. Polymerized HSAs were fluorescently labeled before infusion in the pharmacokinetic analysis. The antibody was diluted 500:1 in 10 mL of each HSA or PolyHSA solution and incubated at 4?C for 2 hours. Standard relationships between concentration and fluores- cence for all solutions were measured after several dilutions using fresh hamster plasma. The fluorescence signals were determined by an automated fluorescence system Fluor- oMax-2 (HORIBA Ltd, Kyoto, Japan) [19].

Data analysis

Results are presented as the mean +- SD. Data within each group were analyzed using analysis of variance for

repeated measurements (analysis of variance and Kruskal- Wallis test). When appropriate, post hoc analyses were performed with the Dunn multiple comparison test. A priori, animal sample size based on MAP was calculated. The calculation of the sample size to detect changes in MAP after small-volume resuscitation hemorrhagic shock in hamsters was based on our previous results [20-22]. We previously found that during small-volume resuscitation, hemorrhagic shock MAP changed by 30% (SD, 12%). Therefore, the number of animals was based on a power analysis using an ? of .05 and a 1-? of .9 and resulted in an estimate of 7 animals to be required to identify differences in MAP. However, as data were collected, statistical analysis was implemented, and following the animal care regulation at our institution, no more animals were included as statistical significance was reached. Microhemodynamic data are presented as absolute values and ratios relative to baseline values. A ratio of 1.0 signifies no change from baseline, whereas lower and higher ratios are indicative of changes proportionally lower and higher than baseline (ie, 1.5 would mean a 50% increase from the baseline level). The same vessels and capillary fields were followed so that direct comparisons to their baseline levels could be performed, allowing for more robust statistics for small sample populations. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software, Inc, San Diego, CA). Changes were considered statistically significant if P b .05.

Table 2 Blood gas parameters

Results

Twenty-four animals were entered into this study; all tolerated the entire protocol without visible signs of discomfort. All animals included in the study passed the Grubbs test, ensuring that all the measured parameter values at baseline were within a similar population (P b

.05). Animals were randomly assigned to the following experimental hemorrhage resuscitation groups: HSA (n = 6; 62.6 +- 4.2 g), PolyHSA_24:1 (n = 6; 64.2 +- 3.6 g),

PolyHSA_60:1 (n = 6; 60.6 +- 4.1 g), and PolyHSA_94:1 (n = 6;

59.8 +- 3.0 g). Systemic data for baseline and shock were obtained by combining all experimental groups. Similarities between groups at baseline and shock were statistically verified among groups (P N .30).

Systemic parameters

Basic blood hematology and blood gas parameters are presented in Table 2. HemorrhAge SIgnificantly reduced the Hct and Hb concentrations compared with baseline. Resuscitation further decreased the Hct and Hb concentra- tions compared with shock. The Hct and Hb concentration decreased as a function of the COP of the PolyHSA solution, which resulted in a larger decrease for the lower MW PolyHSA (PolyHSA_24:1) and in a smaller decrease for the higher MW PolyHSA (PolyHSA_94:1).

Hct (%)		Hb (g/dL)	pH	PaO2 (mm Hg)		PaCO2 (mm Hg)		BE (mmol/L)
Baseline
HSA	47.5 +- 2.4	14.4 +- 0.6	7.34 +- 0.02	60	+- 9	56	+- 8	3.7 +- 2.0
PolyHSA24:1	48.8 +- 3.0	14.8 +- 0.7	7.35 +- 0.02	62	+- 10	57	+- 7	4.1 +- 2.3
PolyHSA60:1	48.7 +- 3.2	14.9 +- 0.9	7.36 +- 0.02	57	+- 6	56	+- 7	4.0 +- 2.0
PolyHSA94:1	48.4 +- 2.4	14.6 +- 0.7	7.36 +- 0.02	62	+- 5	54	+- 2	4.8 +- 1.7
Shock
HSA	28.2 +- 1.1	9.4 +- 0.5	7.24 +- 0.12	95	+- 11	39	+- 7	-4.8 +- 2.2
PolyHSA24:1	28.4 +- 1.0	9.5 +- 0.7	7.21 +- 0.13	97	+- 12	38	+- 6	-5.9 +- 2.4
PolyHSA60:1	28.6 +- 0.9	9.4 +- 0.8	7.22 +- 0.09	94	+- 8	39	+- 7	-4.6 +- 2.0
PolyHSA94:1	28.0 +- 1.4	9.6 +- 0.7	7.22 +- 0.08	92	+- 10	38	+- 7	-4.9 +- 2.4
Resuscitation 60 min
HSA	18.2 +- 1.0	5.6 +- 0.5	7.38 +- 0.04	76	+- 6	46	+- 5	2.3 +- 1.5
PolyHSA24:1	19.3 +- 1.2	6.1 +- 0.5	7.38 +- 0.06	72	+- 9	45	+- 6	2.1 +- 1.6
PolyHSA60:1	21.3 +- 1.4 ?	6.7 +- 0.8 ?	7.37 +- 0.04	76	+- 11	47	+- 5	3.0 +- 1.3
PolyHSA94:1	25.1 +- 1.3 ?, +, ?	7.9 +- 0.7 ?, +, ?	7.35 +- 0.04	93	+- 6 ?, +, ?	43	+- 6	-0.5 +- 1.6 ?, +, ?
Resuscitation 60 min
HSA	18.5 +- 1.4	5.8 +- 0.3	7.35 +- 0.03	76	+- 6	47	+- 6	2.2 +- 1.5
PolyHSA24:1	18.8 +- 1.2	6.0 +- 0.6	7.36 +- 0.04	80	+- 8	46	+- 5	1.8 +- 1.4
PolyHSA60:1	19.3 +- 1.1 ?	6.2 +- 0.5 ?	7.39 +- 0.03	74	+- 10	49	+- 7	3.2 +- 1.6
PolyHSA94:1	25.0 +- 1.3 ?, +, ?	7.7 +- 0.7 ?, +, ?	7.35 +- 0.03	81	+- 8 ?, +, ?	42	+- 6	-0.7 +- 1.5 ?, +, ?
Values are mean +- SD. No significant differences were detected at baseline or during shock between groups. BE indicates base excess. * P b .05 compared with HSA. + P b .05 compared with PolyHSA24:1. ? P b .05 compared with PolyHSA60:1.

Blood pressure and HR at baseline, shock, and after resuscitation are shown in Fig. 2. Blood pressure was significantly decreased after hemorrhagic shock. Small- volume resuscitation with PolyHSA_24:1 and PolyHSA_60:1 increased MAP compared to the hypovolemic shock, although the MAP was still significantly lower than baseline. The MAP of the group resuscitated with PolyHSA_94:1 was not different compared to shock. There were no statistically significant changes in the HR (neither from baseline nor after resuscitation) among the study groups at any time during the experiment.

Blood gas parameters and calculated acid base balance are presented in Table 2. Blood gas parameters (PaO2 and PaCO2) were significantly affected by hemorrhagic shock compared with baseline. Resuscitation with PolyHSA_24:1 and PolyHSA_60:1 recovered blood gases from shock; however, PaO2 remained higher than baseline, and PaCO2 was decreased compared with baseline. arterial blood pH in animals resuscitated with PolyHSA_24:1 and PolyHSA_60:1 was not different compared with baseline. The PaO2, PaCO₂, and arterial pH of the group resuscitated with PolyHSA_94:1 were not different compared with shock and significantly different

120

P < .05

P < .05

P < .05

HSA PolyHSA24:1

PolyHSA60:1

PolyHSA94:1

100

Mean Arterial Pressure, mmHg

compared with baseline. There were no significant changes in blood gases between 60 minutes after resuscitation compared with 90 minutes after resuscitation.

Blood and plasma viscosities were significantly different among resuscitation groups. Blood and plasma viscosities are presented in Table 1. The group resuscitated with PolyHSA_24:1 showed lower blood and plasma viscosities compared with PolyHSA_60:1 and PolyHSA_94:1. In addition, the PolyHSA_60:1 group presented lower blood and plasma viscosities compared with PolyHSA_94:1. Blood and plasma viscosities for the PolyHSA_24:1 and PolyHSA_60:1 groups were lower than baseline viscosity. Baseline viscosity data (blood, 4.2 cP; plasma, 1.2 cP) were obtained from hamsters that did not undergo the shock Resuscitation protocol./a>. Plasma COP was higher for the group resuscitated with PolyHSA_24:1 compared with PolyHSA_94:1. Calculated volume expansion using Hct changes at 60 and 90 minutes after resuscitation was significantly higher for HSA (60 minutes, 53% +- 7%; 90 minutes, 51% +- 9%) and PolyHSA_24:1 (60 minutes, 46% +- 8%; 90 minutes, 55% +- 10%) than PolyHSA_60:1 (60 minutes, 32% +- 7%; 90 minutes, 46% +- 8%) and PolyHSA_94:1 (60

minutes, 17% +- 7%; 90 minutes, 12% +- 6%). PolyHSA_24:1 and PolyHSA_60:1 had significantly higher volume expansion compared with PolyHSA_94:1, as expected due to its lower COP. The HSA, PolyHSA_24:1, and PolyHSA_94:1 maintained and increased their volume expansion between 60 and 90 minutes after infusion, although PolyHSA_94:1 had a low COP. At the end of the experiment, the plasma protein concentration of the groups resuscitated with HSA and

⁸⁰ PolyHSA

24:1

was lower compared with PolyHSA

60:1

and

60

40

500

450

Heart Rate, bpm

400

350

BL Sh R60 R90

Time Point

PolyHSA_94:1, respectively. No difference in plasma protein concentration was measured between the PolyHSA_60:1 and PolyHSA_94:1 groups.

Microvascular hemodynamic measurements are presented in Fig. 3. Arteriolar and venular diameters were statistically vasoconstricted during shock for all experimental groups. Arteriolar diameters in animals resuscitated with PolyHSA_24:1 and PolyHSA_60:1 were significantly different compared with shock but no different compared with baseline. The diameter of arterioles in the group resuscitated with PolyHSA_94:1 was significantly lower compared to resuscitation with PolyHSA_24:1 and PolyHSA_60:1, and no different compared to shock. Arteriolar blood flow during shock was significantly lower compared with baseline in all the experimental groups. In general, resuscitation signifi- cantly increased blood flow compared with shock in all groups, although all the groups were significantly lower compared with baseline. Ninety minutes after resuscitation

P < .05

P < .05

Fig. 2 Mean arterial blood pressure during the hemorrhagic

with PolyHSA_94:1, arteriolar blood flows were significantly

shock resuscitation protocol for HSA, PolyHSA

24:1

, PolyHSA

60:1,

lower compared with PolyHSA_24:1 and PolyHSA_60:1.

Venular diameters were significantly recovered after resus-

and PolyHSA_94:1. Parameters were analyzed before hemorrhage (baseline), 60 minutes after hemorrhage induction (shock), and at 60 and 90 minutes after fluid resuscitation (R60 and R90). All MAPs were significantly lower than baseline during shock and after resuscitation. BL indicates baseline; Sh, shock; R60, resuscitation 60 minutes; R90, resuscitation 90 minutes.

citation compared with shock in all groups. No differences in venular diameters were measured between groups re- suscitated with PolyHSA; however, animals resuscitated with HSA were significantly constricted compared with PolyHSA_94:1 at 60 minutes after resuscitation and compared

1.2

Arteriolar

Venular

1.1

P < .05

P < .05

HSA PolyHSA 24:1

PolyHSA 60:1

PolyHSA 94:1

P < .05 P < .05 P < .05

P < .05 P < .05

P < .05

Diameter, relative to baseline

1.0

0.9

0.8

0.7

1.0

P < .05

P < .05

P < .05

P < .05

P < .05

P < .05

P < .05

P < .05

0.8

0.6

Blood Flow,

relative to baseline

0.4

0.2

0.0

BL Sh R60 R90

Time Point

BL Sh R60 R90

Time Point

Fig. 3 Relative changes with respect to baseline in arteriolar and venular hemodynamics for PolyHSA_24:1, PolyHSA_60:1, and PolyHSA_94:1. The broken line represents the baseline level. Diameters (mean +- SD) for each animal group at baseline were as follows: PolyHSA_24:1 (arterioles [A], 54.6 +- 9.7 um, n = 38; venules [V], 55.8 +- 8.2 um, n = 46); PolyHSA_60:1 (A, 56.7 +- 9.1 um, n = 40; V, 57.6 +- 9.2 um, n = 47),

and PolyHSA_94:1 (A, 55.7 +- 7.2 um, n = 36; V, 54.8 +- 8.5 um, n = 38). n is the number of vessels studied. Red blood cell velocities (mean +- SD) for each animal group at baseline were as follows: PolyHSA_24:1 (A, 4.3 +- 1.2 mm/s; V, 2.1 +- 0.6 mm/s); PolyHSA_60:1 (A, 4.0 +- 1.1 mm/s; V, 2.2 +- 0.9 mm/s), and PolyHSA_94:1 (A, 4.2 +- 0.9 mm/s; V, 2.3 +- 0.8 mm/s). CalculatED flows (mean +- SD) for each animal group at baseline were as follows: PolyHSA_24:1 (A, 10.8 +- 3.2 nL/s; V, 6.5 +- 2.1 nL/s), PolyHSA_60:1 (A, 10.4 +- 3.2 nL/s; V, 6.0 +- 1.9 nL/s), and PolyHSA_94:1 (A, 10.7 +- 2.4 nL/s; V, 6.3 +- 2.3 nL/s). Parameters were analyzed before hemorrhage (baseline), after hemorrhage (shock, 60 minutes after hemorrhage induction), and at 60 and 90 minutes after fluid resuscitation. BL indicates baseline; Sh, shock; R60, resuscitation 60 minutes; R90, resuscitation 90 minutes.

with PolyHSA_24:1 at 90 minutes after resuscitation, respec- tively. Venular blood flow during shock was statistically lower than baseline, and all resuscitation solutions exhibited statistically increased blood flow compared with shock. Venular blood flow after resuscitation with PolyHSA_94:1 was statistically lower compared with PolyHSA_24:1 and PolyHSA_60:1.

Changes in FCD are presented in Fig. 4. Functional capillary density was statistically reduced during shock, whereas resuscitation statistically increased FCD compared with shock in all experimental groups. Functional capillary density was no different between groups. Calculated arteriolar and venular wall shear rates were significantly reduced during shock compared with baseline. Resuscita- tion with PolyHSA_60:1 significantly increased the arteriolar and venular wall shear rates compared with the group resuscitated with PolyHSA_94:1. Calculated arteriolar and

venular wall shear stresses were not different between experimental groups.

Twelve animals were entered into the pharmacokinetic study. All animals included in the study passed the Grubbs test, ensuring that all the measured parameter values at baseline were within a similar population (P b .05). Animals were randomly assigned to the following experimental groups: HSA (n = 3; 63.6 +- 3.6 g), PolyHSA_24:1 (n = 3;

60.1 +- 4.2 g), PolyHSA_60:1 (n = 3; 62.5 +- 4.5 g), and

PolyHSA_94:1 (n = 3; 64.1 +- 3.5 g). Plasma HSA/PolyHSA concentration after a 40% exchange transfusion is shown in Fig. 5. The pharmacokinetic parameter estimates for HSA/PolyHSA are listed in Table 3. Doses received determined at the completion of exchange transfusion were similar among all groups 158 +- 37 mg (HSA), 145 +- 34 mg

(PolyHSA_24:1), 156 +- 48 mg (PolyHSA_60:1), and 161 +- 42

mg (PolyHSA_94:1). Transfusions resulted in total plasma

1.0

Functional Capillary Density, relative to baseline

0.8

0.6

0.4

0.2

0.0

BL Sh R60 R90

Time Point

overload morbidity, rebleeding, and thrombus dislodging by using permissive hypotensive resuscitation.

This study compares the effects of small-volume resusci- tation from hemorrhagic shock and correlates the volume expansion attained with the physical properties of the resuscitation solution. The initial similarity of responses observed with all PolyHSA solutions was caused by BV restoration, and the later results observed at 60 and 90 minutes after resuscitation reflect the effects of volume expansion, microvascular recovery, and the reversal of capillary collapse created during the hypovolemic shock. Volume expansion of PolyHSA was proportional to its COP, in addition the larger-

HSA PolyHSA 24:1

PolyHSA 60:1

PolyHSA 94:1

P < .05

P < .05

P < .05 P < .05

P < .05

sized PolyHSA molecules increased the natural volume

Fig. 4 Relative changes with respect to baseline in capillary perfusion during the shock resuscitation protocol. Functional capillary density was drastically reduced after hemorrhage. FCD Functional capillary density at baseline was as follows: PolyHSA_24:1 (112 +- 12 cm^-1), PolyHSA_60:1 (116 +- 10 cm^-1),

and PolyHSA_94:1 (108 +- 9 cm^-1). Parameters were analyzed before hemorrhage (baseline), after hemorrhage (shock, 60 minutes after hemorrhage induction), and at 60 and 90 minutes after fluid resuscitation. BL indicates baseline; Sh, shock; R60, resuscitation 60 minutes; R90, resuscitation 90 minutes.

HSA/PolyHSA maximum plasma concentrations (C_max) of

3.62 +- 0.52 g/dL (HSA), 3.33 +- 0.62 g/dL (PolyHSA_24:1),

3.41 +- 0.42 g/dL (PolyHSA_60:1), and 3.41 +- 0.42 g/dL

(PolyHSA_94:1), respectively. The volume of distribution suggests a higher distribution within the central compartment for PolyHSA_24:1 and PolyHSA_60:1 formulations compared with HSA and PolyHSA_94:1.

Discussion

The principal finding of this study is that resuscitation with 10 g/dL PolyHSA (PolyHSA_24:1 and PolyHSA_60:1) provided similar recovery of systemic parameters and microvascular hemodynamics. These hyperviscous PEs provided rapid restoration of blood pressure and blood gas parameters. Remarkably, the restoration trend was maintained with PolyHSA_24:1 and PolyHSA_60:1 during the entire observation period. These PolyHSA formulations reinstated microvascular perfusion and recovery from metabolic imbalances after infusion. Hemorrhage after penetrating trauma is a major combat hazard, whose therapy relies on aggressive fluid resuscitation strategies [23,24]. Rapid and large-volume fluid resuscitation targets the recovery of intravascular volume to restore blood pressure and metabolic balance, under the assumption that it will prolong survival. This strategy can resolve the hypovolemic syndrome but may lead to fluid overload disorders that may influence the length of recovery, days requiring mechanical ventilation, and patient mortality [25]. Our small-volume resuscitation strategy, combining HTS with a colloidal PE based on PolyHSA, can prevent

expansion of HSA by increasing its retention in the intravascular compartment. Pharmacokinetic results obtained after exchange transfusion further validate this analysis. The mean residence time in the plasma for PolyHSA_60:1 was 1.4 times higher than of the HSA and PolyHSA_24:1, respectively. In addition, the clearance from the plasma for PolyHSA_60:1 was 0.37 of the HSA clearance and 0.73 of the PolyHSA_24:1 clearance, respectively. This suggests the existence of an optimal molecular size for PolyHSA molecules that increase their circulation time and minimize their clearance. In fact, the pharmacokinetic results show that increasing the molec- ular size to PolyHSA_94:1 did not increase retention or reduce clearance. The pharmacokinetic retention data are in agreement with the basic volume expansion results, calcu- lated based on changes in the Hct and the plasma protein concentration in the hemorrhagic shock experiment.

Volume expansion with HSA requires frequent adminis- tration of HSA to maintain an elevated albumin concentra- tion, increasing the cost of treatment [25,26]. Polymerization of HSA with bifunctional cross-linking reagents can reduce the amount HSA needed to maintain intravascular volume because PolyHSA is larger in size compared with monomeric HSA. Polymerized HSA large size increases, the solution

4

HSA PolyHSA 24:1

PolyHSA 60:1

PolyHSA 94:1

3

Plasma HSA, g/dl

2

1

0

0 10 20 30 40 50

Time, hours

Fig. 5 Pharmacokinetics of PolyHSA_24:1, PolyHSA_60:1, and PolyHSA_94:1. The pharmacokinetic parameters are listed in Table

3. Doses received determined at the completion of exchange transfusion were similar among groups: 158 +- 37 mg (HSA), 145 +-

34 mg (PolyHSA_24:1), 156 +- 48 mg (PolyHSA_60:1), and 161 +- 42

mg (PolyHSA_94:1).

HSA		PolyHSA24:1	PolyHSA60:1	PolyHSA94:1
CL (mL.h-1)	3.60	1.82	1.34	2.22
MRT (h)	24.8	24.9	34.9	31.3
Vss (mL)	89.2	45.3	46.6	69.4
Terminal k (h-1)	0.042	0.041	0.029	0.033
Terminal T1/2 (h)	16.7	16.9	24.0	21.2
Cmax (mg.h-1)	2.92	3.33	3.41	3.67
AUC to Tlast (mg.h.mL-1)	39.6	69.1	87.8	56.5
AUC extrapolated to time infinity (mg.h.mL-1)	46.4	79.8	116.8	72.59
% extrapolated AUC	14.8	13.5	24.8	22.2
AUMC (mg.h.mL-1)	1151	1989	4075	2271
% extrapolated AUMC	43.1	39.2	58.9	55.52
CL indicates plasma clearance; MRT, mean residence time; Vss, volume of distribution at steady state; terminal k, terminal slope; terminal T1/2, terminal half- life; Cmax, maximal concentration; AUC, area under the curve; Tlast, last measurable concentration; AUMC, area under the first moment curve.

viscosity and decreases the solution COP, thereby limiting intravascular volume expansion to the volume infused without moving fluid from the perivascular space because of the high COP of the solutions. The volume expansion attained with PolyHSA_24:1 and PolyHSA_60:1 was not different, suggesting that structural modification of the HSA by the cross-linking process may also affect volume expansion. Theoretically, PolyHSA should remain in the intravascular compartment as long as in the pharmacokinetic study, but hemorrhagic shock-induced changes in vascular permeability may affect volume expansion. The rationale for this analysis is supported by the plasma protein at the end of the experiment. In addition, the differences in Hct after resuscitation also present a snapshot of volume expansion under pseudoequilibrium conditions. Unfortunately, it was impossible to perform long-term pharmacokinetic analysis postresuscitation because ours animal care regulation does not allow us to keep animals hypovolemic beyond the extent of the shock resuscitation protocol. Differences in volume expansion resulted in differences in the Hct and Hb concentration, which also affected the oxygen transport capacity, and whole blood rheological properties.

Table 3 Pharmacokinetic parameters for HSA/PolyHSA solutions

Our small-volume resuscitation strategy from hemor- rhagic shock focuses on restoration of microvascular perfusion, which determines oxygen delivery and washout of metabolic waste products, leading to an improved long- term outcome [27]. To ensure the recovery of microvas- cular perfusion and prevent tissue injury, the biophysical properties of the resuscitation fluid needs to reinstate FCD since FCD reflects the overall function of the microcircu- lation. Microvascular oriented therapies have shown clinical prevention of multiorgan failure [28,29]. On the other hand, resuscitation strategies that aim to restore systemic parameters fail to recover microvascular function, such as vasopressin treatment, which only recovers pressure, shunting blood flow away from the microcircu- lation and decreasing Tissue oxygenation [30]. Given that volume restoration and microvascular function are special during resuscitation from hemorrhagic shock, our novel

PolyHSAs, when used during small-volume resuscitation, produced a Rapid increase in microvascular flow and FCD and sustained recovery of blood pressure. Polymerized HSAs are biocompatible and do not induce RBC aggregation [10]. Our findings suggest that an increase in plasma viscosity, without necessarily restoring blood viscosity to baseline viscosity with PolyHSA, is beneficial during resuscitation. In previous studies, increasing plasma viscosity during anemic conditions was associated with increased capillary pressure, a critical determinant of FCD [17]. The importance of increasing plasma viscosity during anemic and hypovolemic conditions is shown by the sustained recovery of microhemodynamic conditions postresuscitation. Pressure redistribution is caused by arteriolar vasodilatation, likely because of the restoration of shear stress [17]. Shear stress exerted by the flowing plasma on the vascular Endothelial cells influences vessel diameter by the release of vasodilatory autocoids (prostacy- clin, nitric oxide, etc) [31].

In our study, PolyHSA_60:1 produced the most significant resuscitation, by expanding BV and increasing plasma viscosity to 1.8 cP and blood viscosity to 3.2 cP, both values are significantly different than normal blood, where plasma is 1.2 cP and blood is 4.2 cP. The role of plasma viscosity, and consequently, blood viscosity in maintaining systemic blood pressure and blood gases, is highlighted by the results obtained here because increasing plasma viscosity by means of hyperviscous PolyHSA provided consistent restoration of homeostasis, compared with volume resuscitation with conventional PEs. Previous studies evaluated several conventional PEs, including Voluven and Hextend (Hospira, Inc, Lake Forest, IL), in an identical small-volume resuscitation from hemorrhagic shock protocol as described here, and PolyHSA_60:1 showed superior recovery of systemic and microvascular parame- ters compared to these commercial PEs [22]. This study suggests a limit for the benefit attained by increasing plasma viscosity. The upper limit appears to be lower than the plasma viscosity attained with PolyHSA_94:1, although the exact value has not yet been defined. Currently in the

marketplace, the only colloid with high viscosity is Hextend (Hospira, Inc), a high MW HES with a high degree of substitution [32]. However, HES has shown adverse effects on coagulation, mainly on Factor VIII, von Willebrand’s factor, and Platelet function [32]. Polymer- ized HSAs are novel PEs, preserving HSA ligand-binding capacity and compatibility and potentially serving as a substitute for HSA in patients with high blood vessel and glomerular permeability.

Hemorrhagic shock is a critical situation in which rapid correction of hemodynamics can improve outcome. The recovery observed after infusion of HTS is caused by rapid intercompartmental Fluid shifts, and the small volume of hyperviscous PolyHSA stabilized and limited the extravasa- tion of the fluid drawn out of the extravascular space. The gradual and sustained increase of FCD, blood pressure, and flow obtained with the PolyHSA indicates a gradual increase in cardiac output combined with a volume effect, which preserves hemostatic mechanisms, reducing the risk for further bleeding. In conclusion, this study shows that, to recover microvascular function after hemorrhagic shock, the biophysical properties of the fluid used during resuscitation must maintain intravascular volume and increase plasma viscosity. Polymerized HSA potential as a new plasma volume expander is inspired by its tunable intravascular retention and rheological characteristics, controlled by the molar ratio of glutaraldehyde to HSA, and biocompatibility. Although this study has restricted clinical significance, it presents a comprehensive experimental study, with the objective of defining the mechanistic principles for transla- tional developments and that will need to be evaluated in clinical trials. Transfusion of PolyHSA Polymerized HSAs used will reduce the multiple administrations of HSA, thus reducing HSA-associated toxicity and cost. Our results with PolyHSA solutions suggest the existence of an optimal size that maintains intravascular volume, extends circulation time, and minimizes clearance.

Acknowledgments

This work was partially supported by program project P01-HL071064 and grants R01-HL52684, R01-HL62354,

R01-HL078840, and R01-DK070862. The authors thank Froilan P. Barra and Cynthia Walser for the surgical preparation of the animals and Paulo do Nascimento, Jr., MD, PhD for the Clinical interpretation of our findings.

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