The Effects of Resuscitative Fluid Therapy on the Endothelial Surface Layer

REVIEW

published: 07 May 2021

doi: 10.3389/fvets.2021.661660

Frontiers in Veterinary Science | www.frontiersin.org 1 May 2021 | Volume 8 | Article 661660

Edited by:

Pablo Martín-Vasallo,

University of La Laguna, Spain

Reviewed by:

Ivo P. Torres Filho,

United States Army Institute of

Surgical Research, United States

Muhammad Kashif Iqbal,

Cholistan University of Veterinary and

Animal Sciences, Pakistan

*Correspondence:

Lisa Smart

[email protected]

Specialty section:

This article was submitted to

Comparative and Clinical Medicine,

a section of the journal

Frontiers in Veterinary Science

Received: 31 January 2021

Accepted: 16 March 2021

Published: 07 May 2021

Citation:

Smart L and Hughes D (2021) The

Effects of Resuscitative Fluid Therapy

on the Endothelial Surface Layer.

Front. Vet. Sci. 8:661660.

doi: 10.3389/fvets.2021.661660

The Effects of Resuscitative Fluid

Therapy on the Endothelial Surface

Layer

Lisa Smart

and Dez Hughes

School of Veterinary Medicine, College of Science, Health, Engineering and Education, Murdoch University, Murdoch, WA,

Australia,

Department of Veterinary Clinical Sciences, Faculty of Veterinary and Agricultural Sciences, Melbourne Veterinary

School, Werribee, VIC, Australia

The goal of resuscitative ﬂuid therapy is to rapidly expand circulating blood volume

in order to restore tissue perfusion. Although this therapy often serves to improve

macrohemodynamic parameters, it c an be associated with adverse effects on the

microcirculation and endothelium. The endothelial surface layer (ESL) provides a

protective barrier over the endothelium and is important for regulating transvascular ﬂuid

movement, vasomotor tone, coagulation, and inﬂammation. Shedding or thinning of the

ESL can promote interstitial edema and inﬂammation and may cause microcirculatory

dysfunction. The pathophysiologic perturbations of critical illness and rapid, large-volume

ﬂuid therapy both cause shedding or thinning of the ESL. Research suggests that

restricting the volume of crystalloid, or “clear” ﬂuid, may preserve some ESL integrity and

improve outcome based on animal experimental models and preliminary clinical trials in

people. This narrative review critica lly evaluates the evidence for the detrimental effects

of resuscitative ﬂuid therapy on the ESL and provides suggestions for future research

directions in this ﬁeld.

Keywords: endothelium, glycocalyx, shock, ﬂuid therapy, crystalloid, colloid, endothelial surface layer

INTRODUCTION

The importance of microcirculatory function and health of the endothelium has become a large

area of interest for criticalists in the last two decades. Ov er this time, ﬂuid resuscitation strategies

have experienced a shift in perspective. Aggressive “clear” ﬂuid resuscitation was once considered

vital for stabilization of macrohemodynamic variables. However, in certain patient cohorts this

approach has now been shown to either not improve outcome or worsen outcome (

1–4). Though

fast administration of ﬂuid often serves to normalize the commonly measured clinical parameters

during shock, microcirculation may not necessarily beneﬁt from this therapy. There is growing

evidence that ﬂuid resuscitation may actually harm t he endothelium by modifying, or shedding,

the endothelial surface layer (ESL).

The ESL includes a structural scaﬀold, the endothelial glycocalyx (EG), and associated molecules

suspended within a plasma layer. We use the term EG when referring to speciﬁc components of the

structure, or biomarkers of these components, whereas ESL is used when referring to the layer as a

whole. In regards to shedding of the ESL, the term ESL will be used when the evidence supports loss

of the whole layer and the term EG used when the evidence only supports that isolated components

have been shed. Use of the term glycocalyx refers to any type of cell surface glycocalyx and is

not restricted to the endothelium, such as during discussion of glycocalyx shedding biomarkers.

Smart and Hughes Fluids and Endothelial Surface Layer

Resuscitative ﬂuid t h erapy includes a range of ﬂuid choices,

including isotonic crystalloid ﬂuid, synthetic colloid ﬂuid, and

hyperosmolar crystalloid ﬂuid, with the former being the

most common type of ﬂuid used (5). In general, due to the

pharmacodynamic properties of these ﬂuids in either healthy

subjects or shock models (

6–9), the volume of ﬂuid is “large” for

isotonic crystalloid (at least a quarter to half of estimated blood

volume), “moderate” for synthetic colloid (an eighth to quarter

of estimated blood volume), and “small” for hyperosmolar

crystalloids. All three types of ﬂuid resuscitation will be covered

in this review with a particular focus on i sotonic “large-volume”

crystalloid ﬂuid therapy due to t he evidence for its eﬀects on

the ESL. A summary of the evidence reviewed in this article is

provided in Box 1.

STRUCTURE AND FUNCTION OF THE

ENDOTHELIAL SURFACE LAYER

Basic Structure and Function

Most cells in the body are covered in a protective layer of

carbohydrate scaﬀold, which houses many diﬀerent molecules

that serve a variety of functions. This surface layer is called

a glycocalyx. The general structure of the glycocalyx has

similarities between cell ty pes, with only small variations

in individual proteins or carbohydrate molecules. The EG

coats the luminal surface of the endothelium and is a vital

structure for cell signaling and transvascular permeability. It

is composed of proteoglycans, glycosaminoglycans (GAGs),

and glycoproteins (Figure 1). Together with mobile or soluble

components, such as albumin, this compromises the ESL.

Proteoglycans are large molecules that have a cytoplasmic,

transcellular, and extracellular domain (syndecan) or are attached

by a glycosylphosphatidylinositol anchor (glypican-1) (Figure 1)

(

10–12). Their extracellular component, or ectodomain, is

covered by GAG side-chains and performs important roles

that assist with cell to cell, or cell to matrix, interactions (13).

These structures provide a structural scaﬀold for the ESL, in

which many other molecules are housed. The sulfated GAGs

attached to proteoglycans include heparan, chondroitin, and

dermatan sulfate. Heparan sulfate is the most abundant GAG

on syndecans and glypican-1, which is why proteoglycans are

often referred to as heparan sulfate proteoglycans. An additional

GAG, hyaluronan, is not typically associated with a proteoglycan;

instead, it is attached to the endothelium via receptors such as

CD44 or other GAG molecules (Figure 1) (14, 15). Hyaluronan

is a long GAG of varying lengths that weaves its way throug h the

tall “forest” of proteoglycans, with their sulfated GAG “branches”.

These GAG chains contribute to the b a rrier function of the ESL

of excluding large molecules (16). Glycoproteins reside on the

luminal surface of the endothelium, hidden within the ESL, and

include a dhesion molecules such as integrins and selectins (17).

Glycoproteins play an important role in leucocyte traﬃcking

during states of inﬂammation (

18); many of their functions

are only initiated once the ESL has been shed or thinned.

Finally, mobile components residing within the forest of the ESL

include proteins, such as albumin, and anticoagulants, such as

BOX 1 | Summary of the evidence for effects of “clear” uid therapy on

endothelial surface layer (ESL) shedding or modication.

Proposed mechanisms of ESL shedding

Dilution of plasma proteins

Release of natriuretic peptides

Inﬂammatory cytokine release (certain ﬂuid types)

Possible downstream effects

Exacerbation of inﬂammation

Microcirculatory dysfunction

Increased vascular permeability

Increased interstitial edema

Prothrombotic conditions

Proposed strategies that may mitigate ESL shedding

∗

Reduction in dose of clear ﬂuids

Slowing down administration of resuscitative ﬂuids

“Earlier” vasopressor therapy for vasodilatory shock

Adjunctive protein administration (such as plasma)

∗

These proposed strategies are not based on evidence from clinical veterinary

research and require further investigation. These guidelines are opinion of

the authors only, after considering the breadth of evidence available. It is

currently unknown if “glycoprotective” strategies beneﬁt patients. Therapy

should always be tailored to individual patient needs, with prioritization of

reestablishing adequate perfusion and a thorough cost to beneﬁt analysis.

antithrombin and tissue factor pathway inhibitor (15, 19). The

presence of plasma proteins are likely important for maintaining

the normal structure and permeability of the ESL, as well as

providing an anticoagulated surface at the blood interface.

The EG serves a range of functions, many of which are

still being characterized. These functions include maintenance

of a surface barrier that buﬀers circulating leucocytes, inhibits

coagulation, regulates ﬂuid ﬂux, and communicates changes in

vascular wall shear forces. The sulfated GAGs on the surface of

the endothelium generate and maintain a net negative charge,

which repels similarly charged cells from the endothelial surface

(

20). Although albumin has an overall negative charge, it is

likely that the positively charged groups within the molecular

structure are what allow for incorporation of albumin into the

ESL (

21). Maintenance of this negative charge is important for

endothelial integrity; loss of negative charge on the luminal

surface of the endothelium leads to extravasation of albumin

(22–24). In addition to the importance of electrostatic charge,

the ESL provides a barrier to ﬂuid ﬁltration, thus creating a

protein-poor sub-glycocalyx layer and maintaining a colloid

osmotic pressure (COP) gradient favoring ﬂuid retention with in

the vasculature (20).

The traditional Starling hypothesis describes a relationship

between the intravascular and the interstitial colloid osmotic

pressure and implies the importance of this relationship in

determining net transvascular ﬂuid ﬁltration. Many studies

in the last four decades, however, have built a body of

evidence stating that it is mainly the low CO P in the sub-

glycocalyx area that creates the pressure gradient opposing

capillary hydrostatic pressure (

25, 26). The theory is that the

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Smart and Hughes Fluids and Endothelial Surface Layer

FIGURE 1 | The scaffold of the endothelial glycocalyx, within the endothelial surface layer, is provided by proteoglycans, syndecan (four subtypes), and glypican-1.

Glycosaminoglycans are attached to proteoglycans (e.g., heparan sulfate) or the endothelial surface (hyaluronan). Molecules suspended in the plasma of the

endothelial surface layer include proteins such as albumin. These proteins create a protein-poor sub-glycocalyx area that is important for transvascular colloid osmotic

pressure balance. For simplicity, structures within the interendothelial cleft are not represented.

sub-glycocalyx ﬂuid space, within the inter-endothelial cleft,

has a very low concentration of macromolecules. This is due

to the high imperme ability of the ESL to macromolecules

and the rapid ﬂow of water and solutes through the sub-

glycocalyx space (

27). In the steady state with an intact

ESL, net ﬁltration of ﬂuid occurs across the blood vessel

wall, with only a small limitation from intravascular COP.

Increasing intravascular COP does not , and cannot, serve to

reverse net ﬂuid transudation nor does increasing interstitial

COP lead to an increase in ﬂuid transudation (28, 29). A

large drop in hydrostatic pressure, such as that may occur

during circulatory shock, may reverse transvascular ﬂuid ﬂow

to a resorptive state; however, this eﬀect is transient (

29).

These hypot h eses explain why ﬂuid would not be “drawn”

out of the interstitium by increasing intravascular COP, such

as the use of synthetic colloid ﬂuids. Rather, they can

reduce extravasation compared to crystalloid ﬂuid. The COP

of the intravascular space, and even of the sub-glycocalyx

space, is microanatomically and physiologically distant from

the interstitial COP. However, t he ﬂuid dynamics across

the endothelium in disease states with a denuded ESL,

removing the inﬂuence of the sub-glycocalyx lay er, is yet to be

fully characterized.

The EG also plays an important role to changing pressures

and ﬂow within the intravascular space. Proteoglycans, especially

those with heparan sulfate chains, play an important role in

responding to changes in vascular wall shear stress or changes in

intravascular pressure (10, 30, 31). Detection of these mechanical

forces leads to morphological changes in endothelial cells and

release of nitric oxide (

32–35). Shear stress can also lead to a

change in location of proteoglycans or upregulation of their cell

surface expression (

36–38). It is possible that change in location

or upregulation of these molecules during microcirculatory

disturbances may also aﬀect shedding of the extracellular

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Smart and Hughes Fluids and Endothelial Surface Layer

components. This becomes relevant when discussing biomarkers

for EG shedding below.

Shedding

Shedding, or modiﬁcation, of the ESL is a vital step after t issue

injury in order to facilitate leucocyte and platelet adherence

(

39). Shedding reproduced in cell culture or ex vivo models

leads to increased expression of adhesion molecules, increased

leucocyte adhesion, and increased cytokine production (32, 40–

43). This process is likely more complex in vivo, whereby certain

disease states may have varying eﬀects, from selective removal

of glycocalyx components to complete denudation. Although

ESL shedding is a necessary step in localized inﬂammation, it

is becoming clearer that systemic-wide shedding is associated

with severity of illness and poor outcome. There has also

been a growing concern in recent years that interventions

that promote ESL shedding may worsen clinical outcomes for

critically ill patients. Given that ESL shedding is associated with

exacerbating inﬂammation and increasing vascular permeability

in laboratory models, there is theoretical plausibility that limiting

ESL shedding may improve clinical outcome.

Shedding alters capillary perfusion, causing a decrease in

functional capillary density (44), in addition to increased

endothelial permeability (45). A decrease in functional capillary

density means that some vessels within a given area do

not have red blood cells traversing their course, consistent

with microcirculatory dysfunction. This can create regions

of tissue hypoxia. A clear association between ESL shedding

and impairment of microcirculatory blood ﬂow is yet to be

demonstrated in either an in vivo animal model or clinical

study (46). However, given that ESL shedding via artiﬁcial

means can reduce capillary blood ﬂow (44), it is mechanistically

plausible that shedding of the ESL in critical illness plays a

role in altered microcirculation. It has been well-demonstrated

that heterogeneity of capillary blood ﬂow can persist in critical

illness despite normalization of macrohemodynamic variables,

such as blood pressure and cardiac output (47–51). Further,

persistence of altered microcirculatory ﬂow in critically ill

people is associated with severit y of illness and poor outcome

(47, 51–53). Consequently, it appears important to identify

causes of microcirculatory dysfunction that occurs independent

of circulatory shock. More evidence is required to determine

a causal link between ESL shedding and microcirculatory

dysfunction, and whether protection of the ESL can prevent

the lat ter.

There may be downstream consequences to the release of

EG components into circulation. Shed components can stimulate

inﬂammation by acting as danger-associated molecular patterns

or “alarmins.” Soluble heparan sulfate molecules play a key

role in modulating inﬂammation, including leukocyte activation,

increasing production of cytokines, and endothelial activation

(

54, 55). Low molecular weight hyaluronan can also stimulate

production of inﬂammatory mediators (56–59). In contrast,

shed components such as syndecan-1 and−4 ectodomains can

have indirect anti-inﬂammatory eﬀects by facilitating neutrophil

cytotoxicity (

60, 61). The complexity of EG components acting as

eﬀector molecules in the systemic circulation may be analogous

to the systemic inﬂammatory response, where some cytokine

release is beneﬁcial to the host response, whereas a “cytokine

storm” cre a tes pathological consequences.

It is unclear how long it takes for ESL recovery to occur.

Most evidence is based on data from in vitro cell culture or

ex vivo vascular models, which are unable to fully replicate in

vivo conditions. That being said, individual components can be

regenerated within 24 h (

37, 62), but restoration of the structure

itself can take up to 7 days (32, 63). During critical illness,

inﬂammation likely continues the shedding process, delaying

the repair. Further in vivo research is required using real-time

videomicroscopy or similar means in order to characterize the

temporal changes during ESL recovery.

Assessment of Shedding

Shedding of the ESL can be detected by a number of

means. Laboratory studies utilize several methods, including

measurement of circulating components of the EG (proteoglycan

ectodomains and GAGs), dete ct ion of EG components on the

surface of the endothelium, direct visualization of the ESL

via tissue ﬁxation and microscopy, and indirect visualization

via real-time videomicroscopy. Clinical studies usually rely on

measurement of circulating components of the EG, or EG

biomarkers, in serum or plasma s a mples to assess systemic

shedding. The most frequently reported EG biomarkers are

syndecan-1, heparan sulfate, and hyaluronan. There are several

limitations to relying on this kind of assessment of the ESL.

Firstly, studies often only measure a single component of the

EG at a single point in time. That particular component may

have other sources of shedding. For example, syndecan-1 is not

only present on the surface of the endothelium but also on

epithelial cells and leucocytes (

64–68). Also, highly relevant to

intravenous ﬂuid therapy, hyaluronan is abundant throughout

the interstitium and can be “ﬂushed” through the lymphatics

back into systemic circulation (69–71). It is unclear in critical

illness to what degree other sources of glycocalyx shedding

contribute to serum or plasma concentrations. Further, tissue

injury and inﬂammation during critical illness may upregulate

cell surface expression of these biomarkers, especially the

proteoglycans (38, 72–83). Therefore, an increase in circulating

concentration may reﬂect an increase in cell turnover rather

than primarily cell surface shedding. Several recent studies have

demonstrated temporal diﬀerences in the shedding of several EG

components in people presenting to an emergency department

with sepsis, whereby hyaluronan increased early in treatment

whereas sydnecan-1 increased later (

84–86). This raises questions

as to why some biomarkers increase earlier than others. It is

possible that the temporal diﬀerences relate to either alternative

sources of the biomarker or diﬀerences in upregulation.

Ideally, it would be best to visualize shedding of t he EG in vivo

rather than interpreting circulating biomarker concentrations.

These techniques are mostly reserved for use in laboratory

models rather than clinical use. Specialized tissue ﬁxation

techniques can be used to directly visualize the ESL via electron

microscopy, often employed in non-survival rodent models

(

28, 87–91). Given the fragility of the ESL, it is best that the

tissue is preserved via perfusion with ﬁxative prior to de a th,

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Smart and Hughes Fluids and Endothelial Surface Layer

either perfusion of an isolated organ or t he whole body. Tissue

immersion techniques that avoid whole-body perfusion have

also been recently described (92). Anecdotally, it is challenging

to achieve quality images of intact ESL in tissue sections.

Alternatively, components of the EG may be ﬂuorescently

labeled and visualized by confocal microscopy of cell culture

or tissue models (

93). Other laboratory techniques include dye

exclusion with intravital microscopy to estimate ESL thickness

(94–96). Finally, ESL thickness may be estimated in real-time by

sidestream dark ﬁeld microscopy in both large animal models

and clinical research (46, 97–99). This technology approximates

ESL thick ness by measuring the perfused boundary region, which

is the region within the blood vessel that is peripheral to the

ﬂow of red blood cells, or t h e immobile plasma layer. A lthough

this technique shows promise for future use in veterinary clinical

research (100), image acquisition can be technically diﬃcult

and image quality is currently limited for reliably estimating

ESL t hi ckness.

MECHANISMS OF SHEDDING RELEVANT

TO FLUID THERAPY

Inﬂammation and Shock

In the pa tient requiring bolus ﬂuid therapy, it is likely that

alteration of the ESL has already occurred due to the eﬀects of

inﬂammatory mediators. For example, tumor necrosis factor-

α, a potent pro-inﬂammatory cytokine, causes reduction in

thickness of the ESL, shedding of syndecan-1,-4, and GAGs

from the endothelium, as well as upregulation of glycocalyx

components on the endothelial surface (

76, 81, 90, 101). Matrix

metalloproteinases, one of the main perpetrat ors for glycocalyx

shedding, are released by activated leucoytes (17). Tissue injury,

such as ischemia and reperfusion, and production of reactive

oxygen species also cause EG shedding (43, 102–104). Activation

of coagulation may also aﬀect the EG, as both plasmin and

thrombin can cleave syndecan ectodomains (74, 105). Relevant

to sepsis, bacterial components such as lipopolysaccharide and

chemotactic peptides also cause shedding of the EG (43, 77,

81, 106–108). Therefore, it is likely that partial or complete

denudation of the ESL has already occurred before ﬂuid therapy

is administered.

Shedding of the EG in states of critical illness, such as shock,

has been demonstrated in animal models before any ﬂuid therapy

has commenced. In mice, hemorrhagic shock was associated with

a thinner pulmonary ESL, as measured by electron microscopy,

and downregulation of syndecan-1 on the endothelial surface,

compared to a sham model (

109, 110). In a canine hemorrhagic

shock model comparing diﬀerent ﬂuid interventions (111),

plasma hyaluronan concentration was signiﬁcantly increased

across treatment groups after atraumatic blood removal, but

before ﬂuid, compared to baseline (unpublished analysis). An

increase in plasma hyaluronan concentration has also been

demonstrated in a rodent sepsis model, 4 h after Escherichia

coli lipopolysaccharide infusion, where blood pressure was

maintained by norepinephrine administration (

112). There are

limited data on EG shedding in the clinical setting before

ﬂuid therapy; however, serum syndecan-1 concentrations above

healthy control levels has been demonstrated pre-hospital in

human trauma patients, though the temporal relationship to ﬂuid

administration by ﬁrst responders is unknown (

113).

Hemodilution

Bearing in mind that alterations to the ESL may already

exist in critically ill patients, there is growing evidence that

administration of “clear” ﬂuids may propagate ESL shedding.

Several clinical studies in critically ill people have identiﬁed

an association between volume of ﬂuid administered and

EG biomarker concentrations, including increased hyaluronan

(

84), syndecan (114), and heparan sulfate concentrations (115).

Putting aside the confounders of inﬂammation and severity

of illness, bolus ﬂuid therapy may have a direct eﬀect on the

ESL via hemodilution and production of natriuretic peptides.

Ex vivo vascular models provide important data in regards

to this issue, as they exclude the eﬀects of the varying

pharmacodynamic properties of ﬂuid types administered in vivo.

These vascular models have shown that dilution of plasma with

crystalloid reduces ESL thickness. Notably, a mathematica l model

derived from meta-analysis of several studies demonstrated

that dilution of blood with ﬂuid reduced vascular resistance,

independent of hematocrit and COP (116). Also, the decrease in

resistance caused by saline was reduced in magnitude when the

vasculature was pre-treated with heparinase. Heparinase sheds

heparan sulfate side-chains from proteoglyc ans, thus th inning

the glycocalyx (117). From this, it may be inferred that the

decrease in resistance caused by saline infusion is due to not

only changes in hemorrheology but also loss of the ESL. Further,

several studies have shown that perfusion of blood vessels with

crystalloid solution incre ases vascular permeability. One such

study showed an inverse linear relationship between albumin

concentration in the perfusate and hydraulic conductivity,

or permeability, of vasculature (

118). Return to baseline

permeability was not achieved by simply increasing the albumin

concentration back to baseline levels; it required a much higher

albumin concentration. This implies modiﬁcation of the ESL

and its aﬃnity for albumin after crystalloid infusion. Further

experiments showed that perfusion of blood vessels with plasma

was more eﬀective at restoring normal vas cular permeability

after crystalloid infusion (119), compared with an albumin

solution, indicating that substances within plasma are import ant

for maintaining normal transvascular ﬂuid ﬂux. A more recent

study showed that infusion of guinea pig coronary vasculature

with 0.9% saline signiﬁcantly increased ﬂuid extravasation,

compared to low molecular weight (LMW) hydroxyethyl starch

(HES) (Voluven



) and albumin solution (

45). Pretreatment

with heparinase combined with saline infusion did not yield

higher ﬂuid extravasation; stripping the EG before infusion was

just as detrimental as saline infusion alone. Interestingly, no

diﬀerences between ﬂuid treatments in the appearance of the ESL

were appreciated using electron microscopy. Finally, a murine

hemorrhagic shock model compared 15 mL/kg of either fresh

whole blood (FWB), packed red blood cells in lactated Ringer’s

solution (PRBC in LRS), or washed PRBC in LRS, or 75 mL/kg of

plain LRS (

120). Rats that received either washed PRBC or LRS

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had decreased ESL thi ckness compared to baseline, as measured

by intravital microscopy, whereas those that received either FWB

or PRBC did not show a signiﬁcant diﬀerence to baseline. Results

were similar for change in plasma heparan sulfate concentration.

Therefore, it appeared that the presence of protein within the

FWB and unwashed PRBC products was eﬀective at mitigating

ESL shedding.

Natriuretic Peptid es

Another mechanism of EG shedding during bolus ﬂuid therapy

is the action of natriuretic peptides on the endothelium. Atrial

and brain natriuretic peptides (ANP and BNP, respectively) are

released from the cardiac atria and ventricles during stretch of

the myocardium (

121). These peptides counteract the eﬀects

of hypervolemia by decre asing systemic vascular resistance and

causing a natriuresis, among other mechanisms. Natriuretic

peptides also increase vascular permeability (

122, 123). A more

recently discovered role of these peptides is shedding of the

EG. All three major natriuretic peptides (ANP, BNP, and C-

type NP) can shed the EG, as measured by increased syndecan-

1 and heparan sulfate concentrations in the eﬄuent of guinea

pig coronary arteries (88, 124). These studies also demonstrated

increased vascular permeability and visualized denudation of the

ESL via electron microscopy.

Fast intravenous administration of isotonic crystalloid ﬂuid

may stimulate natriuretic peptide release during the bolus phase.

The dose of crysta lloid administered for treatment of shock far

exceeds the volume remaining in circulation after redistribution.

This rapid rise and fall of blood volume during a crystalloid

bolus was demonstrated by Silverstein and others (

7), whereby

the blood volume rapidly rose by 76% during a large bolus

of 0.9% saline before dissipating. Therefore, it is possible that

atria may become “over-stretched” during crystalloid bolus

ﬂuid therapy and release natriuretic peptides, which may then

contribute to systemic EG shedding. Two studies in human

surgical patients have shown a rise in A NP in parallel with

EG biomarker concentrations after ﬂuid loading (125, 126).

These studies showed a signiﬁcant increase in serum syndecan-

1 and hy aluronan concentrations but not heparan sulf ate. One

additional study showed an increase in ANP and all three EG

biomarker concentrations in cardiac surgical patients; in both

those undergoing cardiopulmonary bypass and those undergoing

“oﬀ-pump” procedures (

127). In contrast, one study in people

undergoing hysterectomy (n = 26) showed no signiﬁcant rise

in BNP or biomarker concentrations (syndecan-1 a nd heparan

sulfate) after 25 mL/kg of LRS given over 30 min during surgery

(128). An additional study showed no signiﬁcant rise in BNP or

any EG biomarker concentrations (syndecan-1, heparan sulfate,

hyaluronan) in human surgical patients or healthy volunteers

during a modest ﬂuid load of 3 mL/kg of 20% albumin, though

sample size was small (n = 15 per group) (129). A recent canine

hemorrhagic shock study did not observe a signiﬁcant increase in

plasma ANP concentration after 80 mL/kg of balanced isotonic

crystalloid given over 20 min, however small sample size (n = 6)

and baseline variability may have hampered the ability to detect a

diﬀerence (

111).

Other Considerations Related to EG

Biomarker Type

One of the challenges of interpreting studies that measure EG

biomarker concentrations as the primary assessment of EG

shedding is the variability in choice of biomarker. Across multiple

studies investigating the eﬀect of ﬂuid loading on the EG, it

appears that hyaluronan is consistently increased immediately

after a large volume of crystalloid, or synthetic colloid ﬂuid

in th e setting of hypervolemia (

111, 125, 126). Syndecan-1

concentration signiﬁcantly increases after ﬂuid in humans and

rats (

110, 125, 126, 130–132), but not in all studies (128, 133).

Heparan sulfate has not been shown to signiﬁcantly increase in

humans (12 5, 126, 128) but has been shown to increase in rats

(120, 130, 133). Unpicking t h ese consistencies is diﬃcult due

to diﬀerences across species, study designs, ﬂuid doses, t iming

of intervention and blood s a mpling, and choice of comparator

(other ﬂuid vs. no ﬂ uid vs. sham). Studies may also vary

in whether or not they adjust the biomarker concentration

for the eﬀects of hemodilution, such as indexing to albumin,

hemoglobin, or other tracer concentration in the blood. Although

this may help to account for the diﬀerent pharmacodynamics

of each ﬂuid type, and their variable dilution of blood tracer

components, indexation of biomarker concentrations can create

a margin of error. On closer inspection of individual studies,

inconsistency has been shown within the studies themselves,

showing a signiﬁcant change in one biomarker but not the other

(

125, 126, 133). As mentioned above, the source of circulating

EG biomarkers may not be restricted to th e endothelium

and other sources, such as the interstitium or surface of

other cells, may be contributing. Therefore, a healthy sceptism

should be applied to any conclusion drawn from biomarker

concentrations alone.

COMPARATIVE EFFECTS OF DIFFERENT

FLUID TYPES

Much of t he evidence concerning EG or ESL shedding

after ﬂuid therapy concerns the use of large volumes

of crystalloid ﬂuid. Administering other ﬂuid types

that have less redistribution to the interstitium may

theoretically be associated with less EG shedding. This

section assesses the evidence for the comparative eﬀect of

crystalloid, synthetic colloid, and hy pertonic ﬂuids on the

EG or ESL.

Isotonic Crystalloid Fluid

Multiple rodent studies have shown that administration of large

volumes of cryst a lloid ﬂuid for hemorrhagic shock is associated

with more EG shedding, compared to ﬂuids containing protein

(

109, 110, 120, 130–133). However, it is diﬃcult to compare

interventions in regards to eﬀects on the EG in many of

these studies, as cardiovascular parameters are either not

closely monitored or are diﬀerent between t reatment groups.

Shock itself, without ﬂuid resuscitation, can cause EG shedding

(

109, 110, 131, 134–136), which be c omes a confounder when

comparing resuscitation strategies that provide inequitable blood

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Smart and Hughes Fluids and Endothelial Surface Layer

volume expansion. However, several key studies allow some

assessment of this issue. A murine hemorrhagic shock model

showed that administration of protein-poor ﬂuids, either washed

PRBCs or LRS, was associated with a thinner ESL, whereas

protein-containing ﬂuids (FWB and PRBCs) were not (120).

Cardiovascular parameters after resuscitation appeared similar

across these groups, based on the limited data available. Another

murine hemorrhagic shock model compared seven diﬀerent

ﬂuid strategies: 15 mL/kg of FWB, PRBC, fresh frozen plasma

(FFP) or 5% albumin, 8 mL/kg of 3% saline, 45 mL/kg of

0.9% saline, or 75 mL/k g of LRS (13 0). Shock index, usually

deﬁned by heart rate divided by systolic blood pressure,

was compared before hemorrhage and immediately after ﬂuid

resuscitation within each ﬂuid group. Rats that received either

5% albumin, LRS, 3% saline, or 0.9% saline had a signiﬁcantly

increased shock index after ﬂuid resuscitation, whereas rats

that received FWB, PRBC, and FFP showed no change in

shock index. When comparing only the ﬂuid types that did

not resolve shock, based on shock index, rats that received 5%

albumin had higher ESL thickness and lower EG biomarker

concentrations, compared to the crystalloid groups. Finally,

a murine hemorrhagic shock model used a blood pressure-

targeted resuscitation method for comparing crystalloid ﬂuid

with plasma (

109). Administration of LRS was associated

with a thinner ESL, visualized on electron microscopy, and

decreased endothelial expression of syndecan-1, compared to

administration of plasma.

There is a scarcity of “large animal” models, or those

including pigs, sheep, and dogs, that have assessed ESL shedding

after ﬂuid resuscitation. This may be parti ally due to a

limitation on validated assays available for measuring glycocalyx

biomarkers across these species. Current commercially available

validated options for assessi ng EG shedding are restricted to

circulating hyaluronan concentration. A canine hemorrhagic

shock model did not detect a signiﬁcant diﬀerence in plasma

hyaluronan concentrations when comparing dogs that received

20 mL/kg of FWB (n = 6) with those that received 80

mL/kg of Plasmalyte-148



(n = 6) (

111). Other diﬀerences

in parameters were seen between the crystalloid and colloid

ﬂuid groups in this study, which is discussed further below. In

an ovine endotoxemia model, a pressure-targeted resuscitation

method using vasopressor th erapy was used to compare

either norepinephrine alone or 0. 9% sali ne (40 mL/kg) in

combination with norepinephrine (

137). Although there was

no comparison to resuscitation with a protein-containing ﬂuid,

the comparison of crystalloid ﬂuid to no ﬂuid in the setting

of sepsis provided interesting results. There was no signiﬁcant

diﬀerence in serum hyaluronan concentrations between groups

over time, though sheep that received 0.9% saline showed

a gre at er rate of increase in this biomarker. This greater

increase was not observed directly after the ﬂuid bolus, like

in the aforementioned canine study, but beyond 6 h, which

may have been related to worsening septic shock in this

group. Atrial natriuretic peptide was signiﬁcantly higher at

the end of the ﬂuid bolus in the sheep that received ﬂuid

(see previous discussion on natriuretic peptides). Detection of

between-group diﬀerences was also likely aﬀected by small

sample size in th is study (n = 8 per group), similar t o the

study in dogs.

Synthetic Colloid Fluids

Given t hat crystalloid ﬂuids generally require a larger volume

to expand circulating blood volume to the same extent as

colloid ﬂuids (

6–9), crystalloids cause greater hemodilution

during the ﬂuid bolus and, potentially, greater natriuretic

peptide release. This begs the question if administration

of colloid ﬂuids causes less EG shedding than crystalloid

ﬂuid. Two main types of synthetic colloid are currently in

use: HES and gelatin products. The aforementioned canine

hemorrhagic shock study included both of these ﬂuid types at

20 mL/kg, as comparator ﬂuids to 80 mL/kg of Plasmalyte-

148



(

111). Dogs administered HES had signiﬁcantly lower

plasma hyaluronan concentration immediately after the ﬂuid

bolus, compared to both FWB and crystalloid groups. In

contrast, the group that received 4% succinylated gelatin had

signiﬁcantly increased plasma hyaluronan concentration 40

and 100 min after the end of the ﬂuid bolus, compared to

FWB. Given that inﬂammation may cause EG shedding, these

diﬀerential eﬀects observed between these two colloids may

be due to release of inﬂammatory mediators; HES has been

associated with mitigation of inﬂammation (

138–141) whereas

gelatin has been associated with pro-inﬂammatory eﬀects (142).

However, there were no diﬀerences in plasma concentrations

of inﬂammatory mediators between the two colloid groups in

this study (111). Therefore, the eﬀect of gelatin on hyaluronan

shedding may be a direct eﬀect of the ﬂuid, rather than

pro-inﬂammatory eﬀects, such as washout of the interstitium.

Another hemorrhagic shock study in rats replaced the shed

volume with crystalloid, at either shed volume, two ti mes shed

volume, or three times shed volume, or shed volume with

HES (n = 6 per group) (

141). This study did not identify

any signiﬁcant diﬀerences between groups in response of blood

pressure to ﬂuid resuscitation, organ syndecan-1 expression,

or circulating syndecan-1 concentrati on. A hemorrhagic shock,

stroke volume-targeted resuscitation model in pigs c ompared

balanced crystalloid to HES and found no diﬀerences in post-

ﬂuid serum syndecan-1 or glypican concentrations (6). Blood was

sampled 120 min after commencement of ﬂuids, therefore any

peaks in EG biomarker shedding may have been missed. Given

the limited evidence for diﬀerent eﬀects of colloid ﬂuids on the

EG or ESL, compared to crystalloids, a conclusion cannot be

currently drawn.

Hypertonic Crystalloid Fluids

Hypertonic solutions exert immunomodulatory eﬀects both

in in vitro and animal model studies, including decreased

leucocyte response to pro-inﬂammatory stimuli or circulatory

shock states, when compared to other ﬂuid types (

143–156).

Administration of hypertonic saline to human trauma patients

also reduced inﬂammatory biomarker concentration, compared

to isotonic saline (

157, 158). Several studies have also shown a

reduction in vascular leakage of macromolecules (

147, 151, 155).

Given these eﬀects, it is possible that hypertonic solutions may

have a beneﬁcial eﬀect in regards to reducing ESL shedding.

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Smart and Hughes Fluids and Endothelial Surface Layer

In the rodent hemorrhagic shock model previously discussed

comparing many diﬀerent type of ﬂuids, rats that received 8

mL/kg of 3% saline had a lower increase from baseline in

plasma heparan sulfate concentration, compared to both isotonic

crystalloid groups (45 mL/kg of 0.9% saline or 75 mL/kg of

LRS), and a lower loss of ESL thickness (

130). This diﬀerence

was despite a persistence of an elevated shock index and

hyperlactatemia after resuscitation with 3% saline. Therefore,

although hypertonic saline may have had less impact on the

EG, it may have also provided inadequate ﬂuid resuscitation. A

preliminary randomized clinical trial in people with suspected

sepsis could not demonstrate a diﬀerence between groups in

hyaluronan concentrations after treatment allocation of either

5 mL/kg of 3% saline or 10 mL/kg of 0.9% saline (86).

Serum syndecan-1 concentration decreased after 0.9% saline,

compared to b aseline, whereas serum syndecan-1 concentration

did not change after 3% saline. Though this signiﬁcant diﬀerence

between groups may reﬂect hemodilution, as twice the volume of

0.9% saline was administered, this was not reﬂected in diﬀerences

in hemoglobin concentration. It is unclear what the clinical

signiﬁcance is of this subtle diﬀerence in change in serum

syndecan-1 concentration, whether it reﬂects more syndecan-1

shedding in the hypertonic saline group or not. Limitations of

this study included a low severity of illness and small sample size,

and further studies are needed to clarify the eﬀects of hypertonic

solutions on the EG.

GLYCOPROTECTIVE THERAPIES

The clinical relevance of ESL shedding due to fast or large-

volume ﬂuid administration is yet to be determined. This type

of ﬂuid administration can cause shedding of the ESL, which

may propagate microcirculatory dysfunction, inﬂammation,

and procoagulation. Therefore, there has been much interest

developing in “glycoprotective” ﬂuid strategies or adjunctive

therapies during the resuscitation phase.

Restricting or Avoiding Fluid

Blood volume expansion is th e cornerstone of managing

circulatory shock, with some types of shock an exception

such as cardiogenic shock. Conventionally, this is achieved by

administering large (more than 20 mL/kg) volumes of crystalloid

ﬂuid intravenously as rapidly as possible, or as a bolus (

159, 160).

This serves to increase cardiac preload and improve cardiac

output. The need for this intervention is intuitive for shock

caused by hypovolemia; however, some types of shock have a

component of vasodilation and microcirculatory dysfunction.

This includes states of shock due to a systemic inﬂammatory

response, such as sepsis and blunt trauma. For these types

of shock, improvement of macrohemodynamic variables, such

as cardiac output and blood pressure, may not improve

microcirculatory blood ﬂow in a linear way. This is particularly

pertinent to septic shock, where the role of ﬂuid bolus th erapy

has been called into question, with a movement toward early

vasopressor therapy instead (

161, 162). Further to the lack of

improvement in microcirculatory ﬂow, it has been suggested

that ﬂuid bolus therapy in sepsis can propagate vasodilation,

due to endothelial shear stress, release of nitric oxide, and

direct vasodilatory eﬀects of natriuretic peptides. This may

contribute to some patients becoming refractory to vasopressor

therapy. In an ovine endotoxemia model, administration of 40

mL/kg of 0.9% saline was associated with a signiﬁcantly higher

dose of noradrenaline subsequently administered in order to

maintain blood pressure, compared to sheep that received only

noradrenaline (

137). Given the concern that bolus ﬂuids may

cause harm in sepsis, a preliminary randomized clinical trial

was completed demonstrating feasibility of restricting crystalloid

ﬂuid early in the treatment of sepsis in people (163). Following on

from this, a large multi-center human randomized clinical trial

is underway to compare a restrictive ﬂuid strategy with a more

liberal one in people with early sepsis (164).

It is much less known if restricting crystalloid ﬂuid in

other types of shock may be beneﬁcial. In the setting of

human trauma, discussion concerning restrictive crystalloid

ﬂuid strategies focuses on delayed resuscitation or permissive

hypotension in order to avoid clot disruption and dilutional

coagulopathy before deﬁnitive hemostasis (

4, 165). Although

animal models have shown that ﬂuid resuscitation is likely

beneﬁcial for severe uncontrolled hemorrhage, it increases the

risk of mortality in lower grades of severity of bleeding, compared

to not administering any ﬂuid (166). Hypotensive resuscitation,

in parallel with lower volumes of administered ﬂuids, is also

associated with a reduced risk of mort a lity in human trauma

patients (167, 168), th ough the evidence is mixed (169). This

beneﬁt is likely related to improved coagulation and clot stability;

it is unknown if there are also beneﬁts associated with reducing

endothelial dysfunction. Currently, the recommendation in

human trauma medicine is for limitation of “clear” ﬂuid and

early, yet judicious, administration of blood products. This

includes packed red blood cells, plasma, and platelets in parallel

during resuscitation. The early administration of plasma may

not only serve to limit dilutional coagulopathy but also assist

with repairing the ESL. It is diﬃcult to translate this practice

to veterinary medicine, given the cost of transfusion, limited

availability, and variability in blood banking practices. Given

there are many factors entering into the cost to beneﬁt assessment

of administering plasma or albumin products to individuals,

theoretical beneﬁts of plasma for the ESL are of lesser import ance,

until more is known. Also, caution should be a pplied when

restricting crystalloid ﬂuid to any patient with shock, as the

beneﬁts of a restrictive strategy may be only relevant to certain

patient populations, such as sepsis. This concept was highlighted

by a recent randomized clinical trial in human surgical patients

whereby participants randomized to receive a restrictive ﬂuid

strategy had a signiﬁcantly higher rate of acute kidney injury and

surgical site infection, compared to those randomized to a liberal

ﬂuid strategy (

170).

Slowing Fluid Administration

Fast ﬂuid administration serves to rapidly improve clinical

signs and macrohemodynamic variables, and has been the

bread-and-butter of emergency and critical care medicine

for decades. However, normalization of macrohemodynamic

variables in people does not always translat e to improved

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Smart and Hughes Fluids and Endothelial Surface Layer

microcirculatory ﬂow (47–49, 51). This is termed a lack of

hemodynamic coherence (171). Intravenous ﬂuid delivered

rapidly during the stabilization phase may be contributing to

persistence of distur bed microcirculatory ﬂow. When v iewing

the microcirculation using sidestream dark ﬁeld microscopy

during anesthesia in pigs, fast ﬂuid administration (20 mL/kg/hr)

for 3 h was associated with development of heterogeneity in

capillary blood ﬂow, compared to “standard” anesthesia ﬂuid

rates (5 mL/kg/hr) (

172). Although a direct relationship between

estimated ESL thickness and microcirculatory ﬂow has not

been established in the clinical setting (46), it is possible that

ampliﬁcation of ESL shedding caused by fast ﬂuid administration

may be contributing to microcirculatory dysfunction. It is

possible that slower ﬂuid administration may achieve the same

resuscitation end-points while avoiding some of these deleterious

eﬀects on the endothelium. This concept has been explored

in a handful of studies. In a rodent model where 40% of the

blood volume was removed by atraumati c hemorrhage, rats were

assigned to receive no ﬂuid, rapid ﬂuid (0.9% saline at three times

shed volume over 30 min), or slow ﬂuid (0.9% s aline at three

times shed volume over 12 h) (173). Rats in the fast ﬂuid group

showed higher inﬂammatory cytokine concentrations early in

the study and some markers of worse outcome over the latter

part of the study, including lower mean arterial blood pressure,

higher blood glucose and lactate concentrations, and an increase

in markers of lung injury, compared to rats in the slow ﬂ uid

group. As the rats were euthanased after 24 h, i t is unknown how

their recovery progressed. In a human open randomized clinical

trial, including 50 surgical and 16 septic patients, no signiﬁcant

diﬀerence was found in estimated ESL thickness between patients

that received fast crystalloid administration (5–10 min) and slow

crystalloid administration (20–30 min) (

48). However, an overall

decrease in estimated ESL thickness after ﬂuid administration

was observed. Given that there is some evidence t hat rapid ﬂuid

administration (i.e., within 10 min) may not provide any clinical

beneﬁt over slower ﬂuid administration (i.e., within 20 to 60 min)

(8, 174), larger clinic al trials are justiﬁed to explore eﬀects of ﬂuid

rate on the microcirculation and clinical outcomes.

Choice of Clear Fluid Type

Intense ongoing debate surrounds the choice of ﬂuid for critically

ill patients, including crystalloid, synthetic colloid, and protein-

containing solutions. These conversations surround relative ﬂuid

eﬀectiveness and adverse eﬀects, many of which are still yet to

be fully elucidated for clinical relevance in certain spe ci es or

disease subsets. Adding in the potential beneﬁt or detriment of

certain ﬂuid types to preservation of the ESL and optimizing

microcirculation is still premature. Bearing t hat in mind, the

current evidence supports mitigating hemodilution and avoiding

hypervolemia in order to optimize ESL recovery. There is

theoretical beneﬁt for the ESL to the use of protein-containing

solutions, such as plasma and albumin, rather than “clear” ﬂuids,

but this must be carefully considered against any detrimental

eﬀects of blood products, including ﬁnancial cost. At this stage,

no clear recommendation can be made for veterinary medicine,

and more clinical research is required.

Adjunctive Therapies

Many drugs have been explored as to their protective or

resurrecting actions for the ESL and have been reviewed in detail

elsewhere (

175, 176). These therapies may become viable in the

future for use during the optimization and stabilization phase

of ﬂuid resuscitation (177). Therapies that are in current use

in veterinary medicine that have been shown to have beneﬁcial

eﬀects on t he EG or ESL include albumin solution, plasma,

hydrocortisone, heparin, and N-acetylcysteine.

CONCLUSIONS

The coating of endothelium by the EG and its associated

molecules serves many functions in the body, both int act

and when denuded. Infusion of large volumes of ﬂuid causes

disruption of this layer, which may propagate interstitial

edema and inﬂammation. There is some evidence that slow

ﬂuid administration or restricting the volume of crystalloid

ﬂuid for shock resuscitation may beneﬁt the patient. E xactly

how to do this and for which patient subset this strate gy

beneﬁts in veterinary medicine remains to be identiﬁed. Clinical

veterinary research assessing the eﬀect of glycoprotective therapy

or strategies is currently limited by a lack of validated

EG biomarker assays and aﬀord able, reliable technology for

estimating ESL thickness in vivo. Further work is required

on developing validated, reliable EG biomarker assays and

determining the relationship between these biomarkers and

shedding of the ESL in vivo, especially for dogs and cats.

Despite these current limitations, future research directions

should also focus on strategies limiting crystalloid ﬂuid

volumes, especially in the setting of sepsis and early

vasopressor drug therapy.

AUTHOR CONTRIBUTIONS

LS: preparation of manuscript and ﬁnal approval.

DH: revision of manuscript and ﬁnal approval. Both

authors contributed to the article and approved the

submitted version.

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Conﬂict of Interest: The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be construed as a

potential conﬂict of interest.

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