Shedding of endothelial glycocalyx in severely septic mice leads to leukocyte- endothelial interactions and vascular hyperpermeability

Anesthesiology

Shedding of endothelial glycocalyx in severely septic mice leads to leukocyte- endothelial interactions and vascular hyperpermeability

--Manuscript Draft--

Manuscript Number:

Full Title: Shedding of endothelial glycocalyx in severely septic mice leads to leukocyte- endothelial interactions and vascular hyperpermeability

Article Type: Critical Care Medicine

Section/Category:

Corresponding Author: Akira Ushiyama, Ph.D.

National Institute of Public Health Wako City, Saitama JAPAN Corresponding Author Secondary

Information:

Corresponding Author's Institution: National Institute of Public Health Corresponding Author's Secondary

Institution:

First Author: Hanae Kataoka, D.D.S., Ph.D.

First Author Secondary Information:

Order of Authors: Hanae Kataoka, D.D.S., Ph.D.

Akira Ushiyama, Ph.D.

Yoshihiro Akimoto, Ph.D.

Sachie Matsubara Hayato Kawakami, Ph.D.

Takehiko Iijima, D.D.S., Ph.D.

Order of Authors Secondary Information:

Abstract: Background: The endothelial surface layer (ESL) regulates vascular permeability to maintain fluid homeostasis in vital organs. The glycocalyx (GCX) seems to be a functional component of the ESL, and GCX disorders supposedly trigger pathological hyperpermeability. Since the GCX has a complex and fragile ultrastructure, its function remains unclear. Here, the GCX was visualized in a vital organ, and the function of this component was confirmed in a severe sepsis model.

Methods: A mouse dorsal skinfold chamber technique was used to observe the subcutaneous microcirculation. Wheat germ agglutinin was used to visualize the behavior of the ESL involving the GCX. Morphological changes in the GCX were compared using both intravital microscopy (IVM) and electron microscopy (EM) among three groups: glycosidase administration, LPS-induced sepsis, and a control. The leukocyte-endothelial interactions and the in vivo vascular permeability were also examined.

Results: The illuminated part of the ESL observed using IVM was morphologically confirmed as a GCX structure using EM. This structure was also biochemically confirmed as the GCX. Increased leukocyte-endothelial interactions on the

Suggested Reviewers: Svensen Christer, Dr.

Karolinska Institutet

[email protected]

He is an expertise of fluid dynamics in clinical and basic science. His review would be brought from wide range of knowledge of this field.

Birgitte Brandstrup, M.D., Ph.D.

Hvidovre University Hospital [email protected]

She has accomplished an overview review of fluid management including basic science. We can expect unbiased advise.

Opposed Reviewers:

N ^ATIONAL I ^{NSTITUTE OF} P ^UBLIC H ^EALTH

2-3-6, Minami, Wako City Saitama 351-0197, Japan Tel.+81-48-458-6111 Fax. +81-48-469-1573 Website http://www.niph.go.jp

January, 6, 2016

James C. Eisenach, M.D.

Editor-in-Chief, Anesthesiology

Dear Editor:

Please find enclosed our manuscript titled “Shedding of endothelial glycocalyx in severely septic mice leads to leukocyte-endothelial interactions and vascular hyperpermeability” which we request you to consider for publication as an Original Investigation specialized on “critical care medicine” in Anesthesiology.

The endothelial surface layer (ESL) regulates vascular permeability to maintain fluid homeostasis in vital organs.

The glycocalyx (GCX) seems to be a functional component of the ESL, and GCX disorders supposedly trigger pathological hyperpermeability, resulting in edema formation. Since the GCX has a complex ultrastructure, its function remains unclear. Here, the GCX was visualized in a vital organ, and the function of this component was confirmed in a severe sepsis model.

A part of this work was presented at the International Anesthesia Research Society Meeting (IARS2015) in Hawaii, on March 2015, and at the 10th World Congress for Microcirculation (WCMic2015) in Kyoto, Japan, on September 2015. In IARS2015, we won the Best of Category Award for the Anesthetic Pharmacology category and the Kosaka Award Finalists. In WCMic2015, we were presented the Poster Award.

This manuscript has not been published elsewhere and is not under consideration by another journal. We have approved the manuscript and agree with submission to Anesthesiology. There are no conflicts of interest to declare.

The manuscript has been carefully reviewed by an experienced editor whose first language is English and who specializes in editing papers written by scientists whose native language is not English.

We believe that the findings of this study are relevant to the scope of your journal and are beneficial for all readers if it is published as one of Anesthesiology.

We look forward to hearing from you at your earliest convenience.

Sincerely,

Akira Ushiyama, Ph.D.

Department of Environmental Health, National Institute of Public Health

2-3-6 Minami, Wako, Saitama 351-1097, Japan

Title：

Shedding of endothelial glycocalyx in severely septic mice leads to

leukocyte-endothelial interactions and vascular hyperpermeability

Authors

Hanae Kataoka, D.D.S., Ph.D.

; Akira Ushiyama, Ph.D.

*;

Yoshihiro Akimoto, Ph.D.

; Sachie Matsubara

;

Hayato Kawakami, Ph.D.

; Takehiko Iijima, D.D.S., Ph.D.

Affiliations

1) Department of Perioperative Medicine, Division of Anesthesiology, Showa

University, School of Dentistry, Tokyo, Japan

2) Department of Environmental Health, National Institute of Public

Health, Saitama, Japan

3) Department of Anatomy, Kyorin University School of Medicine, Tokyo,

Japan

Manuscript (Title Page, Abstract, Body, References) OR single file with all manuscript elements, including figures (initial

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4) Laboratory for Electron Microscopy, Kyorin University School of Medicine,

Tokyo, Japan

* Corresponding Author

Akira Ushiyama, Ph.D.

Department of Environmental Health, National Institute of Public Health

2-3-6, Minami, Wako city, Saitama 351-1097, Japan

Phone +81-48-458-6257

E-mail:

Disclosure of funding

This study was supported by Japan Society of the Promotion of Science

(JSPS) KAKENIHI, Grant-in-Aid for Scientific Research (C) 25463145 to TI.

Number of Words

Abstract, 244 words; Introduction, 479 words; Discussion, 1177 words

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Running Head

Glycocalyx shedding and hyperpermeability in sepsis (46 characters)

Declaration of COI

The authors declare no competing interests.

Footnotes

A part of this work was presented at the International Anesthesia Research

Society Meeting in Honolulu, Hawaii, on March 22, 2015, and at the 10

World Congress for Microcirculation in Kyoto, Japan, on September 26, 2015.

What we already know about this topic:

The endothelial surface layer is covered by a glycocalyx (GCX). This

component is thought to be a permeability barrier, and its pathological

breakdown is accompanied by hyperpermeability.

What this article tells us that is new:

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In vivo microscopy and simultaneous ultrastructural examinations have

revealed that the GCX prevents the vascular leakage of macromolecules.

GCX degradation induced by sepsis resulted in the leakage of

macromolecules.

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Abstract

Background: The endothelial surface layer (ESL) regulates vascular

permeability to maintain fluid homeostasis in vital organs. The glycocalyx

(GCX) seems to be a functional component of the ESL, and GCX disorders

supposedly trigger pathological hyperpermeability. Since the GCX has a

complex and fragile ultrastructure, its function remains unclear. Here, the

GCX was visualized in a vital organ, and the function of this component was

confirmed in a severe sepsis model.

Methods: A mouse dorsal skinfold chamber technique was used to observe

the subcutaneous microcirculation. Wheat germ agglutinin was used to

visualize the behavior of the ESL involving the GCX. Morphological changes

in the GCX were compared using both intravital microscopy (IVM) and

electron microscopy (EM) among three groups: glycosidase administration,

LPS-induced sepsis, and a control. The leukocyte-endothelial interactions

and the in vivo vascular permeability were also examined.

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Results: The illuminated part of the ESL observed using IVM was

morphologically confirmed as a GCX structure using EM. This structure was

also biochemically confirmed as the GCX. Increased leukocyte-endothelial

interactions on the endothelium and molecular hyperpermeability to the

interstitium were also observed following GCX shedding in vivo.

Conclusions: We visualized an illuminated part of the ESL layer using IVM

that was confirmed as the GCX using EM. Severe sepsis induced the

morphological shedding of the GCX, increasing the leukocyte-endothelial

interactions. This pathological change consequently led to vascular

hyperpermeability. This study showed that GCX shedding induced by sepsis

is closely correlated with leukocyte-endothelium interactions and vascular

hyperpermeabilty.

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Introduction

A recent advancement of intraoperative fluid therapy is based on

preventing the postoperative accumulation of interstitial fluid

and

titrating intraoperative infusions for individual patients.

This concept is

supported by the emerging revised concept of Starling’s law

and a

reevaluation of the concept of the ‘third space’.

According to the revised

concept of Starling’s law,

filtration (the outward flow of water from the

vessel) should dominate absorption (the inward flow of water from the

interstitium). Infused fluid mostly accumulates in the interstitium and

contributes to edema formation.

This concept is actually supported by the

clinical postoperative outcome.

Postoperative weight gain is closely

correlated with postoperative morbidity, and such weight gain is apparently

caused by fluid overload.

The luminal side of the vessel is covered by the glycocalyx (GCX), which

plays an important role in forming a tight barrier affecting vascular

permeability. The GCX seems to be fragile, and its destruction is thought to

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enhance plasma leakage, leading to edema formation and fluid retention in

various pathological conditions.

This key component in the regulation of

vascular permeability may also play a key role in preventing postoperative

fluid retention.

The endothelial surface layer (ESL), including the GCX, is altered in

response to surgical stress, anesthesia, and other intraoperative factors.

Sepsis and bacteremia may be an appropriate model to reproduce the

derangement of this tight barrier.

The series of events leading to the

activation of endothelial components and leukocyte recruitment can be

pharmacologically reproduced. Endothelial activation triggers an

inflammatory response to infection. Leukocytes are a key player in

interactions with the ESL. Primed leukocytes are activated and adhere to

the endothelium, producing microthromboses, and microvascular

hyperpermeability can cause organ dysfunction during sepsis. The

attenuation and dysfunction of the microvascular endothelium leading to

multiple organ failure is now recognized as underpinning the

pathophysiology of sepsis.

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The GCX is positioned as an interface between circulating cells and the

endothelial surface. As the primary interface between blood and vessel walls,

the GCX may play a buffer role in preventing interactions between flowing

cells and the endothelium, when unnecessary. Once an immunological

process becomes necessary, the GCX may become deformed, opening the gate

for interactions. Although aspects of GCX degradation have been studied

intensively over the last decades, the behavior of the GCX in vivo has not yet

been investigated.

Since the GCX is a component of a very thin layer, putatively less than 500

nm,

most research has utilized electron microscopy (EM) after the fixation

of this structure. To investigate its function in a vital organ, an in vivo model

is needed. We have developed a model using intravital microscopy (IVM) to

examine a mouse dorsal skinfold chamber (DSC) and have observed the

time-dependent behavior of the GCX. We also examined the function of the

GCX using a septic mouse model that putatively reflects the

pathophysiological contribution of the GCX to vascular permeability and

leukocyte-endothelial interactions.

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Materials and Methods

Animals and ethical statement

Experiments were performed using 8-to-10-week-old male BALB/c mice

(Japan SLC, Inc., Shizuoka, Japan) weighing 24-28 g. The mice were kept in

an isolator rack (SuperMouse 1400

; Lab Products Inc., Seaford, DE) under

controlled temperature and humidity (23

C ± 1

C and 50% ± 10%,

respectively) with a 12-h light-dark cycle. They were given free access to

water and standard chow.

All the experimental protocols were approved by the Committee for

Animal Experiments at the National Institute of Public Health (protocol

number 26-002) and were in accordance with all the guidelines and laws for

animal experiments in Japan.

Chemicals

Fluorescein isothiocyanate (FITC)-labeled WGA lectin from Triticum

vulgaris, lipopolysaccharide (LPS) from Escherichia coli O26:B6,

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FITC-labeled dextran (average molecular weight, 40kDa; FITC-dex40),

tetramethyl rhodamine-labeled dextran (average molecular weight, 75 kDa;

TMR-dex75), hyaluronidase from Streptomyces hyalurolyticus, heparinase

III from Flavobacterium heparinum, Lanthanum (III) nitrate hexahydrate,

and glutaraldehyde were purchased from Sigma-Aldrich Co., MO.

Horseradish peroxidase (HRP)-conjugated WGA was purchased from Vector

Laboratories, Inc., Burlingame, CA. Ketamine hydrochloride, xylazine

hydrochloride, N-acetyl-D-glucosamine, and rhodamine 6G were purchased

from Wako Pure Chemicals Industries, Ltd., Osaka, Japan. Tissue-Tek OCT

Compound and 3,3-diaminobenzidine (DAB) were purchased from Sakura

Finetek, Zoeterwoude, the Netherlands, and Nichirei Co., Tokyo, Japan,

respectively.

Identification of ESL

In vivo GCX imaging using WGA-lectin

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For microvascular observations using the DSC technique, a non-metal

DSC assembly was introduced into each of the recipient mice. This procedure

has been described previously by Ushiyama et al.

The DSC implantation

was performed at least 3 days prior to observation to avoid the influence of

acute inflammation because of surgery.

In a pilot study, we compared seven lectins from various plants to

delineate the ESL.

As a result, FITC-WGA was found to have the most

appropriate properties for the present study’s purpose.

The procedure used to create the FITC-WGA model has been described in

a previous article.

Briefly, FITC-WGA was solubilized in saline, and a bolus

of this solution (6.25 mg/kg body weight) was administered via the tail vein

30 minutes prior to obtaining the images. Vascular images within the DSC

were observed using an all-in-one epi-fluorescence microscope (model

BZ-9000; Keyence Co, Osaka, Japan) equipped with a high-sensitivity CCD

camera and a 20× long working distance objective lens (S PlanFL ELWD

ADM 20xC, N.A. = 0.45; Nikon Co., Tokyo, Japan).

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EM examination of WGA-lectin binding

Male BALB/c mice (n = 6) were deeply anesthetized by the intramuscular

injection of a cocktail of ketamine (90 mg/kg body weight) and xylazine (10

mg/kg body weight) and were perfused via the heart using a perfusion pump

at a speed of 7 mL/min with 2.5% glutaraldehyde in phosphate-buffered

saline (PBS; 35 mL per mouse) after perfusion with PBS to remove all the

blood from the circulatory system. After perfusion, the dorsal skin was

excised and immersed in 2.5% glutaraldehyde/PBS, then stored for 24 hours

at 4°C for postfixation. The excised skin was transferred to 10% sucrose/PBS

and was equilibrated for 24 hours. This replacement was continued in a

stepwise fashion up to 20% sucrose/PBS. The skin was embedded with

Tissue-Tek OCT Compound, and sections (thickness, 10 μm) including small

vessels were serially cut in a cryostat (CM1950; Leica Biosystems Nussloch,

Germany). These sections were kept on an MAS-coated slide glass

(Matsunami Glass Corp., Osaka, Japan) for 3 hours at room temperature,

then reacted with HRP-WGA (8 μg/mL) for 24 hours at 4°C in a moisture box.

Sections were stained with chromogen (DAB-H

O

) and were washed with

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PBS and immersed in 1% glutaraldehyde/PBS. After washing with PBS, the

sections were osmicated, then dehydrated through a graded series of ethanol

and embedded in Epon 812. Ultrathin sections were cut, slightly stained

with lead citrate, and examined with EM (JEM 1011; JEOL, Tokyo, Japan).

Electron microscopic images were taken using a KeenView III 1k x 1k CCD

camera (Olympus Co., Tokyo, Japan). Some sections were incubated with

HRP-WGA in the presence of hapten sugar (0.1 M N-acetyl-D-glucosamine;

GlcNAc) to confirm the specificity of the WGA staining. Other sections were

incubated with DAB-H

O

solution only to detect the endogenous peroxidase

activity.

Morphological analysis of GCX under biochemical digestion with glycosidase

Animals were divided into two groups: a control group (n = 10) and a

glycosidase-administration group (n = 10). To confirm whether the WGA

lectin staining was attributable to binding with the hapten sugar (GlcNAc),

we compared the WGA staining with or without the intravenous

administration of glycosidase: a mixture of hyaluronidase (50 units/mouse)

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and heparinase (1 unit/mouse) to mice at 2 hours prior to the observation.

Images were obtained using both EM (n = 3) and IVM (n = 10).

Prior to the observation using IVM, a 100-µL bolus of FITC-WGA (6.25

mg/kg) for staining the GCX and a 100-µL bolus of TMR-dex75 (3% w/v) for

obtaining the blood flow images were injected into the tail vein. Thirty

minutes after the lectin and dextran injections, each mouse was anesthetized

with 1.5% isoflurane and was mounted on a fluorescence microscope

(BZ-9000). The fluorescent images (680 pixels x 512 pixels) were captured for

at least 3 regions of interest per chamber using a fluorescent filter for FITC

and for TMR. To compare the fluorescent intensity of the lectin staining, the

exposure time and the gain level of the camera were set so as to be identical

for all the images.

EM was used to examine the arterioles and venules located in the dorsal

skin. Specimens were prepared from the control group and the

glycosidase-administration group (n = 3 or more per group).

According to the method of Vogel et al.,

lanthanum fixation was used

because the lanthanum ion (La

) binds to the negatively charged glycocalyx.

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Deeply anesthetized mice were perfused via the heart using a perfusion

pump at a speed of 7 mL/min with fixative/staining solution (2%

glutaraldehyde, 30 mM HEPES buffer, 2% La(NO

)

, 5 min) after the

removal of all blood by perfusion with PBS. Diced pieces of the dorsal skin

were immersed at 4°C in the fixative solution (2% glutaraldehyde, 30 mM

HEPES buffer) for 24 hours, then used for EM observations in a manner

similar to the method described above.

Morphological and functional analysis of GCX in LPS-induced septic model

LPS-induced septic model

In this study, severe sepsis was induced by the intraperitoneal

administration of lipopolysaccharide (LPS) (2 mg/kg at 0 and 18 hours)

according to previous studies.

To evaluate the severity of sepsis, we set several physiological endpoints as

follows: 1) reduction of body weight, 2) decrease in blood pressure, 3)

leukocytosis and thrombopenia, 4) decrease in blood albumin level, and 5)

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mortality rate of 40%-50% within 48 hours. The systolic blood pressure (SBP)

and the diastolic blood pressure (DBP) were measured while the mice were

conscious using a computerized tail-cuff system (BA-98A; Softron Co., Tokyo,

Japan) at each of the designated time points. For the biochemical and

hematological analyses, whole blood was collected from the left ventricle of

surviving mice at 48 hours and was analyzed using a biochemical analyzer

(VetScan VS2; Avaxis Inc., Union City, CA) and a fully automated 5-part

differential cell counter (VetScan HM5; Avaxis Inc.), respectively.

Both perfusion fixation for EM and observation using IVM were examined

at 24 hours after the first LPS administration.

IVM examination of ESL and analysis of thickness index

The IVM images obtained for the LPS model and the control model were

used to measure the thickness of the ESL (n = 20 from LPS group, n = 10

from control group).

The images were transferred to BZ image analysis software (Keyence Co.)

for quantification. A straight section of endothelium was chosen to measure

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the thickness. A two-dimensional graph of the intensities of pixels along the

perpendicular to the section was delineated. The width of the plot profile at

the level of the half of the peak was measured (full width at half maximum

[FWHM]). The length was considered as the length of the ESL. We defined

the mean FWHM as the ESL thickness index (μm) for the endothelium.

EM examination of GCX and analysis of thickness

The thickness of the GCX was measured using images obtained from the

LPS group (n = 3) and the control group (n = 3). The images were transferred

to the analysis software (Image J NIH; Bethesda, MD). The area of

lanthanum staining was calculated, and the length of the membrane was

simultaneously measured. The ratio between the two measures, or the

surface area/length measure, was defined as the mean thickness (nm) of the

GCX. All the analyses were performed in a blinded manner by HK and an

assistant.

Observation of adherent and rolling leukocytes in IVM

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The fluorescent marker rhodamine 6G was used to visualize leukocytes in

vivo.

Rhodamine 6G was dissolved in saline at a final concentration of 0.02

mg/mL on the day of the experiment. The solution was filtered through a

0.22-µm membrane filter (syringe-driven filter unit, Millex-GN; Millipore Co.,

Billerica, MA) before each experiment to remove the unsolved substances. A

prepared solution (100 µL) was injected into the tail vein at 5 min before

image recording. During the observation, the animals were anesthetized

with isoflurane inhalation and placed on the stage of a fluorescence

microscope (BZ-9000). Using a fluorescence filter (excitation, 540 nm;

emission, 605 nm; and dichroic mirror, 565 nm), the leukocytes were

illuminated with rhodamine 6G. We randomly choose a region of interest

(ROI) that met the requirements for diameter (about 30 µm for arterioles or

40 µm for venules) and length (100 µm). Images of the flow were recorded for

30 seconds at a video rate of 30 frames/second for each ROI, and the rolling

and adherent leucocytes were counted in the images. Adherent leukocytes

were defined as those that adhered to the endothelium, and rolling

leukocytes were defined as white cells moving on the vessel wall at a velocity

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slower than that of the erythrocytes. The adherent and rolling counts were

calibrated for a vascular diameter of 100 µm.

Analysis of vascular permeability in IVM

The permeability analysis was performed using a method described by

Alfieri et al.,

with some modifications. Seven mice (3 for the septic model, 4

for the control group) were anesthetized with isoflurane inhalation at 24

hours after the first LPS administration. A bolus (200 μL) of a cocktail

containing 0.5% (w/v) FITC-dex40 and 1.5% (w/v) TMR-dex75 was

intravenously injected, and each animal was placed on the stage of a

fluorescence microscope (BZ-9000). The microvasculature in the DSC was

observed through a 20× objective lens, and 5 regions of interest per mouse

were set using the stage mapping function; then, fluorescence images of

FITC-dex40 and TMR-dex75 for the same ROI were captured every 30 min

for up to 120 min. To compare the fluorescent intensities of the different

images, the exposure time and gain level of the camera were standardized

for all the images.

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The image analysis software Image J was used for the off-line analysis of

the fluorescent images. The Image J software assigned an integer value to

the brightness of the fluorescence using an arbitrary 8-bit gray scale (range,

0 to 255) at 3 randomly selected, distinct interstitial areas (900 μm

) for each

ROI. The difference in the value from the first image was considered to be an

index of vascular permeability.

Statistical procedure

The measured data were presented as the mean ± SD, with n indicating

the number of experiments. Comparisons were made using the Student t-test.

P < 0.05 was considered to be significant. The statistical analyses were

performed using SPSS Statistics software (Japan IBM Co., Tokyo, Japan).

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Results

WGA lectin bound to the ESL was identified using electron microscopy

Electron microscopic images of vessels stained with HRP-WGA were

obtained (fig. 1). Images of the HRP-WGA staining showed the presence of

positive staining as a result of peroxidase activity, whereas positive staining

of the peroxidase substrate was not observed without HRP-WGA staining.

These results suggest that the HRP-WGA-positive component was bound to

the luminal surface of the endothelium, which is thought to localize

glycosaminoglycans (GAGs) of the GCX.

Morphological analysis of GCX under biochemical digestion with glycosidase

Morphological images with/without glycosidase administration were

obtained using both IVM and EM (fig. 2, fig. 3).

In vivo images of the microvasculature were obtained using IVM (fig. 2).

The FITC-WGA lectin signal was present along the luminal surface of the

endothelium in the group of control mice (fig. 2A, 2B), whereas the

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intravenous administration of glycosidase attenuated the fluorescence signal,

suggesting that some sugar moieties of ESL were lost (fig. 2C, 2D).

The lanthanum-positive GCX layer and caveola were identified in the

control mice using EM (fig. 3A, 3B). In the glycosidase administration group,

the degradation of the glycocalyx layer was also observed (fig. 3C, 3D). These

observations coincided with the IVM results, suggesting that WGA lectin

recognizes some part of the GAGs.

Septic conditional model

We confirmed that a severe septic conditional model could be properly

established using LPS administration. The body weight and the survival rate

for 48 hours were monitored. The body weight decreased significantly in the

LPS administration group by approximately 15%, compared with that in the

control group. A high survival rate (94%) for the first 24 hours was also

confirmed. The survival rate at 48 hours after LPS administration was

47.4%, although that of the control group was 100%. Both the systolic blood

pressure (SBP) and the diastolic blood pressure (DBP) decreased with time

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after LPS administration. These data are shown as Supplemental Digital

Content 1, Figure 1.

In the surviving mice, blood samples were collected from the heart under

deep anesthesia for examination at 48 hours after the first LPS

administration. We confirmed the presence of leukocytopenia,

thrombocytosis, and low albuminemia, manifesting as severe sepsis. These

data are shown as Supplemental Digital Content 1, Figure 2.

FITC-WGA lectin binding under an intravital microscope and its associated

morphological changes under septic conditions

In vivo vasculature images were recorded using IVM (fig. 4). FITC-WGA

lectin-positive signals were recognized along the luminal surface of the

endothelium in the control mice (fig. 4A, 4B). However, this linear and

marginal FITC-WGA lectin binding to the vessels was intensively reduced in

the septic mice (fig. 4C, 4D). Although ESL was lost in both the

glycosidase-administered and septic condition groups, the extent of the loss

in fluorescent intensity and the thickness of the layer in the septic condition

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group were obviously greater than those in the glycosidase-administered

group.

Using these images, we calculated the thickness index of the ESL (fig. 4E).

The thickness index in the septic condition group decreased to 30%,

compared with that in the control group, meaning that the GCX layer was

disrupted in the septic mice.

Electron microscopy observations of GCX in septic mice

Morphological observations of the GCX under septic and control conditions

were performed using EM (fig. 5). EM images of the capillaries in control

mice revealed a lanthanum-positive GCX layer and caveola (fig. 5A, 5B).

However, these morphological structures were obviously damaged, and most

of the GCX layer had disappeared from the endothelium under septic

conditions (fig. 5C, 5D).

To quantify the thickness of the GCX in the control and LPS-induced

septic mice, we selected postcapillary venules (fig. 6A, 6B) and calculated the

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thickness using image analysis software. The mean thickness of the septic

group was significantly less than that of the control group (P < 0.05, fig. 6C).

Loss of GCX under septic conditions induced leukocyte adhesion in the

subcutaneous microcirculation

In the control mice, most of the leukocytes did not interact with the

endothelium, although only a few leukocytes adhered to the vessel walls

transiently (so called “rolling”). Under septic conditions, however, the

number of adherent leukocytes increased significantly, compared with that

in the control mice, in both precapillary arterioles (fig. 7A, 7B) and

postcapillary venules (fig. 7C). Furthermore, some leukocytes under septic

conditions tended to adhere to the endothelium firmly, exhibiting a constant

resistance to the wall shear force of the flowing blood. This phenomenon may

be attributable to the loss of endothelial GCX, activating adhesion molecules

on the endothelial surface. The video observations are shown as

Supplemental Digital Content 1, Videos 1-4.

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Increased microvascular permeability under septic conditions

LPS-treated septic mice showed a significant increase in the fluorescence

intensity of FITC-dex40 and TMR-dex75 in non-vascular tissue, compared

with the control mice, at each time point (fig. 8).

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Discussion

We have demonstrated that the morphological attenuation of the

endothelial GCX is closely linked to functional changes in vascular

permeability. GCX shedding induced by septic conditions led to the

hyperpermeability of macromolecules and an increase in

leukocyte-endothelial interactions in an in vivo window model.

The endothelial GCX is lined on the luminal surface of the endothelium

and is thought to be an important regulator of endothelial function. The GCX

forms a luminal mesh that provides endothelial cells with a framework to

bind plasma proteins and soluble GAG.

The GCX itself is inactive, but

once plasma constituents are bound, it forms the physiologically active

ESL.

The maintenance of a selective endothelial barrier that regulates fluids,

proteins, and cellular extravasation is essential for normal organ

function.

However, the relevance of the GCX or the ESL to barrier

function has not been explored because the GCX has been regarded as a tiny

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and fragile structure. Many studies have been performed using transmission

electron microscopy (TEM) to determine the GCX thickness,

whereas

IVM studies have mainly focused on pathophysiological GCX functions, such

as molecular permeability, etc.

We would like to emphasize that in the

present study, we were able to confirm the GCX morphology and function

simultaneously under both physiological and pathological conditions using

EM and IVM.

Fluorescently labeled lectin was used to visualize the GCX or ESL directly

in vivo.

We confirmed that FITC-WGA was the most appropriate lectin

for visualizing the ESL in the microvasculature of mouse cutaneous tissue

under IVM in our previous pilot study.

The binding of HRP-WGA to the

luminal surface of the endothelium in both arterioles and venules was

confirmed (fig. 1) using EM. To determine whether this binding was specific

to the WGA lectin, attenuation induced by a glycosidase challenge was

examined using both EM and IVM. The intravital observations showed that

glycosidase (hyaluronidase and heparanase) administration weakened the

FITC-WGA binding to the endothelium, resulting in a partial loss of the ESL

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(fig. 2). Additionally, EM observations showed that glycosidase partially

digests the sugar moieties of GAGs (fig. 3). These results suggest that

hyaluronidase and heparanase are essential for the digestion of

polysaccharide chains in the GCX, but not sufficient for their complete

digestion. Based on this evidence, the GCX has been shown to be degraded

by a variety of enzymes, and these enzymes should be overexpressed to

degrade the GCX completely under pathophysiological conditions.

A systemic inflammatory response to microbial infection can lead to

serious pathological conditions, such as sepsis.

Recent clinical studies have

also shown that the GCX is altered in critically ill patients, particularly

under septic conditions, and concluded that loss of the GCX may predict

microcirculatory dysfunction and hypoperfusion.

Consistent with previous

microscopic observations in glomerular endothelium,

our study also

demonstrated that LPS-induced sepsis attenuated the positive staining of

FITC-WGA to the luminal surface of the endothelium when observed using

IVM, and the degradation of the GCX layer was observed using EM. This

positive layer was significantly reduced under septic conditions (fig. 6). The

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thicknesses estimated using IVM and EM were notably different. The

thicker layers observed using IVM may be attributable to the fact that GAGs

in the GCX are enriched with water and soluble proteins, whereas the

lanthanum-positive layer in the EM might reflect a dehydrated condition

resulting from specimen preparation. In fact, a different fixation method that

preserves the high water content of the GCX layer resulted in measurements

as long as 6 μm for rat fat pad and 11 μm for bovine aorta.

As described

above, TEM can provide information on the charge, composition, and

structure of the GCX; however, results vary greatly depending on the fixation

and staining methods that are used. Therefore, the GCX should be visualized

in vital organs to understand its role, function, and biological significance.

Leukocyte adhesion to endothelial cells is a complex process that involves

the capture of free-flowing leukocytes from the bloodstream, rolling on the

endothelial surface, deceleration, and, eventually, leukocyte immobilization

(firm adhesion). We found that degradation of the endothelial GCX under

septic conditions activated leukocyte-endothelial interactions in not only

postcapillary venules, but also precapillary arterioles. This finding suggests

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that the negative charge of the GCX itself prevents the interaction of

leucocytes with the endothelium and adhesion molecules, such as selectins,

PECAM, VCAMs, and ICAMs. These molecules become more accessible

during inflammation, although they are hidden within the complexed GCX

structure under normal conditions.

Additionally, the expressions of

adhesion molecules are reportedly upregulated under septic conditions.

Hyperpermeability was correlated with septic conditions. In this study, we

examined the permeability of two different molecular weights of dextran:

FITC-dex40 and TMR-dex75. We confirmed that even the very

high-molecular weight TMR-dex75 leaked into the interstitium, similar to

FITC-dex40 (fig. 8). Similar results in mouse ear skin under inflammatory

conditions induced by histamine or IgE treatment have also been reported,

but the GCXwas not mentioned. Sepsis increases vascular permeability

allowing albumin to leak into the interstitium, promoting interstitial edema

and resulting in hypovolemia.

These series of pathological changes

corresponds to the hyperpermeability that was observed following GCX

degradation in the present study. The precise role of the GCX in the

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regulation of water and soluble transport remains unknown. Curry

has

proposed the glycocalyx-junction-break model to explain the function of the

GCX. The GCX forms the principal molecular sieve at the vessel wall, which

is determined by factors such as the size or number of pores. According to

this model, the colloid osmotic forces opposing filtration across continuous

capillaries develop across the GCX, rather than in the interstitial space,

explaining the revised Starling principle.

We have found another interesting morphology using EM.

Lanthanum-positive and caveola-like small vesicles embedded in endothelial

cells were identified on EM images obtained in control mice (fig. 3A, 3B).

Similar structures were observed by Wagner et al.

Using computer-assisted

reconstructions of three-dimensional tomograms, they revealed that free

vesicles in the endothelial cytoplasm play a role as transendothelial channels

that span the luminal and albuminal membranes. Thus, the caveola-like

vesicles shown in fig. 3B might be involved in the regulation of vascular

permeability. Further study is needed to determine the function of this

structure.

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Our study had some limitations. First, this study of subcutaneous

microcirculation may not be representative of the changes that occur in other

organs during sepsis. Unlike other microcirculation models, the DSC model

requires a surgical procedure for chamber attachment. We cannot exclude

the influence of this surgical procedure on the microcirculation. Although the

observation area was not fully physiological, this model has been widely used

for studying microcirculation in various studies,

since it enables

real-time observations of the microcirculation. Second, confocal microscopy

would enable the thickness of the layer to be measured more precisely

because of its improved spatial resolution.

In conclusion, the illuminated area on the ESL in our model was identified

as the GCX. Using this model, we demonstrated that GCX disruption is

closely correlated with leukocyte-endothelial interactions and subsequent

hyperpermeability in septic mice. Our in vivo observation model may

contribute to the development of therapeutic approaches and an improved

understanding of the glycocalyx.

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Acknowledgements

The authors would like to thank Ms. Myrna Harrod for her English

language review. The authors would also like to thank Mr. Yusuke Kan, Ms.

Yukina Miyasaka, Mr. Mao Tanaka, Ms. Masako Osawa, and Dr. Hideyuki

Ochi for their assistance with the experiments, and Dr. Kohji Uzawa and Dr.

Hideki Miyao for their expert advice. This work was supported by JSPS

KAKENHI Grant-in-Aid for Scientific Research (C) 25463145 to TI.

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