Progress in Retinal and Eye Research xxx (xxxx) xxxx

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Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/preteyeres

Advances in the diagnosis and treatment of dry eye

Takashi Kojimaa, Murat Dogrua, Motoko Kawashimaa, Shigeru Nakamuraa, Kazuo Tsubotaa,b,∗
a
Department of Ophthalmology, Keio University School of Medicine, Japan
b
Tsubota Laboratory, Inc., Tokyo, Japan

ARTICLE INFO ABSTRACT

Keywords: The core mechanism of dry eye is the tear film instability. Tear film-oriented diagnosis (TFOD) is a concept to
Dry eye disease clarify the cause of tear film instability by tear film, and tear film-oriented treatment (TFOT) is a concept to treat
Tear film-oriented diagnosis dry eye disease by replacing the lacking components of the tear film layer based on the TFOD. In TFOD, the
Tear film-oriented treatment fluorescein breakup pattern of the tear film is important, and the subtype of dry eye can be judged to some extent
Visual function
from the breakup patterns. Current noninvasive devices related to the dynamic analysis of the tear film and
Oxidative stress
Life style
visual acuity enabled the diagnosis of dry eye, subtype analysis, and the extent of severity. In Asian countries,
secretagogues represent the main treatment in TFOT. Since meibomian gland dysfunction is a factor that greatly
affects the tear breakup time, its treatment is also essential in the dry eye treatment strategy. A newly discovered
dry eye subtype is the short breakup time-type (BUT) of dry eye. The only abnormal finding in this disease is the
short BUT, suggesting a relationship with ocular neuropathic pain and eye strain. Recently, data from many
studies have accumulated which show that dry eye is a life-style disease. In addition to the treatment of dry eyes,
it is becoming possible to prevent the onset by intervening with the daily habits, diet, exercise and sleep, etc. It
has been pointed out that oxidative stress is also involved in the pathology of dry eye, and intervention is being
carried out by improving diet and taking supplements. Future research will be needed to link clinical findings to
the molecular biological findings in the tear film.

1. Introduction diagnostic scheme, aqueous quantity can be examined by the Schirmer

1.1. Concept of new dry eye diagnosis: tear film-oriented diagnosis
The core mechanism of dry eye according to recent consensuses in
Japan and Asia is the instability of the tear film, and the concept to
clarify the cause by focusing on each layer of tear film is referred to as
the tear film oriented diagnosis (TFOD). At present, dry eye diagnostic
criteria in Japanese Dry Eye Society and Asia Dry Eye Society are based
on this concept, and dry eye is diagnosed when tear instability and
subjective symptoms are observed (Shimazaki, 2018).
The tear film is composed of an outermost lipid layer and the lower
aqueous layer. Below the tear film, there is the keratoconjunctival
epithelium holding membrane-bound mucins on the surface. In addi-
tion, secretory mucins exist in the aqueous layer and play a role for
water retention. In the current dry eye diagnosis, tear secretion volume,
tear stability, lipid layer thickness, vital staining, etc. are examined in
order to diagnose dry eye. The concept of TFOD was developed to treat
dry eye disease effectively and efficiently. The difference from previous
methods lies in combining the examinations for this purpose. TFOD
focuses on where the abnormalities are in the tear film. In this
test. Schirmer test is useful to assess tear secretion, but it is problematic
because it is invasive and lacks reproducibility. The evaluation of the
tear meniscus by the strip meniscometry method developed in recent
years and the anterior segment optical coherence tomography (OCT)
are also useful methods for evaluating the amount of aqueous tears on
the ocular surface (Dogru et al., 2006; Ibrahim et al., 2010a, 2012;
Ibrahim et al., 2011; Shinzawa et al., 2018). For the evaluation of the
lipid layer, lipid layer interferometry has been available. One problem
is that there are currently no simple clinical examination methods for
evaluating the ocular surface mucins. This is an area where future de-
velopments are desired. Currently, the evaluation of the epithelial layer
is performed by vital staining methods such as fluorescein or lissamine
green staining. In addition to the conventional aqueous layer, lipid
layer, and epithelial layer tests, the fluorescein breakup pattern can be
used to confirm the abnormalities in each component of the tear film
and to evaluate what is the dominant abnormality.
The fluorescein break up pattern analysis was developed to judge
the abnormalities of the tear film using a physical theory (Yokoi et al.,
2017). Using this method, dry eye can be classified into three subtypes
including the aqueous deficient type, increased evaporation type, and

∗
Corresponding author. Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
E-mail address: tsubota@z3.keio.jp (K. Tsubota).

https://doi.org/10.1016/j.preteyeres.2020.100842
Received 3 November 2019; Received in revised form 19 January 2020; Accepted 24 January 2020
1350-9462/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Takashi Kojima, et al., Progress in Retinal and Eye Research, https://doi.org/10.1016/j.preteyeres.2020.100842
T. Kojima, et al.
the low wettability type, which can be used to infer which tear film
layer has an abnormality. The tear wettability type, which was difficult
to reveal with conventional tests, exhibits a characteristic breakup
pattern, as detailed in 2.5. The hydrophilicity of the ocular surface is
maintained by the membrane-bound mucins present in the outermost
layer of the epithelial layer. When low wettability type is determined by
the fluorescein breakup pattern, it is important to suspect an abnorm-
ality of the membrane-bound type mucins and focus on the relevant
treatment as described later.
The abnormality of multiple tear film layers is often experienced
clinically. In particular, in the case of aqueous layer abnormality, it is
known to cause lipid layer abnormality and mucin abnormality.
However, by judging the most dominant abnormality by TFOD in such a
mixed-type of dry eye disease, treatment of dry eye disease can be ef-
ficiently performed as described later.
1.2. Concept of new dry eye treatment: tear film-oriented therapy
Tear film-oriented therapy (TFOT) (Fig. 1) is a treatment concept for
dry eye recommended by the Japanese Dry Eye Society and Asia Dry
Eye Society (Tsubota et al., 2017; Yokoi and Georgiev, 2018) that tar-
gets the tear film abnormalities revealed by TFOD. If the tear film is
abnormal and meibomian gland dysfunction is present, treatment tar-
geting the meibomian gland is required. If the aqueous layer is ab-
normal, artificial tear eye drops and secretagogue like diquafosol eye
drops may be applied to increase the water content. In addition, punctal
plugs are effective for the purpose of reducing the frequency of eye drop
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 1. Concept of tear film-oriented diagnosis and
therapy.
This concept was created by the agreement of
members of the Japanese Dry Eye Society and the
Asia Dry Eye Society. First, the clinician should in-
vestigate where the abnormality relating to dry eye is
in the tear film and the epithelium, and based on the
results, the corresponding treatment is selected.
Figure courtesy of Japanese Dry Eye Society.
instillations or in case of severe aqueous deficient dry eye. Mucin se-
cretagogues are in the treatment armamentarium for improving mem-
brane-bound mucin and secretory mucins. In addition, treatments tar-
geting the epithelium include autologous serum instillations and
rebamipide eye drops. Dry eye causes ocular surface inflammation due
to ocular surface damage, which can cause tears to deteriorate in sta-
bility. Immunosuppressants and low-dose steroid eye drops are also
suggested as anti-inflammatory treatments in TFOT approach.
The 2017 TFOS DEWS II® Report recommends triaging questions
first, which is expected to exclude other diseases that exhibit symptoms
similar to dry eye (Craig et al., 2017). Then, it is followed by conducting
a questionnaire and one of the diagnostic tests including noninvasive
tear stability, measurement of tear abnormality and ocular surface
staining. Once the diagnosis of dry eye disease is made, the diagnostic
method requires assessment of subtype and severity. The suggested
treatment approach is staged and severity based.
1.3. The importance of treating MGD occurring as a comorbidity of dry eye
MGD may modify DE phenotype. MGD is a common condition as-
sociated with dry eye disease, especially, the evaporative dry eye. In our
multicenter clinic-based study (Kawashima et al., 2017), we reported
that when DE and MGD occur as comorbidities, tear breakup time
(TBUT) is significantly shortened, which indicates increased disease
severity (Vu et al., 2018) (Fig. 2).
Interestingly, the TBUT value was the only ocular surface index that
was greatly influenced by the presence of MGD, regardless of dry eye
Fig. 2. The influence of meibomian gland dysfunc-
tion, friction-related diseases, or both on dry eye and
dry eye subtypes.
We compared clinical signs between patients with
Meibomian gland dysfunction (MGD) and friction-
related disease (FRD), patients with MGD and
without FRD, patients without MGD and with FRD,
and patients without MGD or FRD. Tear film breakup
time (TBUT). Figure and caption reproduced from Vu
et al. (2018) with permission of the copyright holder.
Abbreviations: ADDE, aqueous deficient dry eye.
T. Kojima, et al.
subtype (Vu et al., 2018). These results reinforces the novel concept
that an unstable tear film is the central mechanism of dry eye disease,
and, MGD is significantly correlated with dry eye condition. Therefore,
we should pay attention not only to DE but also dry eye related diseases
such as MGD for DE to be evaluated and treated successfully. In addi-
tion to DE treatment according to the tear film oriented therapy, MGD
treatments such as lid hygiene, lid warming, and meibum expression
are required in order to increase TBUT (Tsubota et al., 2017).
2. Advancements of dry eye diagnosis
2.1. Tear film stability analysis system
According to Asia Dry Eye Society consensus (Tsubota et al., 2017),
for the evaluation of tear film stability, fluorescein breakup time is
useful for evaluating tear film stability, and it has been widely used as a
test that can be performed at any ophthalmic facility. However, it has
been pointed out that water will be added to the ocular surface unless
the water in the fluorescein test paper is sufficiently removed as de-
scribed later (Johnson and Murphy, 2005). In terms of this issue, lipid
layer interferometry, grid xeroscope, and tear stability analysis system
(TSAS), which can noninvasively evaluate the tear film stability, are
useful.
In our experience, we observed that examiners struggle to measure
the corneal topography of patients with severe dry eye and came up the
ideas of serial corneal topography assessment for tear stability analysis.
Tear film stability could be assessed by taking corneal topography
images immediately after and 10 s after eye opening (Goto et al., 2002).
After this study, TSAS, which enabled evaluation of continuous images
of corneal topography, was developed. In this system, it is possible to
analyze the temporal change of the surface regularity index, which is
the index of regularity at the corneal center, and the surface asymmetry
index, which is the index of asymmetry. Using this system, SRI and SAI
have been shown to deteriorate over time in eyes with dry eye (Kojima
et al., 2004) (Fig. 3). In addition, there was also a difference in the
temporal change of the index depending on the type of dry eye. In the
severe aqueous deficient type, the index was high from the beginning,
and the index gradually increased in mild patients. These changes are
believed to reflect the stability of the tear film.
The dynamic analysis of tears was first reported using corneal to-
pography, and then an analysis using aberrometry was reported (Koh
et al., 2006). This examination made it possible to measure the changes
in high-order aberrations continuously from the eye opening. It has
Fig. 3. Representative examination results in TSAS. Note the changes in corneal topography for 10 s after eye opening in normal subject and dry eye patient. While
the topography image is stable for 10 s in the normal subject, it is shown that in the dry eye patient, the irregular astigmatism increases with time. In addition, at the
lower right of each image, there is a graph showing temporal changes of SRI and SAI. In the normal subject, there is almost no change in either index, but in dry eye
patient, it is shown that especially SAI gets worse with time. Figure and caption reproduced from Kojima et al. (2004) with permission of the copyright holder.
Abbreviations: SRI, surface regularity index; SAI, surface asymmetry index.
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
been shown that this test differs depending on the type of dry eye. In
short TBUT type of dry eye with normal tear secretion and no ocular
surface damage, it was reported that high-order aberrations increase
gradually after eye opening. On the other hand, in the aqueous deficient
type with superficial punctate keratopathy, it was revealed that high-
order aberrations show high values immediately after eye opening (Koh
et al., 2008).
The problem with non-invasive tear breakup is that although it
correlates with fluorescein breakup, but it does not evaluate the
breakup of the full thickness of the tear film but the thinning of the tear
film. For this reason, it is necessary to decide whether to use non-in-
vasive breakup or fluorescein breakup depending on the purpose of use.
2.2. Measurement of visual disturbance
A conventional visual acuity test represents the highest vision at a
given examination time. Heretofore, dry eye patients have often com-
plained that it is difficult to see despite good visual acuity. When it
became possible to perform dynamic analysis of the tear film non-
invasively by TSAS, we thought that visual acuity could also be ana-
lyzed dynamically. First, the visual acuity by forced eye opening was
measured under topical anesthesia and it was named as “functional
visual acuity” (Goto et al., 2002). This test has shown that functional
visual acuity was reduced despite the fact that conventional visual
acuity was not affected in dry eye patients. Following this initial report,
it was reported that the same result occurred with continuous visual
acuity testing under natural blinks, and measurement is currently per-
formed under no-anesthesia and non-invasive condition under natural
blink (Kaido et al., 2011). The FVA measurement system is presented in
Fig. 4. Parameters of FVA include initial vision, functional visual acuity,
visual maintenance ratio (VMR), average response time and the blink
frequency. It has been reported in dry eye patients that FVA and VMR
are reduced compared with the normal group (Kaido et al., 2014,
2015).
Not only is FVA useful for diagnosing and screening dry eye (Kaido
et al., 2015), but it is also useful for judging the treatment effect be-
cause improvements in functional visual acuity and VMR have been
reported by eye drops and punctal plug treatment (Kaido et al., 2008,
2012, 2013b; Yamane et al., 2015).
In dry eye patients, complaints of eyestrain such as heavy eyes have
been reported. Dynamic analysis of the tear film and FVA studies show
that dry eye has a large refractive change after eye opening, and it is
speculated that eyestrain is likely to occur. The appearance of the high
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 4. Measurement algorithm of functional visual acuity. A Landolt ring optotype is presented within the instrument and the patient responds to the direction of
opening with a joystick (A). If the reaction is correct, the optotype becomes smaller, and if the reaction is wrong, the optotype becomes larger (B). The results
presented in the print-out (C) are typical of dry eye patients, with a large change in visual acuity for 60 s, and a large difference between initial visual acuity and
functional visual acuity. Red line represents changes of visual acuity for 60 s. Green line represents the functional visual acuity which is the average visual acuity
value for 60 s. Visual maintenance ratio is calculated as the green area divided by the blue area (initial visual acuity). Triangles represent the timing of blinks.
Modified figure and caption reproduced from Kaido et al. (2018) with permission of the copyright holder.

frequency component in the accommodative microfluctuations is more
often in patients with large fluctuations in FVA (Kaido et al., 2017c).
These results may explain that dry eye patients are prone to show
eyestrain due to tear film instability.
Few studies have examined the relationship between FVA and ob-
jective tear film examination. It has been reported that dry eye patients
with corneal epithelial abnormalities show higher values of higher-
order aberrations and coma-like aberrations in the cornea than normal
control subjects and dry eye patients without epithelial abnormalities.
When functional visual acuity was measured in the same patient, it was
shown that the visual maintenance ratio of patients with corneal epi-
thelial abnormalities was lower than that of normal control subjects and
dry eye patients without epithelial abnormalities. These results sug-
gested that a destabilized tear film by dry eye had higher-order aber-
rations and exhibited fluctuations in visual acuity.
The FVA results are affected by multiple factors such as ocular op-
tics, retinal function, and cognitive function. While it is an advantage
that FVA reflects the patient vision in real life, it has the disadvantage
to evaluate the factors affecting the results of the FVA test alone. Future
studies are needed to clarify the cause of abnormal results of FVA test in
combination with other examinations.
2.3. Measurement of tear volume at the tear meniscus
In dry eye outpatients, multiple tests are often performed on the
same day, and the impact of the test on other tests also needs to be
considered. In that respect, it is desirable that the dry eye test is as non-
invasive as possible. Traditionally, Schirmer's test has been mainly used
to measure the tear secretion, but the problem is that it is invasive and
its reproducibility is low (Nichols et al., 2004). On the other hand, it has
been previously reported that the measurement of tear meniscus is
useful for diagnosis of dry eyes (Mainstone et al., 1996). The radius of
curvature of the tear meniscus reflects the retained tear volume and has
been reported to correlate with the fluorescein staining score and lipid
layer interferometry grades (Yokoi et al., 2000, 2004). It is known that
anterior segment OCT is highly reproducible and can non-invasively
examine the ocular surface (Ang et al., 2018). Non-invasive tear me-
niscus height measurement has been reported previously using anterior
segment OCT (Ibrahim et al., 2010a; Tung et al., 2014) (Fig. 5). In this
respect, anterior segment OCT tear meniscus height showed a sig-
nificant correlation with the Schirmer 1 test, fluorescein BUT, fluor-
escein staining score and the Rose Bengal staining score. In addition,
when 0.3 mm of the lower anterior segment OCT meniscus height was
taken as the cutoff value, dry eye diagnosis was possible with a sensi-
tivity of 67% and a specificity of 81%. In addition, it has been reported
that the anterior segment OCT tear meniscus was significantly elevated
after punctal plug treatment, and it could also be used for the evalua-
tion of the therapeutic effect (Ibrahim et al., 2012).
In daily practice, there has been a demand for the development of
an instrument measuring the tear meniscus easily and not requiring any
special device. Strip meniscometry is a device for measuring the
amount of tears using a special water-absorbing material, and can
measure the retained tear volume in the tear meniscus in 5 s (Fig. 6). In
the first report, the in vitro reproducibility, the water absorption, and
the correlation of Strip meniscometry values with the tear functions
were shown (Dogru et al., 2006). In dry eye patients, a significant
correlation was shown between the strip meniscometry value, the tear
film lipid layer interferometry grade, fluorescein BUT, and the vital

4
T. Kojima, et al.
staining scores. The retail version of Strip meniscometry tube (SMTube,
Echo Electricity, Fukushima, Japan) uses rayon and pulp as absorber,
and has a structure in which it is sandwiched by polyethylene. Ac-
cording to the results of evaluating the product version of strip me-
niscometry tubes, it is reported that the cut off value for diagnosing dry
eyes was 4 mm, the sensitivity was 93%, and the specificity was 73%.
Similar results were repeated using the anterior segment OCT and
looking into the correlation between anterior segment OCT and strip
meniscometry value (Shinzawa et al., 2018).
It has also been reported that the strip meniscometry tube is useful
for the evaluation of tears in dogs, cats, rabbits and mice in the veter-
inary field, taking advantage of its minimally invasive advantage
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 5. Tear meniscus height measurement using
anterior segment OCT. Cross section anterior seg-
ment OCT image of tear meniscus in a typical dry eye
patient (A) and a normal subject (B) are shown.
Please note that the tear meniscus height in dry eye
patient is significantly lower than the normal subject.
Figure and caption reproduced from Ibrahim et al.
(2010a) with permission of the copyright holder.
(Rajaei et al., 2018; Shinzawa et al., 2019). However, normal values of
strip meniscometry tube results by animal species and breeds have not
been reported, and further studies are needed.
Since strip meniscometry is a very convenient test, it may also be
used as a self-checking instrument. Recent studies have reported self-
examination to measure diurnal fluctuations in tear volume (Ayaki
et al., 2019). Accurate contact with the tear meniscus is important in
SMTube measurements. It is however necessary to explain the conduct
of testing to patients and how to use them safely for a reduced risk of
keratoconjunctival damage. In the future, SMTube is expected to find
applications for determination of tear protein concentrations and tear
collection for microchip-based tear analyses.
Fig. 6. Structure and use of strip meniscometry
tube.
Strip Menisimetry Tube (A) has a tear absorbent
consisting of nonwoven fabric sandwiched be-
tween 2 sheets of polyethylene (B). The scanning
electron microscope image shows rayon and pulp
sub-fibers in the absorber part of SMTube (C).
Testing from the lateral side is recommended to
avoid irritation to the cornea (D). Binocular
measurement is possible from one Strip
Meniscometry Tube. When the tear meniscus is
gently touched, the amount of tear fluid retained
in the tear meniscus can be measured in about 5 s.
T. Kojima, et al.
2.4. Cellular or tissue analyses using in vivo confocal microscopy
In the past, impression cytology has been performed to evaluate the
ocular surface at the cellular level, and many studies have been re-
ported in ocular diseases such as dry eyes, ocular pemphigoid and
atopic keratoconjunctivitis (de Rojas et al., 1993; Dogru et al., 2008;
Nelson, 1988; Nelson and Wright, 1984; Udell et al., 1986). Impression
cytology is a simple and useful method for observing the surface epi-
thelium, and immunohistochemistry is also possible. However, there is
a disadvantage that only cells in the outermost layer can be observed
and only the collectable cells can be observed. Under these circum-
stances, because in vivo confocal microscopy (IVCM) is minimally in-
vasive and allows repeated observation of the cellular level from the
corneal surface to corneal endothelial cells, we applied it to the clinical
evaluation of the ocular surface epithelium in dry eye disease by de-
vising parameters that could reflect impression cytology assessment.
The main histological finding of a typical epithelium in dry eye
disease is the squamous metaplasia. Our study compared the con-
junctival findings between IVCM and impression cytology in Sjögren's
syndrome regarding on new parameters including the mean individual
epithelial cell area (MIECA) and comparing it with the N/C ratio
(Fig. 7) (Kojima et al., 2010; Wakamatsu et al., 2010). Our findings
showed that the correlation between the parameters of these two tests
was high, and the usefulness of the IVCM was confirmed by the fact that
no significant difference was found between the two tests. In some
cases, there was a discrepancy between the values of the two tests,
owing to the inherent characteristics of each test. For example, im-
pression cytology can be affected due to differences between collected
and non-collectable cells. On the other hand, although IVCM is a
narrow scan, all cells can be observed. In addition, the conjunctiva
contains abundant goblet cells, and it is possible to easily evaluate the
density in impression cytology by using PAS staining. The findings of
goblet cells observed by IVCM are not consistent. One report suggested
goblet cells to be hyper reflective oval bodies (Hong et al., 2010) while
another reported them to be dark round structures (Murube and Rivas,
2003). There is a possibility that there is a change in the image re-
flectivity with IVCM depending on the state whether a large amount of
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 7. Typical conjunctival phenotypic alterations
in a control subject and Sjögren syndrome (SS) pa-
tient.
In the normal subject, relatively small epithelial cells
including abundant goblet cells are shown (A and B),
while enlargement of cells with cleft due to
sloughing cells (red arrow) and pyknotic nuclei were
shown in Sjögren syndrome patient (C and D). Figure
and caption reproduced from Kojima et al. (2010)
with permission of the copyright holder.
secreted mucin is stored in the cell or state whether it has already been
released.
Meibomian gland dysfunction (MGD), a risk factor for dry eye, is a
disease for which IVCM has been proven to be useful. First in 2005,
IVCM was used to observe meibomian gland dilation and obstruction
findings in patients with MGD (Messmer et al., 2005). Later, new
parameters in the observation of MGD were developed and compared
those with normal subjects (Matsumoto et al., 2009). In that paper, it
has been shown that the acinar unit density is lower than that of the
normal subjects and the acinar unit diameter is larger. It has been
shown that these parameters are correlated with meibomian gland
dropout and expressibility grades. In addition, IVCM may be useful for
understanding the pathophysiology of MGD because it can detect pa-
thological findings such as periglandular fibrosis that cannot be de-
termined by ordinary infrared meibography. The diagnosis of MGD
generally entails evaluation of glands by a combination of tests such as
meibomian gland dropout, infrared meibography, expressibility grade,
and dry eye symptoms. Attempts to use IVCM parameters for diagnosis
have been reported. Parameters such as acinar unit density, acinar unit
diameter, and inflammatory cell density have high sensitivity and
specificity and are useful for diagnosis (Ibrahim et al., 2010b) (Fig. 8).
It has also been reported that IVCM is useful for treatment evaluations
with lid hygiene, antibiotics and anti-inflammatory agents. Excessive
fibrosis around the meibomian gland has been reported in severe dry
eye patients with GVHD (Ban et al., 2011).
Currently, the pathophysiology of meibomian gland dysfunction is
unknown, but the effects of aging (Den et al., 2006), inflammation
(Landsend et al., 2018), and sex hormones (Golebiowski et al., 2017)
have been pointed out. As detailed tissue changes in MGD can be
evaluated by IVCM, long-term follow-up of the same patients may re-
veal the mechanism of how MGD develops and why meibomian acinar
unit expands in patients with MGD.
In the literature, IVCM has found another application in the ex-
amination of the lacrimal glands in severe dry eye disease. Lacrimal
gland biopsy, which is included in the diagnosis criteria for Sjögren's
syndrome, is a highly invasive test that can be complicated with
bleeding. We were able to observe the lacrimal gland with IVCM (Sato
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 8. IVCM observation images of a typical meibomian gland dysfunction patient.

(A) shows a typical meibomian gland acinar unit observed in normal subject. Patients with typical MGD have periglandular fibrosis and inflammatory cell infiltration
(B). Finally, when the meibomian gland structure shows atrophy, the meibomian gland acinar unit enlarges as shown in the typical example (C). When comparing the
parameters obtained by IVCM observation, meibomian gland acinar unit density (MGAUD) was significantly lower than in normal subjects (D). In addition, peri-
glandular inflammatory cell density (ICD) was significantly higher in MGD patients compared to normal control (E). Figure and caption reproduced from Ibrahim
et al. (2010b) with permission of the copyright holder.

et al., 2010). It was also found that in cases where exposure of the
lacrimal gland is difficult, measurements could not be made. In fact, in
this study, lacrimal gland was successfully observed in 11 out of 69
primary Sjögren's syndrome patients and 9 out of 22 normal subjects.
However, since it is less invasive as compared to biopsy, it may be more
widely performed if the shape of the IVCM probe cap is improved to
easily access to the lacrimal gland in the future.
2.5. Fluorescein break-up patterns
The tear breakup patterns is a method of evaluating the tear stabi-
lity from physical theory and classifying pathological conditions which
causes tear film instability (Yokoi and Georgiev, 2018).
At the time of eye opening, aqueous component is lifted upward by
the negative pressure of the upper tear meniscus based on Young
Laplace's law. Subsequently, upon upward extension of the lipid layer
based on the Gibbs-Marangoni effect, the liquid layer is lifted based on
viscous drag. At the same time, the liquid layer is thinned by the ne-
gative pressure of the lower tear meniscus, and the tear film is formed
by the end of the aqueous layer redistribution. During this process, it is
presumed that the wettability of the corneal surface may be maintained
by the glycocalyx, in particular, MUC16, the longest membrane-type
mucin. After the formation of the tear film, its evaporation is sup-
pressed by the lipid layer and the water retention function of the se-
cretory mucin, and thus the tear film stability is maintained. Given the
situation in which one of the components is lacking, it is possible to
predict that break-up will occur at some time and some place of the
cornea along with the eye opening.
With regard to the break-up of the representative example, dis-
criminant analysis was conducted and breakup patterns were found to
be pathologically different (Yokoi et al., 2017) (Fig. 9). One of the
important points in this paper was to classify dry eye based only on the
basic type of the break-up pattern. In other words, this classification is a
basic type of dry eye subtype classification based on break-up patterns
clarified by physical theory and clinical evidence. In addition, other
modified forms and complex forms of break-up pattern were also re-
ported (Yokoi and Georgiev, 2018, 2019).
There are arguments about the evaporation suppression effect of the
tear lipid layer (Borchman et al., 2009; Herok et al., 2009; Rantamaki
et al., 2012). It is not known whether the increased evaporation type we
call is actually associated with increased evaporation. In the future,
along with the development of a more accurate clinically usable eva-
poration measurement device, it will be necessary to investigate the
tear evaporation for each classification by breakup pattern.
We believe that this classification may become more practical and
useful after using this classification in a large number of clinical cases. It is
also necessary to build up evidence which connects the break-up pattern
with specific abnormal components of tear film including investigation
into the ocular surface mucins and lipid layer in the future studies.
2.6. A new entity of dry eye subtype: short tear film breakup time-type of
dry eye
Formerly, typical dry eye was thought to have ocular surface ab-
normalities or decreased tear secretion. However, even though patients
complain of dry eye symptoms, we often experienced many dry eye
cases in whom there was normal tear secretion or no ocular surface
abnormalities. Short tear film breakup time-type dry eye (short dry eye)
was first reported in 1995(Toda et al., 1995). In this report, the pre-
sence of short tear film breakup time-type of dry eye in which there was
no abnormality in tear secretion and no vital staining on the ocular
surface was reported. Furthermore, it was reported that the dry eye

7
T. Kojima, et al.
symptoms in Short BUT dry eye were similar to decreased aqueous
secretion type of dry eye accompanied by ocular surface abnormalities.
The importance of tear film stability has been emphasized in Asia Dry
Eye Society Consensus Report (Tsubota et al., 2017) and TFOS DEWS
II® Report (Bron et al., 2017), but at this time the tear film breakup time
was not as important as it is now. The report not only showed the
importance of TBUT measurement, but also changed the concept of dry
eye so far. After this, many studies on short TBUT dry eye have been
reported in Japan and Korea (Ichihashi et al., 2015; Kaido et al., 2016;
Yokoi et al., 2015; Mun et al., 2018; Uchino et al., 2014).
Short TBUT dry eye may be classified as increased evaporation type
in the classification of TFOS DEWS II® Report. It is possible that neu-
ropathic pain components may be involved. Recently, the decreased
wettability type dry eye (Yokoi et al., 2017) is presumed to be a typical
short TBUT type dry eye, and the fluorescein breakup pattern shows a
spot and a dimple breakup pattern. When the normal volunteer without
decrease of tear secretion was divided into two groups with dry eye
symptoms, there was corneal hypersensitivity in the symptomatic group
(Kaido et al., 2016).
Visual function of short BUT type of dry eye patients was found to
show significant fluctuations after eye opening by wavefront aberration
analysis (Koh et al., 2008) and functional visual acuity test (Kaido et al.,
2014). Short TBUT dry eye patients were able to see clearly at the mo-
ment they opened their eyes, but it was shown that it soon became
8
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 9. Typical fluorescein break-up pattern.
In severe aqueous deficient type of dry eye, there is
almost no aqueous layer, and even a blink does not
form a tear film (A, Area break). In the decreased
wettability type, a breakup occurs during eye
opening (B, Spot break). In the aqueous deficient
type, a linear breakup occurs at the inferior part of
the cornea when the aqueous layer is pulled by the
lower tear meniscus (C, Line break). In case with
mild decreased wettability, vertical line-like breakup
occurs by the end of upward movement of fluor-
escein stained aqueous tears (D, Dimple break).
Random break is caused by thinning of the tear film
due to aqueous tear evaporation after movement of
fluorescein stained aqueous tears is completed (E,
Random break). Modified figure and caption re-
produced from Yokoi et al. (2017) with permission of
the copyright holder.
difficult to see. In addition to dryness and ocular pain, short TBUT is
considered to greatly affect the quality of life in terms of visual function.
It is known that continuous refractive fluctuations occur when ac-
commodative responses occur (Campbell et al., 1959). It is reported
that the high frequency component obtained by Fourier analysis of this
fluctuation is related to eyestrain (Maeda et al., 2011). That is, the high-
frequency component of the accommodative response is considered to
represent the tension of the ciliary muscle. Dry eye treatment improved
the high-frequency component of accommodative microfluctuation in
Short BUT dry eye patients using a device that can evaluate this high-
frequency component (Kaido et al., 2017b) (Fig. 10). Although it is
known that dry eye patients complain of ocular fatigue, this symptom
may result from ciliary muscle hypertonia caused by tear film in-
stability and accommodative microfluctuations.
2.7. Advances in MGD diagnosis
The most important elements of MGD diagnosis are obtaining in-
formation from the patient about subjective symptoms and making de-
tailed observations of the eyelid and ocular surface using a slit lamp
microscope. In addition, remarkable progress has been made recently
due to the development of a variety of tests that serve as supplementary
diagnostic and severity assessment methods. Meibography is used to
observe morphology of the MG, and can be utilized to measure decreases
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 10. Fluorescein breakup findings and visual function in typical short TBUT dry eye patient. Before treatment, BUT was almost 0 s and symptoms were severe (A),
but 2 months after upper and lower punctal plug treatment and moisture glasses wear, TBUT improved to 9 s (D). Functional visual acuity showed improvement after
treatment (E) compared to before treatment (B). The high frequency components of accommodative microfluctuations also showed improvement after treatment (F)
compared to before treatment (C).
Abbreviations: BUT, break-up time; HFC, high-frequency component. Figure and caption reproduced from Kaido et al. (2017c) with permission of the copyright
holder.

in the amount of meibum secreted by the glands. Measurements of tear
evaporation rate have shown that this is accelerated in MGD, and ob-
servations made using a confocal microscope have shown enlargement of
the acini and decreased acinar unit density. With respect to tear inter-
ferometry, the LipiView® (TearScience, USA) and DR-1α® (Kowa, Japan)
devices have recently been marketed in Japan. The LipiView measures
the lipid layer at the inferior one-third of the cornea and is able to pro-
vide a quantitative measurement of lipid layer thickness. The DR-1α
allows observation of the entire cornea, including the central region, and
tear dynamics, and is also capable of quantitatively measuring the lipid
layer. The combined use of these devices not only facilitates MGD di-
agnosis, but also allows both the disease severity presented by MGD
patients and the efficacy of treatment to be assessed.
3. Advances in dry eye treatment
3.1. Aqueous layer treatment
cellulose and/or hyaluronic acid to improve the retention of water on
the ocular surface. The most commonly used artificial tear eye drop in
Japan is sodium hyaluronate ophthalmic solution. It has been reported
that with 0.1% and 0.3% sodium hyaluronate eye drops, the healing
rate is significantly higher than in controls in a rabbit corneal wound
healing model (Shimmura et al., 1995). In addition, a clinical study was
conducted in dry eye patients with aqueous deficient dry eye. In this
study, one eye was instilled with 0.1% sodium hyaluronate eye drops,
and the other eye used the same instillation containing vehicle. They
reported that eyes with 0.1% sodium hyaluronate eye drops showed
improvement in subjective symptoms and vital staining scores sig-
nificantly compared to eyes with vehicle instillation (Shimmura et al.,
1995).
Artificial tears have also been reported to be useful in combination
with aqueous secretagogue diquafosol sodium (Kamiya et al., 2012).
Increasing the retention of water secreted by diquafosol is thought to
increase the therapeutic effect.

3.1.1. Artificial tears 3.1.2. Aqueous secretagogues

Artificial tear eye drops contain viscous agents such as methyl Diquafosol sodium is an aqueous secretagogue that acts on P2Y2

9
T. Kojima, et al.
receptors in the cornea and conjunctiva to promote the secretion of
water via an increase in intracellular calcium concentration. There is
also a secretory action of secretory mucin from the conjunctival goblet
cells as described later. It can be prescribed in Asian countries such as
Japan, Korea, Vietnam, and Thailand. Diquafosol ophthalmic solution is
essential treatment component according to the TFOT.
In a recent randomized double blind multi center study, 3%
Diquafosol ophthalmic solution showed significantly higher improve-
ment in Rose Bengal staining score and symptom scores compared with
0.1% hyaluronic acid ophthalmic solution (Takamura et al., 2012).
Effectiveness of 3% Diquafosol ophthalmic solution was reported in
patients with aqueous deficient dry eye such as Sjögren's syndrome
(Koh et al., 2014) and dry eyes after laser in situ keratomileusis (LASIK)
(Mori et al., 2014; Toda et al., 2014).
3.1.3. Punctal plugs
Punctal plug treatment lowers tear clearance and increases reten-
tion on the ocular surface. It is an option if the therapeutic effect of
secretagogue is inadequate. There are many studies showed the efficacy
of punctal plug in dry eye disease (Kaido et al., 2004, 2013a; Kojima
et al., 2014; Liu et al., 2015; Yung et al., 2012). The punctal plugs are
divided into permanent and temporal types. When a permanent type is
inserted, complications such as dropout and migration may occur. Mi-
gration is a complication that must be avoided as it sometimes causes
infections such as canaliculitis and dacryocystitis. We have previously
shown that designs with a gap between the punctal plug and the
plunger leads to migration (Kaido et al., 2009), and all subsequent plugs
have been modified to a gapless type in Japan.
Temporary type of punctal plug is effective for seasonal exacerba-
tion of dry eye and dry eye after laser refractive surgery such as LASIK
(Yung et al., 2012). However, the conventional collagen plug is a rod-
like collagen inserted from the punctal, and the punctal blocking effect
has been reported to be insufficient. The thermosensitive atellocollagen
punctal plug is initially liquid, and when inserted from the punctal, it
solidifies with the body temperature and the punctal is occluded.
However, because it takes time for atellocollagen to solidify, it has been
a concern that it flows from the canaliculus. We previously reported
that in order to maximize the effect of this thermosensitive atello-
collagen punctal plug, a sufficient effect can be obtained by a pre-
heating method, where the plug is preheated at 41 °C and injected in a
somewhat stiffened state (Kojima et al., 2014)(Fig. 11).
Generally, punctal plugs are used for aqueous layer treatment. That
is, by inserting the punctal plug, it is possible to increase the tear film
stability by increasing the amount of water for the aqueous deficient
type of dry eye disease. Until the secretagogue drops were available,
there was no way to treat the underlying cause for the short TBUT-type
dry eye, i.e., the decreased wettability type of dry eye. We have re-
ported treatment as a symptomatic treatment by increasing the tear
volume (Kaido et al., 2012). Although it may cause epiphora, it has
been found that it is possible to greatly improve the symptoms of dry
eye. It is assumed that the use of the punctal plug for decreased wett-
ability type of dry eye is diminishing after secretagogues became
available.
3.2. Ocular surface mucin treatment
3.2.1. Mucin secretagogues
Diquafosol ophthalmic solution is a P2Y2 receptor agonist launched
in Japan in 2010. The P2Y2 receptor is expressed throughout the body
and is present in keratoconjunctival epithelial cells, goblet cells and
meibomian glands on the ocular surface (Cowlen et al., 2003). Diqua-
fosol stimulates the secretion of water from the cornea and conjunctival
epithelium and acts on goblet cells to promote the secretion of secretory
mucin (Fujihara et al., 2002; Murakami et al., 2004). It is also known to
act on corneal epithelial cells to upregulate gene expression of mem-
brane-type mucin (Jumblatt and Jumblatt, 1998). It is thought that
10
Progress in Retinal and Eye Research xxx (xxxx) xxxx
these actions combine to contribute to the tear fluid stability.
Phase 3 randomized double blind multi-center study clinical trial
compared diquafosol ophthalmic solution (3%) with 0.1% hyaluronic
acid eye drop. The fluorescein staining score was equivalent after 4
weeks of treatment. The Rose Bengal staining score in the diquafosol
treatment group showed a significantly larger decrease than the hya-
luronic acid group (Fig. 12) (Takamura et al., 2012). Similar results
have been reported in randomized studies in China and Singapore
(Gong et al., 2015). Studies examining the effects of long-term admin-
istration have reported improvement in subjective symptoms and
findings (Koh et al., 2013).
There are many studies showing the effectiveness of diquafosol
ophthalmic solution for different type of dry eye disease including
Sjogren's syndrome (Koh et al., 2014), short TBUT type dry eye (Kaido
et al., 2013b; Shimazaki-Den et al., 2013), dry eye after LASIK (Mori
et al., 2014; Toda et al., 2014) and contact lens-related dry eye
(Nagahara et al., 2015, 2017).
Rebamipide is a quinolinone derivative with mucin producing ac-
tivity that has been released in Japan since 2012. It has been reported
that goblet cells increase after two weeks of instillation (Kato et al.,
2017). Rebamipide has also been shown to promote the synthesis of
MUC16 (Yamasaki et al., 1987). It also has the effect of promoting re-
covery of tight junctions and increasing the microvilli. These effects
may be due to promotion of differentiation and induction of the epi-
thelium. According to the results of a randomized double-masked,
multicenter placebo-controlled phase II study, 1% and 2% rebamipide
significantly improved the corneal fluorescein score, conjunctival lis-
samine green score and symptom scores compared to placebo
(Kinoshita et al., 2012). Comparison with 0.1% hyaluronic acid was
performed in a phase 3 randomized and multicenter study (Kinoshita
et al., 2013). Conjunctival lissamine green staining score was sig-
nificantly improved compared to 0.1% hyaluronic acid. Moreover,
foreign body sensation and eye pain significantly improved compared
to 0.1% hyaluronic acid. In addition, 2% rebamipide has been reported
to be effective in contact lens-related dry eye (Igarashi et al., 2018), dry
eye after vitreoretinal surgery (Kato et al., 2016) and persistent corneal
erosion (Kashima et al., 2012), etc.
3.3. Ocular surface epithelial treatment
3.3.1. Rebamipide
In dry eye, ocular surface epithelial damage causes instability of the
tear film. Furthermore, there is a vicious cycle where instability of the
tear film exacerbates epithelial disorders. Membrane-type mucin on the
ocular surface is localized to microvilli in the ocular surface epithelium,
and the membrane-type mucin changes the ocular surface from hy-
drophobic to hydrophilic. In dry eye, it is known that the structure of
the microvilli is damaged, and the density of the microvilli is reported
to be correlated with the tear film stability and dry eye symptoms
(Cennamo et al., 2008).
Rebamipide has been reported to promote epithelial wound healing
in the rat exorbital lacrimal gland excision model as compared to
control (instillation of substrate only without Rebamipide) (Kimura
et al., 2013). It has also been found that after elimination of ocular
surface mucin with N-acetylcystein, treatment with rebamipide in-
creases mucin-like substance in the cornea and conjunctival epithelium
over controls (Urashima et al., 2004).
Furthermore, it has been reported that it is effective for improve-
ment of the epithelial disorder not only in dry eye but also in lid wiper
epitheliopathy and superior limbal keratoconjunctivitis (Itakura et al.,
2013; Takahashi et al., 2014).
3.3.2. Autologous serum
Autologous serum (AS) eye drops are used for the purpose of sup-
plying components to the ocular surface that cannot be obtained by
other topical eye drops. Since the therapeutic effect on Sjögren
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 11. Change of thermosensitive atelocollagen punctal plug by temperature rise.

The thermosensitive atelocollagen punctal plug comes in a syringe and is injected from the punctal using a blunt needle (A). Thermosensitive atelocollagen solidifies
completely in 24 h (B). Temporal temperature change and solidification of thermosensitive atelocollagen were investigated using an absorbance meter (C, D, E).
Figure and caption reproduced from Kojima et al. (2014) with permission of the copyright holder.

syndrome patients was reported by Fox et al., in 1984 (Fox et al., 1984),
the efficacy has been reported for various ocular surface diseases.
Fig. 13 shows a general method of preparation and storage of AS eye
drops.
An increase in goblet cells and an improvement in conjunctival
squamous metaplasia using AS were reported in Sjogren's syndrome
patients (Tsubota et al., 1999). Autologous serum eye drops were pre-
scribed for patients with severe dry eyes due to graft-versus-host disease
and who did not respond to other treatments, and reported safety and
efficacy (Ogawa et al., 2003). Autologous serum eye drops were also
prescribed for neurotrophic corneal ulcers, and reported improvement
in corneal sensation as well as improvement in ulcers (Matsumoto et al.,
2004). Dry eye after laser refractive surgery is considered to be caused
by surgery induced corneal nerve damage, and it has been reported that
AS was effective for this condition (Noda-Tsuruya et al., 2006).
In our randomized control study (RCT), after a 2-week washout
period, we randomly divided patients into two groups (artificial tears
group and autologous serum group) and compared the two groups be-
fore and after treatment (Kojima et al., 2008). As a result, compared
with artificial tears, the autologous serum group had significant im-
provement in the subjective symptom scores, the vital staining scores,
and the TBUT. Cochrane Database Systematic Review showed that
serum instillation may be effective for dry eye treatment. However, it
has been concluded that there is a lack of evidence and well-planned,

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T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 12. Summary of randomized double blind multi-center clinical trial comparing 3% diquafosol sodium and 0.1% hyaluronic acid ophthalmic solution.
In the clinical trial, after a washout period by vehicle eye drop, patients were randomly assigned to the diquafosol group and the hyaluronate group (A). The
fluorescein staining score was not significantly different between the two groups during the 4-week treatment period (B), but the Rose Bengal staining score was
significantly lower in the diquafosol group at the 4-week endpoint (C). Regarding the subjective symptom score, the score regarding the heaviness was significantly
lower in the diquafosol group than in the hyaluronate group at 4-week endpoint (D). Figure and caption reproduced from Takamura et al. (2012) with permission of
the copyright holder.

Occasionally, dry eye may be refractory to treatment and is sus-

Fig. 13. Method for making and storing autologous serum eye drops. Although
autologous serum eye drops are made under a clean-bench operation, education
of patients is also important in paying attention to the expiration date and usage
guidelines. Figure and caption reproduced from Higuchi et al. (2018) with
permission of the copyright holder.
large, high-quality RCTs are needed, as there are various deficiencies in
previous RCT studies (Pan et al., 2013, 2017). Moreover, because the
dry eye questionnaires used in each study are different, a unified
standardized questionnaire should be used.
pected to involve neuropathic ocular pain (NCP). In such cases, AS eye
drops are effective through their neuro-regenerative properties
(Dieckmann et al., 2017). Autologous serum includes nerve growth
factor (NGF), and NGF has been reported to improve allodynia and
hyperalgesia in the results of basic studies using NCP animal models so
far (Cirillo et al., 2010; Colangelo et al., 2008). Clinical studies have
also reported that AS eye drops were effective in improving corneal
nerve morphology in NCP patients (Aggarwal et al., 2019).
Autologous platelet rich plasma has been reported to contain higher
concentrations of biologically relevant factors (EGF, NGF and PDGF)
than autologous serum and has efficacy for dry eye and refractory
ocular surface diseases (Anitua et al., 2015, 2016; Merayo-Lloves et al.,
2016). A randomized control trial will be required to clarify its effec-
tiveness in the future. A study comparing autologous serum eye drop
and platelet rich plasma eye drop is also needed.
Since AS eye drops are prepared from the serum of a patient, it has
disadvantages such as the risk of contamination, the difference between
various components, and the possibility of the components becoming
denatured over time. In addition, the need for blood collection for each
preparation is also a heavy burden on patients. In order to solve these,
there is an idea to use the active ingredient contained in serum as a
substitute for AS eye drops. The serum contains many vitamins, growth
factors such as the epidermal growth factor, nerve growth factor, and
the insulin like growth factor. In addition, proteins such as albumin,

12
T. Kojima, et al.
lactoferrin and lysozyme are also abundantly contained.
Our group first focused on albumin, and it became clear that al-
bumin suppresses apoptosis of human corneal epithelial cells through
suppression of caspase 3 (Higuchi et al., 2007). In a preliminary clinical
study, it was shown that 5% albumin instillation significantly improved
fluorescein staining, rose bengal staining, TBUT, and symptoms. Later,
a study comparing 5% albumin and 0.1% hyaluronic acid eye drops in
persistent epithelial defects and sterile corneal ulcers suggested that the
results in both groups were comparable, but it was suggested that al-
bumin instillation could reduce the recurrence of sterile corneal ulcer
(Schargus et al., 2015).
The selenoprotein contained in the serum is a selenium-transfer
plasma glycoprotein contained in serum or extracellular fluids such as
milk. Selenoprotein P was present in tears and reduced in dry eye pa-
tients. Selenoprotein P is involved in the control of oxidative stress. In
experiments involving a rat dry eye model, fluorescein staining was
significantly lower compared to the PBS group, and the production of
oxidative stress marker 8-OHdG was suppressed (Higuchi et al., 2010).
Selenoprotein P has been shown to improve dry eyes by reducing oxi-
dative stress. It will be necessary to confirm its effectiveness in humans
in the future.
Even today, AS eye drops are used in many countries. Research on
active ingredients contained in AS is also in progress, but at present no
definitive ingredient has been found. AS eye drops effects may be due to
presence of several active ingredients. Based on the research results
obtained from AS eye drops, the development of new drops are ex-
pected in the future.
3.4. Anti-inflammatory treatment
Anti-inflammatory treatment is also important in therapeutic stra-
tegies employing TFOT. Inflammation damages the outer surface epi-
thelium, which may cause changes in keratoconjunctival wettability,
tear film instability, and deterioration of subjective symptoms due to
ocular surface nerve damage.
3.4.1. Rebamipide
Control of inflammation plays an important role in the treatment of
dry eye. Rebamipide has been reported to suppress conjunctival TNF-α
expression and suppress macrophage infiltration in a corneal epithelial
wound healing model (Tajima et al., 2018). Rebamipide eye drops were
applied for atopic keratoconjunctivitis patients with dry eyes, and that
IL-8, eosinophil cationic protein and total IgE in tears decreased, and
allergic subjective symptoms were alleviated (Ueta et al., 2014). Re-
bamipide may be effective in dry eyes with inflammation, and further
studies are needed including elucidation of the mechanism of action.
3.4.2. Cyclosporine
T cell plays an important role in dry eye onset. Cyclosporine is
prescribed as an eye drop targeting inflammation. Cyclosporin A (CsA)
reduces T cell activation via IL-2. Several studies have reported im-
provement in symptoms, tear stability and tear secretion (Sall et al.,
2000; Stevenson et al., 2000; Stonecipher et al., 2005). An important
aspect of the therapeutic effect of CsA is to control the progression of
the disease and to obtain continuous improvement of corneal fluor-
escein for 24 months (Rao, 2011). Moreover, according to the Cochrane
Database Systematic Review, increased goblet cell density was found to
be a major therapeutic attribute of topical cyclosporine (de Paiva et al.,
2019). Problems in continuing CsA instillation are symptoms associated
with eye drops such as burning sensation. The combined use of steroid
eye drops has been reported to reduce the side effects of CsA (Sheppard
et al., 2014), and the combination of steroid eye drops may be useful.
3.4.3. The role of lactoferrin on tear film and ocular surface inflammation
in dry eye disease
Tear lactoferrin is produced in the acinar cells of the lacrimal gland
13
Progress in Retinal and Eye Research xxx (xxxx) xxxx
and secreted into the tears. The normal tear lactoferrin levels vary
between subjects ranging from 0.63 to 2.9 g/l with an average 1.42 g/l
which have been shown to decrease with increased age and in dry eyes
associated with Sjögren's syndrome (Flanagan and Willcox, 2009).
Early in-vitro research from our group suggested a protective role
for lactoferrin in ultraviolet induced oxidative stress and cell death in
corneal epithelial cells (Shimmura et al., 1996). Initial clinical research
from our group disclosed that there were significant correlations be-
tween tear lactoferrin and epidermal growth factor and tear function
tests in Sjögren syndrome dry eyes such as tear function index, Rose
Bengal staining scores and Schirmer test. The decline in tear lactoferrin
in this disease was thought to be due to dysfunction of the lacrimal
gland while the pathogenesis of the decrease was attributed to the
absence of neural stimulation in the lacrimal gland (Ohashi et al.,
2003).
Our initial attempt using enteric coated lactoferrin in the treatment
of dry eye disease entailed administration of 270 mg/day of oral enteric
lactoferrin capsules to 20 Sjögren's syndrome patients with dry eyes for
1 month (Dogru et al., 2007). This study showed that the mean corneal
sensitivity, tear film breakup time value, central tear film lipid layer
thickness as observed by lipid layer interferometry, keratoconjunctival
vital staining scores, squamous metaplasia grades, and goblet cell
densities were significantly worse before lactoferrin treatment, im-
proving significantly and concomitantly with dry eye symptomatology
after 1 month of treatment. This study fell short of studying the changes
in a larger population and showing the alterations of inflammatory
changes in the tear film with treatment which need to be clarified in
future studies. Lactoferrin has been shown to inhibit the production of
various cytokines, such as tumor necrosis factor α and interleukin 1β,
via receptor-mediated signaling pathways. Lactoferrin can suppress
inflammation by downregulating tumor necrosis factor α and upregu-
lating interleukin 10 in rat adjuvant-induced arthritis (Dogru et al.,
2007). Future trials may focus on the changes of these markers with
treatment. Aged mice which were on a 2% lactoferrin diet for 6 months
had significant decreases in the infiltration of inflammatory cells and
inflammatory cytokine expressions in the lacrimal gland (Fig. 14)
(Kawashima et al., 2012).
In conclusion, recent dry eye research disclosed new rapid and cost-
wise methodology to quantify tear lactoferrin which needs to be tested
in broader populations of subjects with dry eye disease and still requires
description of test sensitivity, specificity and cut-off values. The testing
which requires only a small volume of tears promises to be a milestone
and a leap in tear diagnostics of severe dry eyes. Further research with
oral lactoferrin supplements and topical drops needs randomized con-
trolled trials on larger populations with dry eyes. The corneal and
conjunctival bioavailability of lactoferrin eye drops still remains an
issue which needs further clarification.
3.5. Treatment of meibomian gland dysfunction
Meibomian gland dysfunction (MGD) occurs in 64.6% of the elderly
patients with ocular discomfort (Shimazaki et al., 1995). Dry eye (DE)
and MGD are common comorbidities (Lemp et al., 2012), and a recent
epidemiological study indicated that the comorbidity rate of these
conditions is 12.9% (Arita et al., 2019). It is important to treat MGD via
both prescriptions issued by physicians and patient self-care. Innova-
tions in MGD treatment methods are currently ongoing. One such study
known as the TFOS MGD Report (Geerling et al., 2011) reported that
improvements in daily habits such as lid hygiene (mentioned below),
application of a hot compress, and dietary modifications are re-
commended for the basic treatment of MGD. Meibum is believed to
harden in cases of MGD as the melting point of patients’ meibum is
known to be elevated. Thus, the use of a hot compress to warm the
eyelids raises the temperature to the melting point of meibum, in turn
promoting melting and increased secretion. Eyelid hygiene is also ef-
fective, and is listed together with application of a hot compress as one
T. Kojima, et al.
Fig. 14. Morphological changes in lacrimal glands after dietary lactoferrin in
aged mice.
We compared the morphology of the lacrimal gland tissue in the dietary lac-
toferrin group and the normal dietary group for 6 months. A to D show he-
matoxylin and eosin stained photographs. It was shown that the lactoferrin
group (B, D) had less cell infiltration as compared to the normal group (A, C).
Immunostaining using an anti-CD45 antibody also showed that CD45 positive
cell infiltration was attenuated in the lactoferrin group (F) than in the normal
bait group. Observation of the organelle by transmission electron microscopy
showed irregular shapes and irregular sized vesicles (G) in the normal group. In
contrast, uniform and regular shaped vesicles were observed at an apical side of
cells in the lactoferrin group. Figure and caption reproduced from Kawashima
et al. (2012) with permission of the copyright holder.
of the internationally accepted standards for MGD treatment. Ideally,
patients should perform these self-care routines twice a day (morning
and night). Although lid hygiene is recommended for early-stage MGD
based on a study showing that this leads to improvements in subjective
symptoms and TBUT, it is also important that lid hygiene is maintained
at all stages of the disease, even in cases of advanced MGD with dis-
placed MGs (Kaido et al., 2017a). In addition, it has been suggested that
Demodex, parasites which reside around the eyelids, may also
14
Progress in Retinal and Eye Research xxx (xxxx) xxxx
contribute to the pathophysiology of this disease, again highlighting the
importance of lid hygiene (Kawakita et al., 2010).
Although the use of diluted baby shampoo was previously thought
to be effective for lid hygiene, compliance has been poor given that it is
a strong irritant. Thus, Tsubota developed a patient-friendly lid-hygiene
lotion that is less irritating. A study was conducted in relation to the lid
hygiene using eye shampoo on patients with symptoms of dry eye who
regularly wore heavy makeup, finding that both subjective and clinical
symptoms showed improvement (Okura et al., 2015) (Fig. 15). We are
also awaiting the clinical introduction of an eye shampoo/lid hygiene
method that utilizes an eye brush (Tanabe et al., 2019).
Effective pharmacotherapy for MGD includes lipid replacement
treatment in the form of small doses of Tarivid ophthalmic ointment
(Goto et al., 2006). Combination therapy consisting of dry eye treat-
ment (artificial tears, hyaluronic acid eye drops, diquafosol ophthalmic
solution [Diquas®], rebamipide eye drops [Mucosta ophthalmic sus-
pension®]), and lipid treatment is effective in the improvement of both
subjective and clinical symptoms. Diquas® may also be utilized for di-
rect improvement of MGD (Ikeda et al., 2018). In cases of MGD ac-
companied by microbial infection, the predominant microbes that are
detected in MG lipids are Propionibacterium acnes (P. acnes) in young
patients and Staphylococcus in elderly patients. In such cases, antibiotic
eye drops (cephalosporin antibiotic ophthalmic solution [Bestron®]) are
effective. Meibomian gland dysfunction has been implicated in in-
flammation of the eyelid margin, suggesting that the normal bacterial
flora may be involved in this inflammation. The action of microbial
lipases on MG lipids, which are mainly esters, frees the fatty acids,
which in turn causes inflammation of the eyelid margins. As a result,
the MG duct epithelium keratinizes and becomes blocked. Oral tetra-
cycline or doxycycline is effective to treat blepharitis-related MGD by
suppressing the production of microbial lipase (Geerling et al., 2011).
Topical azithromycin is also characterized by fewer side effects than
oral antibiotics and relatively high penetration to the eyelid, and its
efficacy has been reported for MGD with blepharitis (Haque et al.,
2010). Other common treatments include the use of a steroid-con-
taining (0.1% Fluorometholone®) antimicrobial ophthalmic solution
and long-term administration of low-dose tetracycline and macrolide
drugs. These antibiotics exhibit good tissue transfer to the MGs, and, in
addition to their antimicrobial effect on the eyelid margins and within
the MGs, exert secondary effects such as a suppressive action on bac-
terial lipase (tetracycline) and an anti-inflammatory action (macrolide).
Expression of the meibum is also an effective MGD treatment
method, and the efficacy of expression when combined with lid hygiene
and hot compresses has been reported (Aketa et al., 2019). Recently,
new treatment methods have been introduced, including the use of the
LipiFlow device, which performs both hot compresses and expression
mechanically, the use of the Maskin probe, which removes material
blocking duct openings (Nakayama et al., 2015), and Intense Pulsed
Light (IPL) therapy (Craig et al., 2015). In addition, studies have sug-
gested that vitamin D may be an effective MGD treatment (Arita et al.,
2017), and vigorous blinking may be useful in improving symptoms
(Kawashima and Tsubota, 2013).
Although MGD is an extremely serious disease, it is liable to be
taken less seriously than is warranted when encountered in daily clin-
ical practice. With the recent development of diagnostic tests and de-
vices, the development of novel treatment methods, and other advances
in the field of MGD, however, awareness of the importance of this
disease is increasing. Although much about MGD remains unknown, as
our knowledge increases, so too will the development of treatment
options and therapeutic strategies.
3.6. Antiaging approach
Dry eye prevalence increases with aging as a result of systemic
changes (Kawashima et al., 2013a). Although the basic treatment for
dry eye is ophthalmic solution, making improvements in environmental
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 15. Lid hygiene using eye shampoo.

After removing the makeup, apply the eye shampoo around the eyelashes, wash it, and finally wash the face (A). In a study targeting women with chronic eye
discomfort caused by heavy use of eye makeup and poor eye hygiene habits, eye shampoo improved subjective symptoms (B), meibomian gland orifice obstruction
(C), and meibum secretion compared to controls (D). Figure and modified caption reproduced from Okura et al. (2015) with permission of the copyright holder.

factors and daily habits are also important methods of treating and
preventing the disease. Studies published in recent years have gradually
increased our understanding of the lifestyle-related factors that are
related to dry eye. This has, in turn, led to a gradual shift from focusing
on treatment alone to initiating more aggressive attempts to prevent
dry eye onset, not relying solely on ophthalmic solution-based treat-
ment strategies, but rather more actively pursuing intervention strate-
gies based on preventative medicine (Kawashima et al., 2010, 2013a;
Kawashima and Tsubota, 2011; Tsubota et al., 2010, 2012) (Fig. 16).
The results of a 2011 dry eye survey conducted by the Dry Eye
Society on office workers (the Osaka Study) obtained new data re-
garding dry eye; dry eye prevalence among office workers is 65% and
that age is a risk factor for the condition (Uchino et al., 2013). As people
age, tear production decreases, and those with metabolic syndrome
have been shown to have a significantly reduced tear volume than those
without metabolic syndrome (Kawashima et al., 2014a) (Table 1).
3.7. Lifestyle intervention
We would like to emphasize the concept that dry eye is a lifestyle
disease. In recent years, it has been reported that there are a number of
lifestyle and environmental factors that influence diseases associated
with aging, including metabolic syndrome, and that oxidative stress is
involved as a background factor. Multiple dry eye mouse models and
clinical studies have provided support to the notion that oxidative stress
plays a major role in dry eye onset and pathophysiology. A lifestyle-
intervention approach consisting of exercise and diet therapy is the
basic treatment and preventative method for type II diabetes and

Fig. 16. Tear function and morphological change by CR effect. (A) Cotton thread test. AL, ad libitum group; CR, caloric restriction group, *p = 0.02. (B) Fluorescein
score, **p = 0.01. (C) Tear protein secretion increasing rate (% of before stimulation) by Carbachol stimulation. Figure and caption reproduced from Kawashima
et al. (2010) with permission of the copyright holder.

15
T. Kojima, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Table 1
Volume of tear secretion in MetS, pre-MetS, and non-MetS subjects.
MetS pre-MetS non-MetS total p-value (MetS vs. non- p-value (MetS vs. pre-
MetS) MetS)

n (men:women) 47 (44:3) 56 (55:1) 264 (171:93) 367 (270:97) – –

Age 40–49 years 15 34 179 228 – –
Age 50–64 years 32 22 85 139 – –

Tear volume by Schirmer test 11.0 ± 9.7 16.4 ± 10.2 18.5 ± 11.9 17.2 ± 11.7 0.000 0.007
Age 40–49 years 15.2 ± 10.8 17.6 ± 11.3 20.5 ± 11.8 19.7 ± 11.8 0.095 0.493
Age 50–64 years 9.0 ± 8.6 14.6 ± 8.2 14.4 ± 11.1 13.2 ± 10.3 0.015 0.021

Prevalence of lacrimal gland hypofunction (Schirmer 16 (34.0%) 10 (17.9%) 45 (17.0%) 71 (19.3%) 0.009 0.071
value ≤ 5 mm)

MetS, metabolic syndrome.

Statistical analysis: t-test.
A Schirmer value of <5 mm indicated lacrimal gland hypofunction according to the dry eye workshop definition. Table reproduced from Kawashima et al. (2014a)
with permission of the copyright holder.

Fig. 17. Comparative analysis of MET score and dry eye disease.
Each column shows the mean ± SD. P values were analyzed by Tukey's multi-
comparisons following ANOVA. DED: dry eye disease. Figure and caption re-
produced from Kawashima et al. (2014b) with permission of the copyright
holder.
Abbreviations: DED, dry eye disease.
metabolic syndrome, and one underlying mechanism that may explain
the success of this approach is a reduction in oxidative stress. In addi-
tion, the results of a cross-sectional study indicated that those in the
non-dry eye group exercised more than those in the dry eye group,
though it should be noted that this was based on a questionnaire
(Kawashima et al., 2014b) (Fig. 17). This study elucidated the fact that
those who exercised more had a higher tear volume (Kawashima et al.,
2014b). Based on these results, exercise is thought to be effective
against dry eye. Indeed, animal experiments have already shown that
exercise not only leads to systemic improvements, but also contributes
to improvements in tear volume (Sano et al., 2014).
In addition to the known fact that the desk work engaged in by
office workers is a risk factor for dry eye, it has also been hypothesized
that a “sedentary lifestyle” and “sympathetic dominance” may also
influence risk. The World Health Organization (WHO) has listed a se-
dentary lifestyle as a health risk, alongside smoking and obesity
(https://www.who.int/en/news-room/fact-sheets/detail/physical-
activity). It has been reported that remaining seated for long periods of
time while working leads to sympathetic dominance, which in turn
increases the risk of hypertension, diabetes, and cancer. One sub-ana-
lysis included in the Osaka Study showed that the amount of time spent
seated daily was significantly longer in the under 5-s TBUT (abnormal)
group, than in the over 5-s TBUT group (Kawashima et al., 2014b). The
guidance provided to dry eye patients has long been to “take sufficient
breaks (rest the eyes) when engaged in VDT (visual display terminal)
work or driving for long periods of time.” However, it is now considered
important that individuals spend time away from work-related activ-
ities moving or engaging in light exercise. If the disruption of auto-
nomic balance and resulting sympathetic dominance has a negative

Fig. 18. Time points at which the test parameters

were evaluated.
In the normal and abdominal breathing groups, the
R-R interval, tear meniscus volume, salivary amylase
activity, pulse, and blood pressure were investigated
before, immediately after, 15 min after, and 30 min
after the breathing activity was completed. Figure
and caption reproduced from Sano et al. (2015) with
permission of the copyright holder.

16
T. Kojima, et al.
Fig. 19. Increase in tear meniscus volume by abdominal breathing. Comparison
of tear secretion between the normal breathing group and the abdominal
breathing group, tear secretion increased significantly after 15 min in the ab-
dominal breathing group compared to the control breathing and the initial time
point (before) in the abdominal breathing group. Figure and caption re-
produced from Sano et al. (2015) with permission of the copyright holder.
effect on dry eye, then an interventional approach that leads to dom-
inance of the parasympathetic nervous system should be considered.
One evidence-based method of activating the parasympathetic nervous
system is abdominal breathing. We previously reported that 3 min of
abdominal breathing activates the parasympathetic nervous system and
increases tear volume (Sano et al., 2015) (Figs. 18 and 19). Therefore,
this may be an effect method of dry eye self-care that is simple, safe,
and does not require the use of any tools or devices.
In addition, recent evidence has indicated that depression has a
strong relationship with dry eye, and particularly dry eye symptoms
Fig. 20. Comparison of subjective happiness score (SHS) score between objective and subjective classifications. SD = standard deviation. Figure and caption
reproduced from Kawashima et al. (2015) with permission of the copyright holder.
17
Progress in Retinal and Eye Research xxx (xxxx) xxxx
(Kim et al., 2011b) (Labbe et al., 2013) (Vehof et al., 2014). Although
the causal relationship between depression and dry eye remains un-
known, it is thought that dry eye and the state of one's emotions have a
mutual effect on one another. Also, psychogenic factors such as de-
pression, anxiety, and stress are thought to cause changes in an in-
dividual's perception of pain. For example, patients with depression
have been shown to exhibit a lower pain threshold. In addition, a study
of other diseases has shown that those with depression report more
intense pain than those without depression, even when they have the
same disease and disease stage (Kim et al., 2011a). In the Osaka Study,
we found that there is an inverse relationship between the subjective
symptoms of dry eye and the level of subjective happiness: subjective
happiness was lower in the group with subjective symptoms of dry eye
but no other findings of the disease, whereas subjective happiness was
higher among those with findings of dry eye but no subjective symp-
toms (Kawashima et al., 2015) (Fig. 20). Finally, it has further been
indicated that those with dry eye commonly have sleep disorders
(Kawashima et al., 2016b), that there is a relationship between dry eye
and sleep disorders/mood disorders (Ayaki et al., 2015, 2016b), and
that dry eye treatment leads to an improvement in mood disorders
(Ayaki et al., 2016a).
Evidence for the effectiveness of exercise therapy on depression has
already been established, and based on this, exercise as a treatment for
dry eye could be an important lifestyle intervention (Sano et al., 2018).
It may be possible to extend the use of exercise therapy to patients with
strong subjective symptoms of dry eye despite clinical findings showing
improvements as a result of ophthalmic solution therapy, and patients
who would like to discontinue the use of ophthalmic solution due to its
inconvenience. Dry eye intervention based on positive psychology may
be effective in ameliorating subjective symptoms (Kawashima et al.,
2018), though the mechanisms of action underlying this are still being
studied. Furthermore, animal experiments have demonstrated that
happy, content mice experience reductions in tear volume when placed
under stress, indicating that psychology may also influence clinical
findings (Sano et al., 2019). Enriched environment (EE) was an effec-
tive intervention to prevent and alleviate stress-induced decreased tear
secretion. Furthermore, we found that brain-derived neurotrophic
factor (BDNF) expression is related to tear secretion and to the pa-
thology of dry eye disease (Fig. 21).
T. Kojima, et al.
Multiple studies into dietary habits have shown that dry eye is less
prevalent among those with a higher intake of polyunsaturated fatty
acids (PUFAs), but that an increased omega-6/omega-3 ratio is related
to a higher incidence of dry eye (Miljanovic et al., 2005). dry eye is
commonly accompanied by inflammation, and it has been indicated
that the anti-inflammatory properties of omega-3 are useful in ameli-
orating the subjective symptoms of dry eye (Miljanovic et al., 2005).
Indeed, a study by our group also reported that omega-3 intake is ef-
fective in the treatment of dry eye (Kawakita et al., 2013). Interestingly,
multiple studies have reported lower omega-3 PUFAs values in patients
with depression. A diet rich in fish, nuts, and other sources of omega-3
18
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 21. Enriched environment prevents and alle-
viates stress-induced decreased tear secretion.
(a) Standard environment (SE) and enriched en-
vironment (EE) (b) Basal tear secretion was not af-
fected by EE housing (n = 6). (c) EE housing pre-
vented decreased tear secretion compared to SE
housing (n = 6; **P < 0.01 by Student's t-test). (d)
EE prior to stress alleviated stress-induced decreased
tear secretion (n = 6; **P < 0.01,*P < 0.05 by
Student's t-test). (e) EE introduced following stress
was associated with faster recovery compared with
SE housing (n = 6; **P < 0.01 by Student's t-test).
Figure and caption reproduced from Sano et al.
(2019) with permission of the copyright holder.
may therefore be effective against dry eye. A recent multicenter, ran-
domized double blind study reported significant improvements from
baseline for OSDI, corneal and conjunctival vital staining score, and
tear break-up time for each arm (omega-3 PUFAs and oleic acid).
However, there were no significant between group differences. Since
oleic acid is a peroxisome proliferator-activated receptor agonist with
anti-inflammatory activity, omega-3 PUFAs may also possess the po-
tential benefit to improve ocular inflammation in dry eye disease (Dry
Eye et al., 2018).
Recent reports have indicated that lactoferrin (Kawashima et al.,
2012), probiotics, and functional foods that contain astaxanthin (Huang
T. Kojima, et al.
Fig. 22. Suppressive effect on ROS generation and decrease in tear secretion by
three common edible berries.
A: ROS generation in the lacrimal gland, tissue homogenates were incubated
with each berry extract. Bilberry extract (BBE), blackcurrant berry extract
(BCE) and maqui berry extract (MBE). ROS production was assessed using the
ROS-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescin diacetate.
(n = 5 lacrimal gland) *P < 0.05, versus BBE or BCE at 10 μg/mL. B: Effects on
tear secretion and corneal staining in a rat blink-suppressed dry eye model.
(n = 6 rats) *P < 0.05, versus the vehicle. All data represent the mean ± SD. C:
Typical tear secretion patterns measured by a cotton thread (upper) and corneal
staining (lower). Punctate staining appeared in the whole corneal surface with
the vehicle treatment. The arrow shows the wetted length by tear secretion.
Figure and caption reproduced from Nakamura et al. (2014) with permission of
the copyright holder.
et al., 2016) are effective in improving dry eye, highlighting the im-
portance of dietary interventions in dry eye patients. It has also been
reported that royal jelly intake can lead to increased tear volume (Inoue
et al., 2017), and that the intake of supplements containing a combi-
nation of lactoferrin and WB2000 is an effective method of improving
dry eye (Kawashima et al., 2016a).
After the Osaka Study was concluded with a key message that “dry
eye is a lifestyle disease”, we conducted a randomized controlled study
of a combined lifestyle intervention approach including exercise, diet,
and mood interventions in dry eye patients over a two-month period.
Although limitations included the fact that the study period was short
and that it was impossible to conduct a follow-up study of the patients,
this study produced hopeful results, demonstrating a significant im-
provement in subjective symptoms (Kawashima et al., 2018). In the
future, we would like to conduct a more detailed study into this.
Evidence that things that are potentially good for the body as a
whole are also good for dry eye is gradually accumulating. In the future,
treatment and preventative measures for dry eye are likely to in-
corporate the use of localized treatment of the tear layer such as oph-
thalmic solution with comprehensive interventional options for the
entire body and brain. Now, in addition to detailed interviews with and
examinations of patients designed to determine their lifestyle, risk
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
factors, and the type of dry eye they are suffering from, we are also
employing a lifestyle intervention approach tailored to each individual
patient.
3.8. Neuropathic pain treatment
It is widely accepted that improved tear stability is effective in
improving the clinical symptoms of dry eye. However, it has been found
that there are dry eye patients existing who are resistant to improve-
ments in subjective symptoms. Recently, the concept of neuropathic
ocular pain(NOP) has been found to apply to some cases of dry eye.
Treatment strategy is determined following identification of the site of
the pain, but in cases of peripheral pain, additional treatment is pro-
vided for dry eye and MGD and the treatment outcome is then assessed.
In cases of centralized pain, where it has been determined that tear
dynamics require improvement, the first-line treatments are ophthalmic
solutions and punctal plug insertion, as is performed as part of dry eye
treatment. In cases of short-BUT dry eye, 3% diquafosol sodium oph-
thalmic solution is effective against corneal hypersensitivity (Kaido
et al., 2018). Since patients with chronic pain often have either ag-
gressive pain or neuropathic pain, it is believed that objective findings
such as reduced tear volume and increased evaporation are in-
compatible (Galor, 2019). Significant improvement of symptoms of
allodynia and photoallodynia was found after treatment with 20% au-
tologous serum tear within 3.6 months of treatment in NOP patients. In
addition to improvement in patients’ symptoms, IVCM clearly demon-
strates a significant increase in corneal subbasal nerve density, as well
as decrease in nerve reflectivity and tortuosity. They concluded that the
ability of autologous serum tear to regenerate corneal nerves has been
associated with the improvement of the symptoms of NOP (Aggarwal
et al., 2015).
Currently, there is no evidence-based treatment of centralized,
peripheral, or combined-type pain. However, there have been reports
that, in addition to drugs that act on the central nervous system such as
pregabalin and duloxetine, nerve blockers (Duerr et al., 2019) and
acupuncture (Dieckmann et al., 2017) are also effective. Our group has
reported that cognitive behavioral therapy leads to an improvement in
the subjective symptoms of dry eye (Sano et al., 2018), and we are
observing an increased number of cases in which strength-based in-
tervention, a type of positive psychology, is effective (Matsuguma et al.,
2018).
Given that eye pain patients have a multitude of background char-
acteristics, no standard treatment has been established. The use of
acupuncture and moxibustion in addition to nerve blockers in refractive
cases is currently being investigated. Thus, treatment options that are
tailored to individual patients through cooperation with other medical
departments would seem to be advantageous.
3.9. Antioxidants
Dry eye is caused by reduction of tear secretion due to lacrimal
glands hypofunction. Our research group obtained data that the un-
balanced antioxidant status causes the functional decline of the tear
secretion capacity due to damage of lacrimal gland. Deficiency of
Cu,Zn-superoxide dismutase (SOD1), a ubiquitous cytosolic enzyme
which catalysis the oxygen free radicals, in mice leads to the decreased
production of tears, and an extensive lipid and DNA oxidative stress
damage in the lacrimal gland (Kojima et al., 2012). Transgenic mice
which impaired activity of electron transport chain in mitochondria
exhibits overexpression of reactive oxygen species (ROS) induced de-
creased production of tears accompanied by lacrimal inflammation
(Uchino et al., 2012). In addition, the rat blink-suppressed dry eye
model, simulate a mode with excess computer users by repeatedly ex-
posing rats to stressful conditions, showed persistent disordered tear
dynamics due to lacrimal gland dysfunction with the excessive forma-
tion of ROS (Nakamura et al., 2014). These studies showing that to
T. Kojima, et al.
modify the generated ROS production from lacrimal glands may have a
great potential for managing or preventing dry eye syndrome and
suggested that the pharmacological modulation of LG dysfunction may
be a prospective treatment.
Anthocyanins are common plant polyphenolic pigments. Like other
flavonoids, anthocyanins are also expected to have antioxidative with
potential benefits for human health including vision and eye (Tsuda,
2012). Anthocyanins-rich berries and berry ex-tracts have been used as
food supplement for the treatment of eye diseases based on the findings
of animal and human studies (Ohguro et al., 2012; Matsumoto et al.,
2005). However, a suitable source of berries or the active anthocyanins
that attenuates dry eye symptoms have yet to be examined in detail.
Scavenging effect of ROS in lacrimal glands wad evaluated among
common edible berries for natural ingredients, bilberry, blackcurrant
berry and maqui berry using homogenized rat lacrimal gland
(Fig. 22A). All berry species dose-dependently suppressed ROS gener-
ated. The suppression of ROS was significantly stronger with maqui
berry than with blackcurrant berry or bilberry. To further assess the
effects of dietary supplementation of maqui berry, edible berries with
potent antioxidative property, on dry eye, murine and human clinical
trial were conducted. In a rat blink-suppressed dry eye model, the
prevention of a reduction in tear secretion and increase in corneal
20
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 23. Effect of mixed supplement with probiotics
Enterococcus faecium WB2000, on tear production
and ROS formation from the lacrimal gland in a rat
dry eye model.
(A) Tear secretion and (B) ROS formation from the
lacrimal gland. The supplement components were
dissolved in 2% DMSO at 0.4 mg/ml (10 mg/kg) or
2 mg/ml (50 mg/kg) and orally administered at 1 ml
daily for 2 days. The 2% DMSO served as the vehicle
control. Right panel: Tear production was measured
using a cotton thread. Left panels: The length of
wetting (arrows) in the cotton thread by tear secre-
tion in the vehicle (top) and 50 mg/kg groups
(bottom). Data are presented as the mean ± SD.
(n = 6 per group) *P < 0.05, **P < 0.01,
***P < 0.001 versus the vehicle. Figure and caption
reproduced from Kawashima et al. (2016a) with
permission of the copyright holder.
Abbreviations: DCF, dichlorofluorescein.
staining was significant stronger with orally maqui berry extract
(Fig. 22B and C). A randomized double-blind placebo-controlled clin-
ical trial was undertaken on probable dry eye patient for 4 weeks
(Yamashita et al., 2018). This clinical trial resulted in significant in-
crease in Schirmer value in the maqui berry group compare to placebo
group.
There is accumulating evidence that microbiome is related to health
and pathogenesis of various diseases (de Vos and de Vos, 2012). In-
testinal dysbiosis exacerbates dry eye in the Sjögren syndrome model
mice with desiccating stress, and Sjögren syndrome patients' ocular
surface and systemic severity are negatively correlated with microbial
diversity (de Paiva et al., 2016). Probiotics are microbial food supple-
ments that can potentially benefit for wide variety of health and have
been used widely throughout the world (Williams, 2010). Although the
beneficial pleiotropic effects of probiotics are reported, the underlying
mechanisms are multifactorial and still are not thoroughly understood.
Reduction of oxidative stress have recently been proposed as possible
underlying mechanisms (Wang et al., 2017; Mishra et al., 2015). To
examine the effect of dietary supplementation of probiotics on dry eye,
we investigated the efficacy of the combined supplement with probio-
tics Enterococcus faecium WB2000, which was isolated from the feces of
a healthy human and is contained in the Japanese gastrointestinal
T. Kojima, et al.
Fig. 24. The efficacy of the combined supplement with probiotics Enterococcus
faecium WB2000 in dry eye patient.
Rate of increase in the Schirmer test value in human subjects in the supplement
and placebo groups. Combined dietary supplement (2 capsules/day; 20 parti-
cipants) and placebo (vehicle; 19 participants) groups and treated once daily for
8 weeks. Data are presented as indicates a mean ± SD. P values versus placebo
group. Figure and caption reproduced from Kawashima et al. (2016a) with
permission of the copyright holder.
Fig. 25. The relation between tear evaporation rate from ocular surface
(TEROS) and environmental humidity.
Data was obtained from a subject applying TEROS measurement goggles. Please
note that the evaporation rate dramatically decreases when environmental
humidity is low. Figure and caption reproduced from Tsubota and Yamada et al.
(1992) with permission of the copyright holder.
agent, on dry eye signs and symptoms in human subjects and murine
dry eye model (Kawashima et al., 2016a). In a rat blink-suppressed dry
eye model, dose dependent the prevention of a reduction in tear se-
cretion and reduction ROS generation from lacrimal gland were ob-
served in probiotics containing supplement (Fig. 23). Randomized,
double-blind, placebo-controlled trial in human patients treated once
daily for 8 weeks resulted in the rate of increase in the Schirmer value
was greater in the supplementation group and no adverse events oc-
curred (Fig. 24).
Antioxidant consumption by common edible agent, such as antho-
cyanins or probiotics, may be a good candidate for treatment for patient
with dry eye.
3.10. Other treatment modalities
3.10.1. The past, present and future of dry eye spectacles in the
management of dry eye diseases
Early work showed that dry eye patients develop symptoms in
21
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 26. Modern dry eye glasses.
The glasses have a cover integrated with the frame of the glasses. In addition, it
has a tank for containing water, and it is possible to put water into it to further
increase the humidity around the eyes. Figure and modified caption reproduced
from Ogawa et al. (2018) with permission of the copyright holder.
windy or dry environments due to increased tear evaporation from the
ocular surface (Tsubota and Yamada, 1992). Employing a tear eva-
poration rate analyzer, the TEROS, Tsubota and colleagues reported in
1992 that the relative contribution of tear evaporation to overall tear
dynamics was greater in dry eyes than normal eyes. The authors had
then devised a correlation equation between tear evaporation values
and environmental humidity proposing that the lesser the humidity, the
greater the evaporation from the ocular surface (Fig. 25) (Tsubota and
Yamada, 1992) (Tsubota, 1998). Our study also suggested that the
greater the humidity surrounding the eye the better the tear film layer
and that it is important to decrease any air flow by enclosing the space
surrounding the eyes with side panels or goggles (Tsubota et al., 1994).
The initial prototype of dry eye spectacles had small and moistened
triangular sponges attached to special side panels of modified eye-
glasses. The idea was to increase the periocular humidity due to eva-
poration from moist side sponges. The impracticability of preparing
sponges, attaching them to side panels and applying eye drops on them
led to the development of better versions with moist water tanks built in
the side frames (Fig. 26) (Ogawa et al., 2018). These spectacles provide
15.2% higher humidity over the corneal surface compared to the room
humidity. This work also demonstrated that experimental wind ex-
posure significantly elevated the dryness symptom scores and tear
evaporation rates in eyes without spectacle wear whereas no significant
changes in symptom scores, the tear film break up times and tear
evaporation rates were observed when these spectacles were worn.
These spectacles also perform better than the conventional glasses in
relation to dry eye symptom and tear stability. Relief of dry eye
symptomatology may be attributable to the decrease of the tear film
hyperosmolarity which justifies further studies assessing osmolarity
changes associated with the use of these spectacles. The design and
weight of these spectacles are extremely important because the moist-
urizing effect is lost if the spectacles do not have a good fit and slip
down on the nose. Eyeglass position should be checked and the me-
chanism for humidity maintenance should be explained to the dry-eye
patients when prescribed. The use of lightweight lenses, correctly sized
ear wires, and properly cut side panels tailored to each dry eye patient
T. Kojima, et al.
are essential issues where we hope to see new technological improve-
ments.
Humidity control in previous dry eye glasses has relied on passive
evaporation of water from wet sponges and water tanks. Recently, ul-
trasound moisture glasses, which we developed, have built-in ultra-
sound atomizing transducer, and can actively control humidity
(Fig. 27). In our preliminary study, it was shown to improve the
symptoms and BUT of subjects with dry eye symptoms (Onomura et al.,
2018). Development of lenses with antifogging properties or water re-
pellant chemicals remain as unexplored future research territories.
3.11. Future directions and conclusions
With advances in dry eye diagnosis, it has become possible to detect
the abnormalities of each tear film layer and this strategy is referred to
as TFOD. As a result, dry eye can now be divided into aqueous deficient,
decreased wettability, and increased evaporation type. At present,
wettability of the ocular surface seems to be associated with membrane
type mucin 16. However, research combining clinical and molecular
analysis is poor and there is no clear evidence. In addition, it has been
clarified that the symptoms of dry eye, though to a greater or lesser
extent, involve the neuropathic pain component. Therefore, it has be-
come important to evaluate the ocular surface hypersensitivity in ad-
dition to tear film oriented diagnosis. Currently, there are a few ex-
amination methods that can be used clinically, and future research and
development should be necessary. When dry eye and MGD develop as
comorbidities, the tear breakup time is reduced regardless of the dry
eye subtype. Although the treatment of MGD is very important, there is
no fundamental treatment to improve the keratinization of meibomian
gland duct, and further research is necessary.
In terms of treatment, a new treatment strategy, the TFOT, which
applies to tear film abnormalities revealed by TFOD, is popularly applied
in Japan and Asia, and made individualized medical treatment tailored
to the patient's dry eye conditions. TFOT concept has made a great
progress with the advent of the aqueous/mucin secretagogue. Currently,
dry eyes associated with neuropathic pain are difficult to treat, and
strategies to approach the corneal nerve and central nerves directly and
thus the tear film will require further investigation in the future.
Until now, dry eye was considered to be an ocular surface disease,
22
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 27. An overview of ultrasonic moisture glasses.
(A) An atomizing transducer (A ′) and a water tank
are provided on the side of the glasses (A). Water
mist is shown blowing from atomizing transducer
(B). A schematic of ultrasonic moisture glasses is
shown (C). The glasses were worn on a mannequin
and the ambient humidity was measured (D). Figure
and modified caption reproduced from Onomura
et al. (2018) with permission of the copyright holder.
but recent basic and epidemiological studies have revealed that dry eye
is a life-style disease. In relation to dry eye treatment and in addition to
ophthalmic solution-based strategies, life-style interventions such as
diet therapy, exercise, and anti-aging approaches may be effective for
dry eye treatment. In the future, life-style interventions might become
the first line treatment in dry eye disease. Further research will be
needed to determine the role of life style interventions not only on the
treatment efficacy but also on the prevention of dry eye disease onset.
Funding
The research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declarations of competing interest
Takashi Kojima reports personal fees from Staar Surgical, Santen
Pharmaceutical Co., Ltd., Johnson & Johnson, Otsuka Pharmaceutical
Co., Ltd., Carl Zeiss Meditec, Echo Electricity Co., Ltd., and Alcon
Japan, outside the submitted work.
Murat Dogru reports personal fees from Santen Pharmaceutical Co.,
Ltd, Otsuka Pharmaceutical Co., Ltd, and Echo Electricity Co., Ltd., and,
outside the scope of the paper, receives research grants from Echo
Electrical Co., Kobayashi Pharma and I.com, Inc.
Kazuo Tsubota reports consultancies and research funding received
from Santen Pharmaceutical Co., Ltd., Wakamoto Pharmaceutical Co.,
Ltd, and Otsuka Pharmaceutical Co., Ltd.; research funding received
from Kowa Company; patent holder for the method and the apparatus
used for the measurement of functional visual acuity (US patent no: 255
7470026), inventor of JINS Moisture eye glasses by JINS, Inc., patent
holder; and outside the submitted work, he reports the position of CEO
of Tsubota Laboratory, Inc. a company in the dry eye field; con-
sultancies for Laboratoires Thea and Novaliq GmbH; investor in Tear
Solutions and Tissue Tech, Inc.; research funding from Rohto
Pharmaceutical Co., Ltd., Pfizer, R-Tech Ueno, Ophtecs, Alcon Japan,
and AMO Japan K.K.
Motoko Kawashima and Shigeru Nakamura report no financial in-
terests.
T. Kojima, et al.
Acknowledgements
The authors express our sincere thanks to Ms. Catherine Oshima for
generous administrative support and editing of the manuscript.
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Further reading
Landsend, E.C.S., Utheim, O.A., Pedersen, H.R., Aass, H.C.D., Lagali, N., Dartt, D.A.,
Baraas, R.C., Utheim, T.P., 2018. The level of inflammatory tear cytokines is elevated
in congenital aniridia and associated with meibomian gland dysfunction. Invest.
Ophthalmol. Vis. Sci. 59, 2197–2204.