The Ocular Surface 16 (2018) 45e57

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The Ocular Surface

journal homepage: www.theocularsurface.com

Review

The impact of diabetes on corneal nerve morphology and ocular

surface integrity
Maria Markoulli a, *, Judith Flanagan a, b, Shyam Sunder Tummanapalli a, Jenny Wu a,
Mark Willcox a
a
School of Optometry & Vision Science, University of New South Wales, Sydney, Australia
b
Brien Holden Vision Institute, Sydney, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Diabetes mellitus is a chronic disease that results from inadequate insulin production or ineffective in-
Received 18 January 2017 sulin utilization. It is one of the most common systemic diseases worldwide with increasing prevalence.
Received in revised form Diabetes mellitus is associated with premature mortality, macrovascular complications such as cardio-
3 October 2017
vascular disease, and microvascular complications, including nephropathy leading to kidney failure,
Accepted 26 October 2017
potentially blinding diabetic retinopathy, and diabetic neuropathy. While the retinal complications of
diabetes are well recognized by eye care professionals, the effects on the ocular surface are poorly un-
Keywords:
derstood. Recent studies have reported on the association between peripheral neuropathy and corneal
Corneal erosions
Corneal nerves
neuropathy, showing the latter to be of predictive value for the systemic disease. Corneal neuropathy can
Corneal neuropathy lead to loss of corneal sensation and can ultimately result in neurotrophic ulcers and significant visual
Diabetes morbidity. The epithelial fragility and poor wound healing that result from reduced epithelial adhesion to
In vivo confocal microscopy the underlying basement membrane in diabetes, together with corneal neuropathy, are thought to in-
Neuropathy crease the susceptibility to persistent corneal erosions and infection, as well as to increase the risk of
Neuropeptides post-surgical complications. The aim of this article is to review the impact of diabetes on corneal nerve
Ocular microbiome morphology and ocular surface integrity. Changes in the tear film and ocular surface microbiome are
Ocular surface
highlighted in discussion of the mechanisms that underpin ocular surface changes that increase the
Tears
susceptibility to corneal erosion and infection.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction
1.1. Overview
The World Health Organization (WHO) defines diabetes mellitus
as “a chronic disease that occurs when the pancreas does not pro-
duce enough insulin, or when the body cannot effectively use the
insulin it produces.” [1] It is one of the most common systemic
diseases in the world, with the American Diabetes Association
estimating that 25.8 million children and adults, approximately 8.3%
of the population of the United States of America, have the condition
[2]. Worldwide, diabetes is thought to affect 415 million people [3]
and its prevalence continues to rise [4], with estimates that dia-
betes will affect more than 640 million people by the year 2040 [3].
* Corresponding author. School of Optometry & Vision Science, The University of
New South Wales, Sydney, NSW, 2052, Australia.
E-mail address: m.markoulli@unsw.edu.au (M. Markoulli).
The WHO classifies diabetes into three main categories [5], with
other sub-categories also described in the literature. The first
category, type 1, comprises 5e10% of the diabetic population, with
an age of onset usually before 30 years; the underlying cause is
destruction and loss of the secretory function of insulin-producing
pancreatic b-cells. The genetic characterization of maturity-onset
diabetes in the young (MODY) reviewed by Tuomi et al. [6] fea-
tures defective control of insulin secretion and introduces another
type of insulin deficiency.
The second category, type 2 diabetes, affects approximately 95%
of the diabetic population with increasing prevalence [3], described
by some as the pandemic of the 21st century [7]. This form of
diabetes is a result of inadequate insulin production or utilization
[8]. Risk factors include family history, being overweight, older age,
ethnicity, gestational diabetes (shortage of insulin or pregnancy
hormones), impaired glucose tolerance or impaired fasting glucose,
hypertension, and abnormal cholesterol levels. In many instances,
there is uncertainty regarding the diagnosis of type 2 diabetes, for

https://doi.org/10.1016/j.jtos.2017.10.006
1542-0124/© 2017 Elsevier Inc. All rights reserved.
46 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57

Abbreviations NADPH nicotinamide adenine dinucleotide phosphate

NF-kB nuclear factor-kB
AGE advanced glycation end products NGF nerve growth factor
AMP adenosine monophosphate NO nitric oxide
CGRP calcitonin gene-related peptide NPY neuropeptide Y
COX cyclooxygenase NT-3 neurotrophin-3
DAG diacylglycerol PKC protein kinase C
DPN diabetic peripheral neuropathy PMNs polymorphonuclear leukocytes
eNOs nitric oxide synthase PRK photorefractive keratectomy
Epi-LASIK epithelial laser in situ keratomileusis ROS reactive oxygen species
IGF-1 insulin-like growth factor SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
IGFBP3 insulin-like growth factor binding protein 3 electrophoresis
IGF-1R insulin-like growth factor receptor TGF-b transforming growth factor-b
IL-1 interleukin 1 TIMP tissue inhibitors of metalloproteinase
LADA latent autoimmune diabetes in adults TNF-a tumor necrosis factor- a
LASEK laser epithelial keratomileusis VEGF vascular endothelial growth factor
LASIK laser in situ keratomileusis VIP vasoactive-intestinal peptide
MMPs matrix metalloproteinases WHO World Health Organization
MODY maturity-onset diabetes in the young ZO zonula occludens
Naþ/Kþ ATPase sodium potassium adenosine triphosphatase

example, in those who are not overweight and do not have meta- wound healing [31,32], increased susceptibility to persistent
bolic syndrome and in those with residual b-cell function [6]. This epithelial erosions [26], and hence propensity to corneal infection
confusion has led to the introduction of latent autoimmune dia- [33,34].
betes in adults (LADA) [9], thought to occur in 2e12% of all cases of
diabetes [10]. 1.2. Aim
The third diabetic category is known as pre-diabetes [11e13],
and is considered to be a substantial risk factor for progression to The aim of this article is to review the impact of diabetes on
type 2 diabetes and associated complications [14]. It is thought to corneal nerve morphology and ocular surface integrity. Changes in
affect between 10 and 15% of the population [11]. the tear film and ocular surface microbiome are highlighted in
Diabetes is associated with premature mortality and with sys- discussion of the mechanisms that underpin these ocular surface
temic complications such as cardiovascular disease, and micro- changes that increase the susceptibility to corneal erosion and
vascular complications including retinopathy, nephropathy and, infection.
neuropathy [8], leading to organ and tissue damage in approxi- A literature review was conducted through PubMed Central
mately one third to one half of people with diabetes [15]. These using the following keywords: diabetes AND peripheral neuropa-
complications can have a significant impact on both immediate and thy, diabetes AND corneal neuropathy, diabetes AND corneal ero-
long-term healthcare costs [16]. Chronic hyperglycemia is the core sions, diabetes AND wound healing, diabetes AND tear film,
causative mechanism for each of these complications as a result of diabetes AND ocular surgery, diabetes AND contact lens wear and
changed metabolism, which in turn results from advanced glyca- diabetes AND microbiome. Articles were included up to January
tion end (AGE) products, abnormalities in signaling cascades, and 2017.
elevated production in reactive oxygen species (ROS), as well as
abnormalities in the stimulation of hemodynamic regulation sys-
tems [17]. By controlling hyperglycemia, this cascade can also be 2. Corneal neuropathy
controlled and the complications delayed [18e20].
In the diabetic eye, damage to the retinal capillaries leads to Corneal nerves bundles, derived from the nasociliary branch of
progressive capillary occlusion, which in turn results in retinal the ophthalmic division of the trigeminal nerve, enter the
ischemia. Gradually, ischemic retinal tissue elaborates signals to
stimulate new vessel proliferation. New vessels are prone to
leakage of intravascular fluid and are also prone to hemorrhage,
increasing the risk of blindness from retinal detachment secondary
to a fibrovascular membrane, and macular edema [21].
While the retinal complications of diabetes are well-recognized
by eye care professionals, the impact on the ocular surface is poorly
understood, despite the fact that up to 70% of people with diabetes
suffer from diabetic keratopathy during the course of their disease
[22e25]. This lack of understanding may be due to the varied
presentation of ocular surface disease in diabetes, ranging from dry
eye disease [26], through to neurotrophic ulcers (Fig. 1) [27]. Recent
work has revealed reduced corneal nerve density to be progressive
with duration of diabetes [28]. This change in morphology results
in functional changes such as loss of corneal sensitivity [29,30] and Fig. 1. A neurotrophic ulcer as a result of diabetes (courtesy of the L.V. Prasad Eye
is thought to contribute to subsequent events resulting in poor Institute, Hyderabad, India).
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
peripheral cornea in a radial fashion parallel to the corneal surface
in the stroma and converge in an area known as the inferior whorl
(Fig. 2) [35]. The large nerve bundles then divide, taking a 90-
degree turn to penetrate Bowman's layer to form the corneal sub-
basal nerve plexus between Bowman's membrane and the basal
epithelial cells [36]. The small nerve fibers terminate in free nerve
endings in the corneal epithelium [36]. Unlike nerves elsewhere in
the body, it is possible to image the corneal nerves in vivo using
in vivo confocal microscopy due to the transparency of the cornea
[37]. Such imaging offers useful insight into the systemic impact of
diabetes [28,38,39].
Chronic hyperglycemia induces several pathological pathways
that result in nerve damage that leads to neural loss in peripheral
nerves, including the corneal nerves [40]. The following section
reviews the pathogenesis of diabetic neuropathy in order to better
understand the pathophysiology of corneal neuropathy.
2.1. Pathogenesis of diabetic neuropathy
A number of pathological pathways have been identified, which
intersect and complement each other in the process of diabetic
nerve damage, with both metabolic and vascular factors acting
synergistically in all stages of the disease [41,42]. These mecha-
nisms and pathways are summarized in Fig. 3 and include oxidative
stress, AGE products, the polyol pathway and protein kinase C (PKC)
activation [43e45].
2.1.1. Oxidative stress
Oxidative stress refers to the state in which the production of
ROS exceeds the ability of any surrounding antioxidants to reduce
ROS to non-toxic substances. Oxidative stress can lead to tissue
damage [46]. In diabetes, elevated levels of extracellular glucose
increase the oxidative metabolism of glucose in mitochondria [44].
Excessive entry of glucose causes surplus transport of electrons,
generating highly reactive ROS known as superoxides [44]. These
superoxides cause a dangerous imbalance in the mitochondrial
electron transport chain, accompanied by reduced scavenging ca-
pacity of antioxidant enzymes [44]. The larger mitochondrial vol-
ume in nerve fibers renders axons more susceptible to
mitochondrial hyperglycemic injury with a depletion of energy,
Fig. 2. In vivo confocal microscopy image of the inferior corneal whorl.
47
causing conduction block and demyelination of axons [45,47e50].
Neurotrophic factors such as neurotrophin-3 (NT-3) and nerve
growth factor (NGF) are also reduced in concentration by mito-
chondrial damage [51,52]. In the diabetic cornea, 8-
hydroxydeoxyguanosine has been measured in the diabetic rat
cornea as a marker of oxidative stress and has been found to be
increased relative to control rat corneas [53]. This indicates that the
apoptosis of corneal cells seen in diabetes may be related to
oxidative stress.
2.1.2. AGE products
Protein glycation leading to the formation of AGE products plays
an important role in the pathogenesis of diabetic peripheral neu-
ropathy [54,55]. Glycation is the non-enzymatic covalent binding of
a protein or lipoprotein with a sugar molecule such as fructose or
glucose [56]. These non-enzymatically glycated proteins slowly
form crosslinked protein products with altered structure and
function known as AGE products [44]. Myelin proteins of the nerves
are glycated in diabetes, resulting in structural and functional
damage of the peripheral nerves and axonal degeneration [55]. AGE
deposition has been reported in perineurial collagen and in the
axoplasm of nerve fibers and Schwan cells of human diabetic pe-
ripheral nerves, suggesting that the enhanced glycation may play a
role in the development of diabetic neuropathy [57,58]. In the
cornea, the accumulation of AGE products may be responsible for
the observed increased autofluorescence [59]. A higher expression
of AGE products has been reported in the diabetic rat cornea,
indicating that this may be implicated in the development of dia-
betic keratopathy as a result of apoptosis [53].
2.1.3. The polyol pathway
Hyperglycemia causes increased levels of intracellular glucose
in nerve cells, leading to saturation of the normal glycolytic
pathway. Excess glucose is therefore shunted into the polyol
pathway, known also as the sorbitol-aldose reductase pathway
[60]. The excess glucose is then converted to sorbitol and fructose
by the enzymes aldose reductase and sorbitol dehydrogenase. In
the process of reducing high intracellular glucose to sorbitol, aldose
reductase consumes and depletes the cofactor nicotinamide
adenine dinucleotide phosphate (NADPH) [61]. In addition, the
nerve cell membrane is relatively impermeable to sorbitol and
fructose, which tend to accumulate within the nerve cell, increasing
osmotic stress [62]. This excess accumulation of sorbitol and fruc-
tose leads to reduced levels of free nerve myoinositol, a sugar
alcohol that is required for the normal nerve function [63]. A
reduction in myoinositol decreases membrane sodium potassium
adenosine triphosphatase (Naþ/Kþ ATPase) activity, which slows
down nerve conduction velocity and brings about nerve structural
breakdown. The polyol pathway is known to play a role in diabetic
cataract formation [64], but its role in corneal neuropathy is not yet
understood.
2.1.4. Protein kinase C activation pathway
Elevated intracellular glucose stimulates the synthesis of an
endogenous protein kinase C (PKC) activator, diacylglycerol (DAG)
[65,66]. Activation of PKC can affect Naþ/Kþ ATPase activity, and
other enzymes that are crucial in cellular membrane potential,
nerve conduction, and nerve regeneration [67e69].
2.2. Diabetic peripheral neuropathy
Diabetic neuropathy is characterized by progressive loss of pe-
ripheral nerve axons in a distal to proximal pattern, resulting in
pain, decreased sensation, and eventually complete loss of sensa-
tion [70]. A simple classification based on anatomical and
48 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
Fig. 3. Pathophysiology of diabetic neuropathy: the underlying mechanisms of nerve damage as a result of hyperglycemia.
pathophysiological characteristics originally proposed by Thomas
[71] and modified by Boulton [72] and Vinik [73,74] classifies dia-
betic neuropathies as peripheral, autonomic, proximal and focal.
Diabetic peripheral neuropathy is the most common neuropathic
presentation in diabetes, comprising 75% of all diabetic neuropa-
thies [75], with painful diabetic peripheral neuropathy being pre-
sent in approximately 26% of patients with diabetes [76]. Following
a 25-year follow-up, approximately 50% of patients with diabetes
were found to have diabetic peripheral neuropathy [77]. Foot ul-
ceration occurs in 7% of patients with diabetic peripheral neurop-
athy compared with 1% of individuals with diabetes but without
neuropathy [33]. Lower limb amputation is required in advanced
cases [33,72,76,78]. There is currently no U.S. Food and Drug
Administration (FDA)-approved therapy that prevents or reverses
diabetic peripheral neuropathy. Attention is therefore targeted at
detection in order to identify those at increased risk of developing
sequelae.
Diabetic peripheral neuropathy affects both the small and large
peripheral nerve fibers. The current gold standard for diagnosis of
diabetic peripheral neuropathy is vibration perception using bio-
thesiometry [79]. However, biothesiometry measures large nerve
fiber function rather than small nerve fiber function, with changes
to the latter being more prevalent in type 1 diabetes [80] and being
an earlier indicator of peripheral neuropathy [81]. Objective and
sensitive assessment of small nerve fiber damage is via skin punch
biopsy to examine nerve morphology [82]. Biopsy, however, is
invasive and non-repeatable [83], and the procedure can place
patients at risk of poor wound healing and infection [84]. The lack
of a sensitive, non-invasive, and repeatable endpoint to measure
changes in peripheral nerves is a major factor limiting clinical trials
for the treatment of diabetic peripheral neuropathy [33,85,86].
Morphological changes in the corneal subbasal nerve plexus
correlate with changes in the peripheral nerves and hence might
provide an alternate measure for diabetic peripheral neuropathy
[33,87e89].
2.3. Corneal neuropathy as an alternate measure of peripheral
neuropathy
The corneal subbasal nerve plexus is the main object for study
when assessing corneal nerve density using in vivo confocal mi-
croscopy. It is arranged in a single plane parallel to the corneal
surface, whereas stromal nerves are distributed throughout the
stroma in three dimensions, making their density difficult to
estimate [36]. In vivo confocal microscopy illuminates and images a
single point of tissue (400  400 mm) simultaneously in the same
plane. The parameters measured include: nerve fiber bundle den-
sity (number of nerve fiber bundles per mm2), nerve branch density
(number of nerve fiber branches per mm2), total nerve fiber length
(per mm2), number of nerve interconnections (per mm2), nerve
fiber width and nerve fiber tortuosity [37]. In healthy people
without diabetes, these variables have been found to be stable over
a three-year period [90], except in contact lens wearers, in whom
reduced nerve density compared to non-lens wearers has been
reported [91]. Age has also been shown to reduce corneal nerve
fiber density [90,92,93]. A recent review by Cruzat et al. highlighted
the corneal diseases that can affect corneal nerve parameters [94].
In diabetes, a reduction in corneal nerve fiber length, corneal nerve
fiber density, and nerve fiber branch density has been found with
increasing neuropathic severity in both type 1 and type 2 diabetes
(Fig. 4) [89,95]. Edwards and colleagues [83] showed that study
participants with diabetic peripheral neuropathy had significantly
reduced corneal nerve fiber length and branch density compared to
controls and participants with diabetes but without diabetic pe-
ripheral neuropathy [83]. Pritchard et al. reported in a longitudinal
study that reduction in corneal nerve fiber length is predictive of
the development of diabetic peripheral neuropathy [96], while
Misra and colleagues reported that subbasal nerve density changes
precede other clinical and electrophysiology tests of neuropathy
[39]. These findings are supported by a meta-analysis that included
13 studies and 1680 participants and found that corneal nerve fiber
density, nerve branch density and nerve fiber length were signifi-
cantly reduced in people with diabetic peripheral neuropathy [97].
In the animal model, a study on the corneal dendritic cells in
streptozotocin-diabetic mice identified greater corneal infiltration
with dendritic cells compared to controls, as well as a significant
negative correlation between the number of dendritic cells and
corneal nerve fiber density [98]. The close proximity of dendritic
cells with the subbasal nerve plexus and their function to recog-
nize, process, and present antigens indicates that the dendritic cells
contribute to the development of corneal neuropathy [98].
While diabetic neuropathy might be associated with reduced
nerve migration [99], that is, the movement of nerves relative to the
inferior whorl, recent studies have demonstrated that corneal
nerves in people with diabetes can regenerate after interventions to
improve diabetic control [40,88,100]. Significant improvements in
nerve fiber length were seen 12 months after simultaneous
pancreas and kidney transplantation in a group of 15 patients with
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
Fig. 4. A reduction in corneal nerve fiber density has been reported in patients with diabetes relative to healthy controls.
type 1 diabetes [78], and significant improvements in intra-
epidermal nerve fiber density were seen after 24 months of strict
glycemic control and changes in lifestyle [101]. These studies sug-
gest that corneal nerve assessment may be a suitable index to
evaluate interventional efficacy.
While diabetic corneal neuropathy has significant implications
for the diagnosis of diabetic peripheral neuropathy, it also has
significant local impact on the ocular surface itself. The following
section reviews the downstream effect of diabetic corneal neu-
ropathy on the integrity of the ocular surface and how neuropathy
increases the susceptibility to injury and infection.
3. Ocular surface integrity in diabetes
Corneal nerves provide trophic support to the corneal epithelial
cells by releasing soluble mediators that stimulate epithelial cell
growth, mitosis, differentiation, and migration [36]. Epithelial cells,
in turn, provide trophic support to corneal neurons by secreting
growth factors that promote neurite extension [36]. In diabetes,
there is a reduction in the concentration of these mediators [38],
leading to a disruption in epithelial integrity, and a flow-on effect
that causes further neuron loss, epithelial fragility, thinning of the
epithelium and reduced epithelial cell density [38]. This vicious
cycle leads to the formation of recurrent corneal erosions and
eventually results in diabetic neurotrophic keratopathy [30].
Neurotrophic keratopathy is a severe manifestation of ocular
surface dysfunction secondary to corneal denervation with an
estimated prevalence of 5/10,000 individuals [27]. Corneal nerve
damage leads to neurotrophic keratopathy both directly (through
loss of trophic support) and indirectly (through dry eye from
reduction of tear and mucin production) [102]. Neurotrophic ker-
atopathy is classified into three stages. Stage 1 is characterized by
rose bengal staining of the inferior palpebral conjunctiva, a
reduction in tear break-up time, and superficial corneal staining. In
longstanding cases, superficial vascularization, stromal scarring,
and epithelial hyperplasia can follow. In stage 2, epithelial break-
down occurs, with an epithelial defect surrounded by loose
epithelium that is hazy and edematous. Stage 2 presents with a
characteristic smooth and rolled appearance as the defect ages
without normal epithelial growth. Stage 3 is characterized by
stromal involvement, with potential for stromal melting and
perforation [36]. Persistent epithelial defects are often unrespon-
sive to conventional treatment, including patching, preservative-
49
free tears, or soft contact lens bandages [103]. More recent thera-
pies have been reviewed by Shih et al. [104] and offer promising
alternatives to protect against the loss of corneal sensitivity and the
subsequent development of corneal ulcers.
To understand the cascade of events that leads to the manifes-
tation of neurotrophic keratopathy, we review in the sections
below how diabetes affects the corneal epithelium and the tear
film. Given that dry eye disease predisposes the ocular surface to
greater surface cell desquamation [105], the co-existence of dia-
betes and dry eye disease places individuals at risk of thinning of
the corneal epithelium [38]. When combined with a poor tear film,
the risk of epithelial damage is increased, disrupting the normal
barrier function of the epithelium [106]. Ocular surgery and contact
lens wear provide an additional challenge to an already compro-
mised ocular surface. The following sections also review the impact
of these challenges on diabetic ocular surface integrity.
3.1. Corneal epithelial wound healing in diabetes
Epithelial cells are pleomorphic and irregularly arranged in
diabetic eyes, with reduced epithelial cell proliferation and inade-
quate adhesion of cells to an abnormal basement membrane [26].
These irregularities translate to a cornea that is prone to persistent
epithelial erosions with a weakened epithelial barrier [106,107].
When corneal neurons are damaged in diabetes, epithelial cells
swell, lose microvilli, and produce abnormal basal lamina [108,109].
Epithelial cells slough off into tears at an accelerated rate and
corneal staining appears within hours of denervation [36,110].
Delayed wound healing has been shown in the streptozotocin rat
model in diabetes [111]. A 5-mm wound in the center of the cornea
healed in 100% of the healthy rats within 48 h, while only one-third
of the diabetic rats healed within the same time [111]. Furthermore,
a diminished activation of endothelial growth factor receptor was
found in the rat diabetic corneas compared to healthy controls as
well as a different distribution in the expression of the tight junc-
tion proteins [111]. The rat diabetic corneas had sparser b-catenin in
the apical cornea and minimal zonula occludens-1 (ZO-1) in the
central cornea [111]. The authors concluded that there was delayed
but not absent formation of cell-cell junctions in diabetic corneas.
These epithelial changes in diabetic corneas are thought to be due
to the direct effects of hyperglycemia on the cellular metabolism,
the accumulation of AGE products on the basement membrane
[112] and oxidative stress [36,113,114].
50 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
3.2. Tear film biochemistry in diabetes
The tear film is critical to the protection, cellular regeneration,
and wound healing of the ocular surface. These mediators are
released by both the corneal nerves and the epithelial cells as
trophic support.
In diabetes, it is thought that there is a change in the distribution
of these mediators, leading to a disruption in epithelial integrity,
and a flow on effect that causes further neuron loss and ultimately
neurotrophic ulcers [30,115,116]. An understanding of these un-
derlying mechanisms may contribute to the understanding ocular
surface disease in diabetes, as well as to the development of
treatments and preventative measures. The following sections re-
view the biochemistry of the tear film and its influence on the
ocular surface in diabetes.
3.2.1. Neuropeptides and nerve growth factor
Neuropeptides, including substance P, insulinelike growth
factor 1 (IGF-1), calcitonin gene-related peptide (CGRP), neuro-
peptide Y (NPY) and vasoactive-intestinal peptide (VIP) [117], are
released from sensory nerve endings in the cornea, conjunctiva and
lacrimal gland [118]. Substance P and CGRP are the most abundant
neuropeptides in trigeminal ganglion neurons [119]. Neuropeptides
can modulate the infiltration and activation of immune cells and
interact with endothelial, immune and mast cells [120] to play an
inflammatory role by inducing blood vessel dilation, leukocyte
extravasation, immune cell activation, and synthesis and release of
several cytokines [117,121]. In the process, neuropeptides induce
neurogenic inflammation symptoms, which manifest as dry eye
disease [120]. In isolation and in combination with other constit-
uents, substance P plays a role in the maintenance and nutrition of
the cornea by promoting migration and proliferation of corneal
epithelial cells [122e126]. Neuropeptides VIP and NPY can be anti-
inflammatory, inhibiting T-cell proliferation and helper T-cell type
1 response while moderating the release of cytokines, chemokines
and NO [117].
Substance P levels in the central and peripheral nervous system
have been found to be significantly decreased in type 1 diabetes,
particularly in patients with diabetic peripheral neuropathy [127].
In the tear film, the concentration of substance P has been corre-
lated with the duration of diabetes and severity of diabetic reti-
nopathy [118], as well as corneal nerve fiber density [128,129]. In
support of this, eyes with corneal hypoesthesia have also been re-
ported to have decreased concentrations of substance P compared
to healthy control eyes [130]. Of note, adding non-steroidal anti-
inflammatory drops such as diclofenac can further reduce the
concentration of substance P in tears [131]. This has implications for
patients with corneal neuropathy requiring concurrent diclofenac
or other non-steroidal anti-inflammatory drops. Diclofenac can
inhibit the cyclooxygenase (COX)-2 pathway of arachidonic acid
metabolism, ultimately blocking prostaglandin release [132]. With
a reduction in substance P, corneal epithelial migration can
decrease and neurotrophic keratopathy may develop, resulting in
further corneal complications in the case of people with diabetes
[118]. In one published case study, a patient with diabetes was
treated with collagen cross-linking for keratoconus and then
treated with non-steroidal anti-inflammatory drops (nepafenac),
and presented 6 weeks post-operatively with corneal melting,
perforation, and iris prolapse [133].
In contrast to these reports on substance P, a study using the
diabetic mouse model found that while the concentrations of
neuropeptides CGRP and VIP were elevated in the irides, there was
no change in these levels in the corneas of the diabetic mice [134].
The authors argue that the absence of change in these corneal
neuropeptides suggests that trophic peptides in corneal sensory
nerves are not related to the keratopathy seen in diabetes [134].
This study, however, measured the neuropeptides only 3 months'
after disease onset.
Topical treatment with derivatives of substance P and IGF-1
have been shown to help heal epithelial defects and restore
ocular surface integrity in neurotrophic keratopathies, including in
diabetic keratopathy [23,102]. In addition, topical NGF can restore
corneal sensitivity and ocular surface integrity after corneal neu-
rotrophic keratitis [135,136]. NGF is a neurotrophin and is essential
for the survival and growth of sympathetic and sensory neurons
and for differentiation of neurons in the central nervous system.
NGF also restores the function of damaged neurons and stimulated
mucin production by goblet cells [137]. There are receptors for NGF
on corneal and conjunctival epithelial cells [136].
3.2.2. Insulin
Insulin was identified in the tear film, and insulin receptors were
found on the human ocular surface by Rocha and colleagues in
2002 [138]. More recently, evidence was found for extra pancreatic
insulin production by the lacrimal gland [139]. Whether the insulin
found in tears is a product solely of the lacrimal gland or whether it
is pancreatic in origin is unknown [139]. Insulin supports the
metabolism and growth of exocrine glands, such as the lacrimal
gland, and plays a role in maintaining corneal and lacrimal
epithelial cells, as well as meibomian gland cells [140], possibly
exerting a mitogenic stimulus in a similar manner to other growth
factors on the ocular surface [138,141,142]. Specific mechanisms of
insulin action on the ocular surface, and how this action relates to
glucose in tears is unclear [138]. In diabetes, a reduction in insulin-
derived neurotrophic support has been proposed to contribute to
the corneal denervation observed in diabetic keratopathy [143].
Several animal studies have reported that topical insulin promotes
re-epithelialization and improved corneal sensitivity in diabetic
rats with keratopathy [144e146]. Insulin-like growth factor binding
protein 3 (IGFBP3) has been found to be upregulated 3-fold in type
2 diabetes, leading to insulin-like growth factor receptor (IGF-1R)
activation which, in turn, may lead to disruption of cellular adhe-
sion, contributing to the pathogenesis of diabetic corneal compli-
cations [147].
3.2.3. Glucose
An increased concentration of glucose in the tears of patients
with diabetes has been reported, with the tear glucose level
correlating with the blood glucose level [148,149]. This correlation
has led researchers to develop ocular surface glucose sensing de-
vices for non-invasive monitoring of blood glucose levels [150]. It is
not currently known whether the increased glucose concentration
in tears plays a role in hyperglycemic damage to corneal nerves in
patients with diabetes.
3.2.4. AGE products
Elevated levels of glucose in tears of individuals with diabetes
[148,149] might result in the glycation of tear proteins, resulting in
AGE products. In a study of the basal tears of individuals with
diabetes and controls by Zhao et al., total AGE-modified proteins
were elevated in those with diabetes, both with and without reti-
nopathy [151]. The concentration of the tear film AGE modified
proteins correlated with the AGE modified hemoglobin and the
post-prandial blood glucose levels [151]. Western blots of the two-
dimensional gels indicated that many protein species were AGE
modified, more so in the diabetes samples. Zhao and colleagues
suggest that these results indicate that tear film AGE-modified
proteins may be used as a diagnostic biomarker for diabetes
[151]. It is not known, however, if glycation of tears impacts on tear
film function or corneal nerves.
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
3.2.5. Abundant proteins
Grus and colleagues collected the tears from 255 controls and
260 people with diabetes but without anterior eye complications
and analyzed the samples using sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE), hence investigating the
more abundant proteins [152]. The authors reported significantly
more ‘peaks’ in people with diabetes compared to controls, and
more so in those with longer disease duration, indicating more
proteins of higher concentration. This is in accordance with a study
by Stolwijk et al., who compared the tears of patients with and
without diabetic retinopathy [153]. While total protein content did
not differ compared to healthy controls, lysozyme was significantly
greater in those with retinopathy and pre-albumin was found to be
present in lower concentrations [153].
3.2.6. Inflammatory mediators
Tumor necrosis factor-a (TNF-a) is an inflammatory cytokine
associated with insulin resistance [154]. TNF-a is a potent mediator
of leukostasis and is involved in the apoptotic pathway of retinal
neurons and vascular endothelial cells in diabetic retinopathy,
contributing to the breakdown in the blood-retinal-barrier. Cos-
tagliola et al. measured the concentration of TNF-a in the tears of
people with and without type 2 diabetes using the Schirmer strip.
Participants with proliferative diabetic retinopathy had signifi-
cantly greater levels of TNF-a in their tears compared to those with
non-proliferative diabetic retinopathy, and healthy controls [155].
These authors suggest that TNF-a plays a role in the pathophysi-
ology of diabetic retinopathy and suggest that TNF-a may be used
as a means by which to screen for diabetic retinopathy [155].
Matrix metalloproteinases (MMPs) are a family of zinc-
dependent endopeptidases found in tissues and fluids throughout
the body. MMP-9 has an affinity for collagen VII, which is present in
the anchoring fibrils within the corneal basement membrane and is
vital for corneal regeneration and wound healing [156]. Regulation
of MMPs involves endogenous tissue inhibitors of metal-
loproteinase (TIMPs) [156]. The ratio of MMP-9 to TIMP-1 is
important in maintaining homeostasis [157]. Excess MMPs in tissue
or tears lead to ulceration and perforation in extreme cases [158].
Elevated MMP-9 levels in tissue have been shown to occur in
recurrent corneal erosions [159], dermatological ulcers and in
diabetic retinopathy [22,160].
3.3. Diabetes and dry eye disease
In a healthy eye, superficial damage to the ocular surface is
rapidly healed, with contributions to the healing process coming
from tissue and the tear film. In aqueous deficient dry eye, the
amount of tears is reduced which may increase the risk of corneal
erosion formation [36].
Approximately 50% of people with diabetes complain of dry eye
symptoms [161e163]. With longer disease duration, reduced
corneal sensation results in greater tolerance to dry eye disease,
reflecting the progression of the neuropathy [141]. Interestingly,
dry eye signs and symptoms have also been found to correlate with
the degree of peripheral neuropathy and severity of diabetic reti-
nopathy [26, 162,164].
There is evidence that tear film parameters are altered in pa-
tients with diabetes, with a reduction in tear break-up time and tear
secretion [165]. Factors thought to contribute to reduced tear pro-
duction in diabetes include microvascular damage to the lacrimal
gland from hyperglycemia, reduced binding sites for androgens and
estrogens on the lacrimal gland [166], reduced lacrimal innervation
due to autonomic neuropathy, lacrimal gland insufficiency [167],
and reduced reflex tearing due to impairment of corneal sensitivity
[162]. Reduction in tear film stability and tear break-up time may be
51
due to reduction in mucin production by goblet cells. Reductions in
corneal innervation result in reduced goblet cell function [30].
Dogru et al. and Inoue et al. have confirmed that diabetic patients
have decreased basal tear production and lacrimation, with the
cotton thread test and Schirmer test indicating lacrimal gland
involvement [30,168]. Dogru et al. further demonstrated that poor
diabetic control and peripheral neuropathy caused significantly
lower tear lacrimation, postulating the theory that peripheral
neuropathy affects the lacrimal gland [30]. A higher osmolarity has
also been reported in patients with diabetes compared to controls
[169,170], although the reverse was found in a study by Beckman
et al. [171] This is thought to be due to a reduction in lacrimal gland
secretion or increased tear evaporation.
Immortalized human meibomian gland epithelial cells can be
stimulated by insulin in a dose-dependent manner to proliferate
and accumulate lipid, while excess glucose causes progressive cell
loss and alterations to cell morphology [140]. These findings sup-
port the hypothesis that insulin deficiency and hyperglycemia are
toxic for the meibomian gland epithelial cells, placing individuals
with diabetes at risk of meibomian gland dysfunction and dry eye
disease [140].
3.4. Ocular surface integrity after ocular surgery in diabetes
3.4.1. Cataract surgery
A common cause of visual impairment in diabetes is the for-
mation of a cataract, with a three-to four-fold increase in preva-
lence of cataract in people with diabetes under the age of 65
[172,173]. As a result, phacoemulsification is commonly per-
formed in patients with diabetes. Besides the well-known risks of
post-operative progression of diabetic retinopathy and the devel-
opment of macular edema, as reviewed by Haddad et al. [174],
people with diabetes also have a greater risk of ocular surface
complications and have an increased 30-day post-procedure
readmission following cataract surgery compared to people
without diabetes [175]. Complications include superficial corneal
staining [22], corneal erosions [176], delayed wound healing [22]
and corneal edema [177], implying damage to the endothelium
[178,179]. Diabetes as a risk factor for endophthalmitis following
cataract surgery has been both supported [180,181] and refuted
[182,183].
Subbasal nerve density has also been found to be reduced
following cataract surgery [184]. Given the already reduced nerve
density in diabetes, surgery may further compromise the cornea,
particularly when combined with reduced tear secretion [165] and
an abnormal basement membrane [22]. Jiang et al. reported that
individuals with diabetes undergoing phacoemulsification are
more prone to developing dry eye disease than their non-diabetic
counterparts [185]. They found that 7 days post-operatively, 17%
of the patients with diabetes developed dry eye disease, as indi-
cated by tear film break-up time, Schirmer scores, and corneal
staining, compared to only 8% of the control patients [185]. While
the control group had a 0% incidence after 1 month post-
operatively, the group with diabetes took 3 months to return to
baseline levels [185]. These findings are supported by a retrospec-
tive clinical study of the prevalence of dry eye in dogs post-cataract
surgery, where the risk was greatest 2-weeks post-operatively in
the case of diabetes [186].
These findings indicate that the post-operative care of patients
with diabetes should be customized and care given during surgery
to minimize damage to the epithelium. Given the role of substance
P and IGF-1 in promoting epithelial migration [121], Chikamoto and
colleagues investigated the use of drops containing these sub-
stances on the prevention of corneal staining following cataract
surgery [187]. When compared to saline eye drops, the substance P
52 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
e IGF-1 combination resulted in significantly less staining 2 days
and 7 days following cataract surgery, with no difference found by
the two-week point [187]. As corneal staining represents the loss of
the epithelial barrier, quick recovery is important for the preven-
tion of infection. Autologous serum has been used with similar
effect [188]. Similarly, in a randomized, double-blind study
comparing the effect of oral aldose reductase inhibitor on post-
surgical corneal staining in diabetes, an improvement in corneal
staining, corneal sensitivity and symptoms was noted compared to
placebo, while measures of the tear film did not show a difference
[189]. Topical aldose reductase has similarly been shown to
improve corneal epithelial barrier function [190]. Given the role
that aldose reductase plays in the activation of the polyol pathway,
its inhibition may reduce the damage to the corneal nerves and
hence allow normal wound healing to take place. The findings of
these studies are promising and may help to reduce the ocular
surface complications seen after routine surgery.
3.4.2. Refractive surgery
Refractive surgery offers a model for assessing the impact of
interfering with the corneal subbasal nerve plexus, via incision or
obliteration. In laser in situ keratomileusis (LASIK), a hinged flap of
corneal epithelium and stroma is created with a keratome. Once the
flap is folded back, a laser is applied to remove precise amounts of
corneal stromal tissue and the flap is then returned to its original
position without sutures. In procedures such as epithelial laser in
situ keratomileusis (Epi-LASIK), laser epithelial keratomileusis
(LASEK), and photorefractive keratectomy (PRK), the laser is used to
refigure the curvature of the corneal surface of the cornea, hence
requiring removal of the epithelium.
In the year 2000, the FDA declared diabetes to be a relative
contraindication to LASIK [191]. Patients with diabetes who un-
dergo LASIK have been found to be at a significantly greater risk of
post-operative corneal complications when compared to a group of
people without diabetes [192]. Fraunfelder and Rich first reported a
complication rate of 47% in patients with diabetes compared to 6.9%
in those without diabetes, with the most common complications
being erosions and persistent epithelial defects [192]. This study
also reported poorer visual outcomes in the group with diabetes
[192]. The complications reported by Fraunfelder and Rich appear
to be related to the poor wound healing discussed above and may
be indicative of coexisting corneal neuropathy. Fraunfelder and
Rich, however, did not report on the severity of diabetes in their
study. In a study by Halkiadakis et al. of 24 patients with well-
controlled diabetes, 3 developed an epithelial defect post-
operatively [193], indicating good short-term safety of LASIK in
diabetes. The age group in this study, however, was 10 years
younger than that reported by Fraunfelder and Rich. In a study by
Cobo-Soriano and et al., a lower proportion of complications was
reported, with visual outcomes being no different than in the
control group [194]. This study had a younger average age group
and a lower range of myopia than that reported by Fraunfelder and
Rich, which may explain the reported differences. A review by
Spadea and Paroli recommended that LASIK be considered in pa-
tients with well-controlled diabetes and with no ocular manifes-
tations of diabetes [195]. Patients with diabetes should be informed
of this risk and be monitored closely post-operatively.
3.4.3. Other forms of ocular surgery
Other forms of ocular surgery have also been associated with
complications when diabetes is a comorbidity. Trabeculectomy has
been associated with corneal epithelial defects, dellen, and fila-
mentary keratitis [196]. The greatest risk factors for these compli-
cations post-trabeculectomy are pre-existing corneal staining and a
history of diabetes [196]. These findings are in agreement with
reports that diabetes is a risk factor for corneal complications
following vitrectomy [197,198] and laser photocoagulation. Dogru
and colleagues reported on the corneal response to argon green
focal/grid laser photocoagulation using an applanation contact lens
and coupling fluid in a group of patients with diabetes and a group
without diabetes [199]. The group with diabetes had lower corneal
sensitivity, lower tear film break-up time, and a greater extent of
corneal staining following the procedure, a finding supported by
Ozdemir et al. [176] Dogru et al. attribute this to the use of the
coupling fluid in conjunction with the co-existence of dry eye dis-
ease [199]. The potential of topical anesthetic to prevent cell
migration [200] may further add to this result.
These findings indicate that regardless of the form of ocular
surgery, greater vigilance for corneal complications may be
required post-operatively when a patient presents with a history of
diabetes.
3.5. Complications with contact lens use in diabetes
Consensus is lacking among practitioners regarding the safety of
contact lenses for patients with diabetes, with some considering
diabetes as a contraindication to lens wear [201]. When the actual
prescribing habits were surveyed in the United Kingdom, 2% of
patients fitted with contact lenses had diabetes [201]. Of the
practitioners surveyed, 55% felt that contact lens wear was safe for
patients with diabetes, while 42% responded ‘maybe’ to the ques-
tion of safety and 3% felt that contact lens wear was not safe in
these patients [201]. The majority of practitioners felt that daily
wear was the appropriate modality with more regular aftercare
visits recommended for this patient group, compared to those
without diabetes [201].
The complications of contact lens use in patients with diabetes
have been reviewed by O'Donnell and Efron [26,202]. Studies on
diabetic contact lens complications tend to be in patients with
advanced disease and when contact lenses are worn in the
extended wear modality [203,204]. To address the uncertainty
among practitioners, O'Donnell et al. conducted a prospective study
in which people with and without diabetes were fitted with contact
lenses for a 12-month period [205]. Over the observation period, no
significant differences were found between the groups in terms of
bulbar redness, corneal staining, corneal thickness, or corneal
sensitivity [205]. O'Donnell et al. reported no compromise to the
corneal integrity with contact lens wear by patients with diabetes
[205], findings supported by March et al. [206] To establish the
effect of contact lens wear on corneal hydration in people with
diabetes, O'Donnell and Efron fitted 23 people with and 23 people
without diabetes with thick, low water content soft contact lenses
and measured corneal thickness before and after contact lens wear
[207]. They again found no difference in corneal thickness between
groups and no difference was found in the rate of recovery, leading
these authors to conclude that the presence of diabetes should not
prevent clinicians from fitting their patients with diabetes with
contact lenses [207]. Similarly, a 2011 paper found no difference in
the corneal thickness of people with diabetes, regardless of contact
lens wear status, although endothelial density was significantly less
in the group wearing contact lenses [208].
In the future, contact lenses may be increasingly used to detect
glucose levels in the tears, hence being useful as a means of diabetic
metabolic control [150,209e212]. The available evidence indicates
that with current materials, daily wear of contact lenses is safe in
people with diabetes. However, practitioners should be aware of
the impact that diabetes has on the ocular surface in general and
advise patients accordingly. Careful examination of the cornea and
tear film is needed during fitting and follow-up to identify prob-
lems early.
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
4. The ocular surface microbiome in diabetes
The combination of corneal neuropathy, poor wound healing,
and dry eye places people with diabetes at increased risk of
persistent epithelial defects [213], recurrent erosions [22,214,215],
neurotrophic keratopathy [216], and possibly at higher risk of mi-
crobial keratitis [141,217e219].
Recent studies have suggested a role for the human gut micro-
biome in the development of diabetes [220], with declines in Fir-
micutes and an increases in Bacteroidetes [221,222].
A study that investigated microbial diversity of the conjunctiva
in a rat model of streptozotocin-induced type 1 diabetes [223]
found that onset of diabetes was associated with reduced
complexity of the conjunctival microbiota, the disappearance of
species of staphylococci, Aerococcus viridans and Klebsiella pneu-
monia, but the appearance of enterococci, Kocuria kristinae,
Enterobacter aerogenes, and Proteus vulgaris. [223] A study using a
streptozotocin mouse diabetes model showed that duration of
diabetes increased the risk of developing endogenous bacterial
endophthalmitis after injection of Klebsiella pneumoniae into the
mouse tail vein [224]. The study also showed that longer duration
of diabetes in these mice increased likelihood of developing
endophthalmitis [224].
Most, but not all [225], studies have shown significant increases
in microbes on the conjunctiva of diabetic patients. Habib et al.
[226] found that there was an increase in bacterial diversity on the
conjunctiva with type 2, but not type 1, diabetes, and patients with
type 2 diabetes were significantly more commonly colonized by
Staphylococcus aureus [226]. Conjunctival swabs from people with
diabetes contain a greater frequency of coagulase negative staph-
ylococci [227], Staphylococcus aureus, enterococci, certain strepto-
cocci, and Gram-negative bacteria including Klebsiella sp [228],
Enterobacter sp. and Escherichia coli [229,230]. Conjunctivas are
more frequently colonized by microbes of patients with diabetic
retinopathy [227,231]. The increased bacterial load on the con-
junctiva may be related to severity of disease and perhaps to con-
trol of glucose levels over time.
5. Summary
Diabetic corneal neuropathy affects epithelial integrity, tear film
function, corneal sensitivity, and the healing response by reducing
the trophic support that is critical for ocular surface homeostasis.
The severity of diabetic keratopathy can vary from dry eye symp-
toms to visual impairment from corneal ulceration [26]. This review
highlights the need for clinical vigilance in detecting ocular surface
disease when examining patients with diabetes.
Understanding the mechanisms resulting in the reduced corneal
nerve density and the mechanisms contributing to the suscepti-
bility to corneal erosion and infection in diabetes is critical, not only
for prevention but also for insight into the systemic disease. The
cornea may be a simplistic model for understanding how diabetes
affects other organs. There is a need to better characterize the
relationship between the neural, biochemical, and structural
changes that occur in early diabetes. By understanding these re-
lationships, we can better prevent and predict the development of
diabetes-related complications. By modulating these changes, we
may be able, in the future, to slow down the progress of diabetic
ocular surface disease and other diabetes-related complications.
Funding
No financial support was received for this work.
The authors have no proprietary or commercial interests in any
concept or product discussed in this article.
53
Acknowledgements
The authors wish to acknowledge Morag Stewart, Timothy Chan,
Jonathan Tolentino, Carmen Duong for their assistance with this
manuscript.
References
[1] Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes
mellitus and its complications. Part 1: diagnosis and classification of diabetes
mellitus provisional report of a WHO consultation. Diabet Med 1998;15:
539e53.
[2] Charnogursky G, Lee H, Lopez N. Diabetic neuropathy. Handb Clin Neurol
2014;120:773e85.
[3] International Diabetes Federation. IDF diabetes atlas. seventh ed. 2015.
http://www.diabetesatlas.org/.
[4] Seidell JC. Obesity, insulin resistance and diabetesea worldwide epidemic. Br
J Nutr 2000;83(Suppl 1):S5e8.
[5] WHO. Expert committee on diabetes mellitus. 1980.
[6] Tuomi T, Santoro N, Caprio S, Cai M, Weng J, Groop L. The many faces of
diabetes: a disease with increasing heterogeneity. Lancet 2014;383:
1084e94.
[7] Ginter E, Simko V. Type 2 diabetes mellitus, pandemic in 21st century. Adv
Exp Med Biol 2012;771:42e50.
[8] World Health Organization, International Diabetes Federation. Definition and
diagnosis of diabetes mellitus and intermediate hyperglycemia: report of a
WHO/IDF consultation. Geneva, Switzerland. 2006.
[9] Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR. Anti-
bodies to glutamic acid decarboxylase reveal latent autoimmune diabetes
mellitus in adults with a non-insulin-dependent onset of disease. Diabetes
1993;42:359e62.
[10] Kobayashi T, Tamemoto K, Nakanishi K, Kato N, Okubo M, Kajio H, et al.
Immunogenetic and clinical characterization of slowly progressive IDDM.
Diabetes Care 1993;16:780e8.
[11] Colagiuri S. Epidemiology of prediabetes. Med Clin North Am 2011;95:
299e307.
[12] Atkinson MA. The pathogenesis and natural history of type 1 diabetes. Cold
Spring Harb Perspect Med 2012;2.
[13] Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical
interventions in type 1 diabetes. Nature 2010;464:1293e300.
[14] Buysschaert M, Bergman M. Definition of prediabetes. Med Clin North Am
2011;95:289e97.
[15] Cade WT. Diabetes-related microvascular and macrovascular diseases in the
physical therapy setting. Phys Ther 2008;88:1322e35.
[16] Alva ML, Gray A, Mihaylova B, Leal J, Holman RR. The impact of diabetes-
related complications on healthcare costs: new results from the UKPDS
(UKPDS 84). Diabet Med 2015;32:459e66.
[17] Nowotny K, Jung T, Hohn A, Weber D, Grune T. Advanced glycation end
products and oxidative stress in type 2 diabetes mellitus. Biomolecules
2015;5:194e222.
[18] Nunan D, Mahtani KR, Roberts N, Heneghan C. Physical activity for the
prevention and treatment of major chronic disease: an overview of sys-
tematic reviews. Syst Rev 2013;2:56.
[19] Madden KM. Evidence for the benefit of exercise therapy in patients with
type 2 diabetes. Diabetes Metab Syndr Obes 2013;6:233e9.
[20] Brownlee M. Biochemistry and molecular cell biology of diabetic complica-
tions. Nature 2001;414:813e20.
[21] Sheetz MJ, King GL. Molecular understanding of hyperglycemia's adverse
effects for diabetic complications. JAMA 2002;288:2579e88.
[22] Schultz RO, Van Horn DL, Peters MA, Klewin KM, Schutten WH. Diabetic
keratopathy. Trans Am Ophthalmol Soc 1981;79:180e99.
[23] Abdelkader H, Patel DV, McGhee C, Alany RG. New therapeutic approaches in
the treatment of diabetic keratopathy: a review. Clin Exp Ophthalmol
2011;39:259e70.
[24] Didenko T, Smoliakova G, Sorokin E, Egorov V. Clinical and pathogenetic
features of neurotrophic corneal disorders in diabetes. Vestn Oftalmol
1999;115:7e11.
[25] Vieira-Potter VJ, Karamichos D, Lee DJ. Ocular complications of diabetes and
therapeutic approaches. Biomed Res Int 2016;2016.
[26] O'Donnell C, Efron N. Diabetes and contact lens wear. Clin Exp Optom
2012;95:328e37.
[27] Sacchetti M, Lambiase A. Diagnosis and management of neurotrophic kera-
titis. Clin Ophthalmol 2014;8:571e9.
[28] Pritchard N, Edwards K, Russell AW, Perkins BA, Malik RA, Efron N. Corneal
confocal microscopy predicts 4-year incident peripheral neuropathy in type
1 diabetes. Diabetes Care 2015;38:671e5.
[29] Cousen P, Cackett P, Bennett H, Swa K, Dhillon B. Tear production and corneal
sensitivity in diabetes. J Diabetes Complicat 2007;21:371e3.
[30] Dogru M, Katakami C, Inoue M. Tear function and ocular surface changes in
noninsulin-dependent diabetes mellitus. Ophthalmology 2001;108:586e92.
[31] Friend J, Thoft RA. The diabetic cornea. Int Ophthalmol Clin 1984;24:111e23.
[32] Yoon KC, Im SK, Seo MS. Changes of tear film and ocular surface in diabetes
54 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
mellitus. Korean J Ophthalmol 2004;18:168e74.
[33] Pritchard N, Edwards K, Shahidi AM, Sampson GP, Russell AW, Malik RA,
et al. Corneal markers of diabetic neuropathy. Ocul Surf 2011;9:17e28.
[34] Schein OD, Glynn RJ, Poggio EC, Seddon JM, Kenyon KR. The relative risk of
ulcerative keratitis among users of daily-wear and extended-wear soft
contact lenses. A case-control study. Microbial Keratitis Study Group. N Engl J
Med 1989;321:773e8.
[35] Patel DV, McGhee CN. Mapping of the normal human corneal sub-basal
nerve plexus by in vivo laser scanning confocal microscopy. Investig Oph-
thalmol Vis Sci 2005;46:4485e8.
[36] Muller LJ, Marfurt CF, Kruse F, Tervo TM. Corneal nerves: structure, contents
and function. Exp Eye Res 2003;76:521e42.
[37] Oliveira-Soto L, Efron N. Morphology of corneal nerves using confocal mi-
croscopy. Cornea 2001;20:374e84.
[38] Cai D, Zhu M, Petroll WM, Koppaka V, Robertson DM. The impact of type 1
diabetes mellitus on corneal epithelial nerve morphology and the corneal
epithelium. Am J Pathol 2014;184:2662e70.
[39] Misra SL, Craig JP, Patel DV, McGhee CN, Pradhan M, Ellyett K, et al. In vivo
confocal microscopy of corneal nerves: an ocular biomarker for peripheral
and cardiac autonomic neuropathy in type 1 diabetes mellitus. Investig
Ophthalmol Vis Sci 2015;56:5060e5.
[40] Efron N. The Glenn A. Fry award lecture 2010: ophthalmic markers of dia-
betic neuropathy. Optom Vis Sci 2011;88:661e83.
[41] Cameron NE, Eaton SE, Cotter MA, Tesfaye S. Vascular factors and metabolic
interactions in the pathogenesis of diabetic neuropathy. Diabetologia
2001;44:1973e88.
[42] Feldman EL, Stevens MJ, Greene DA. Pathogenesis of diabetic neuropathy.
Clin Neurosci 1997;4:365e70.
[43] Ahsan H. Diabetic retinopathy-biomolecules and multiple pathophysiology.
Diabetes Metab Syndr 2015;9:51e4.
[44] Babizhayev MA, Strokov IA, Nosikov VV, Savel'yeva EL, Sitnikov VF,
Yegorov YE, et al. The role of oxidative stress in diabetic neuropathy: gen-
eration of free radical species in the glycation reaction and gene poly-
morphisms encoding antioxidant enzymes to genetic susceptibility to
diabetic neuropathy in population of type i diabetic patients. Cell Biochem
Biophys 2015;71:1425e43.
[45] Yagihashi S, Mizukami H, Sugimoto K. Mechanism of diabetic neuropathy:
where are we now and where to go? J Diabetes Investig 2011;2:18e32.
[46] Betteridge DJ. What is oxidative stress? Metabolism 2000;49:3e8.
[47] Duby JJ, Campbell RK, Setter SM, White JR, Rasmussen KA. Diabetic neu-
ropathy: an intensive review. Am J Health Syst Pharm 2004;61:160e73. quiz
75e76.
[48] Vincent AM, Edwards JL, McLean LL, Hong Y, Cerri F, Lopez I, et al. Mito-
chondrial biogenesis and fission in axons in cell culture and animal models of
diabetic neuropathy. Acta Neuropathol 2010;120:477e89.
[49] Vincent AM, McLean LL, Backus C, Feldman EL. Short-term hyperglycemia
produces oxidative damage and apoptosis in neurons. FASEB J 2005;19:
638e40.
[50] Hamid HS, Mervak CM, Munch AE, Robell NJ, Hayes JM, Porzio MT, et al.
Hyperglycemia- and neuropathy-induced changes in mitochondria within
sensory nerves. Ann Clin Transl Neurol 2014;1:799e812.
[51] Zherebitskaya E, Akude E, Smith DR, Fernyhough P. Development of selective
axonopathy in adult sensory neurons isolated from diabetic rats: role of
glucose-induced oxidative stress. Diabetes 2009;58:1356e64.
[52] Tomlinson DR, Fernyhough P, Diemel LT. Role of neurotrophins in diabetic
neuropathy and treatment with nerve growth factors. Diabetes
1997;46(Suppl 2):S43e9.
[53] Kim J, Kim CS, Sohn E, Jeong IH, Kim H, Kim JS. Involvement of advanced
glycation end products, oxidative stress and nuclear factor-kappaB in the
development of diabetic keratopathy. Graefes Arch Clin Exp Ophthalmol
2011;249:529e36.
[54] Aubert CE, Michel PL, Gillery P, Jaisson S, Fonfrede M, Morel F, et al. Asso-
ciation of peripheral neuropathy with circulating advanced glycation end
products, soluble receptor for advanced glycation end products and other
risk factors in patients with type 2 diabetes. Diabetes Metab Res Rev
2014;30:679e85.
[55] Ryle C, Donaghy M. Non-enzymatic glycation of peripheral nerve proteins in
human diabetics. J Neurol Sci 1995;129:62e8.
[56] Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation and the
pathogenesis of diabetic complications. Ann Intern Med 1984;101:527e37.
[57] Sugimoto K, Nishizawa Y, Horiuchi S, Yagihashi S. Localization in human
diabetic peripheral nerve of N ε -carboxymethyllysine-protein adducts, an
advanced glycation endproduct. Diabetologia 1997;40:1380e7.
[58] Sugimoto K, Yasujima M, Yagihashi S. Role of advanced glycation end
products in diabetic neuropathy. Curr Pharm Des 2008;14:953e61.
[59] McDermott AM, Xiao TL, Kern TS, Murphy CJ. Non-enzymatic glycation in
corneas from normal and diabetic donors and its effects on epithelial cell
attachment in vitro. Optom St Louis Mo 2003;74:443e52.
[60] Stavniichuk R, Shevalye H, Hirooka H, Nadler JL, Obrosova IG. Interplay of
sorbitol pathway of glucose metabolism, 12/15-lipoxygenase, and mitogen-
activated protein kinases in the pathogenesis of diabetic peripheral neu-
ropathy. Biochem Pharmacol 2012;83:932e40.
[61] Chung SS, Ho EC, Lam KS, Chung SK. Contribution of polyol pathway to
diabetes-induced oxidative stress. J Am Soc Nephrol 2003;14:S233e6.
[62] Oates PJ. Aldose reductase, still a compelling target for diabetic neuropathy.
Curr Drug Targets 2008;9:14e36.
[63] Greene DA, Sima AA, Stevens MJ, Feldman EL, Lattimer SA. Complications:
neuropathy, pathogenetic considerations. Diabetes Care 1992;15:1902e25.
[64] Huang SP, Palla S, Ruzycki P, Varma RA, Harter T, Reddy GB, et al. Aldo-keto
reductases in the eye. J Ophthalmol 2010;2010:521204.
[65] Hempel A, Maasch C, Heintze U, Lindschau C, Dietz R, Luft FC, et al. High
glucose concentrations increase endothelial cell permeability via activation
of protein kinase C alpha. Circ Res 1997;81:363e71.
[66] Geraldes P, King GL. Activation of protein kinase C isoforms and its impact on
diabetic complications. Circ Res 2010;106:1319e31.
[67] Obrosova IG. Diabetes and the peripheral nerve. Biochim Biophys Acta
2009;1792:931e40.
[68] Farmer KL, Li C, Dobrowsky RT. Diabetic peripheral neuropathy: should a
chaperone accompany our therapeutic approach? Pharmacol Rev 2012;64:
880e900.
[69] Lehning EJ, LoPachin RM, Mathew J, Eichberg J. Changes in Na-K ATPase and
protein kinase C activities in peripheral nerve of acrylamide-treated rats.
J Toxicol Environ Health 1994;42:331e42.
[70] Said G. Diabetic neuropathy-a review. Nat Clin Pract Neurol 2007;3:331e40.
[71] Thomas PK. Classification, differential diagnosis, and staging of diabetic pe-
ripheral neuropathy. Diabetes 1997;46(Suppl 2):S54e7.
[72] Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global burden
of diabetic foot disease. Lancet 2005;366:1719e24.
[73] Vinik AI, Mehrabyan A. Diabetic neuropathies. Med Clin North Am 2004;88:
947e99.
[74] Vinik A, Ullal J, Parson HK, Casellini CM. Diabetic neuropathies: clinical
manifestations and current treatment options. Nat Clin Pract Endocrinol
Metab 2006;2:269e81.
[75] Smith HS, Argoff CE. Pharmacological treatment of diabetic neuropathic pain.
Drugs 2011;71:557e89.
[76] Davies M, Brophy S, Williams R, Taylor A. The prevalence, severity, and
impact of painful diabetic peripheral neuropathy in type 2 diabetes. Diabetes
Care 2006;29:1518e22.
[77] Pirart J. Diabetes mellitus and its degenerative complications: a prospective
study of 4,400 patients observed between 1947 and 1973. Diabete Metab
1977;3:97e107.
[78] Tavakoli M, Mitu-Pretorian M, Petropoulos IN, Fadavi H, Asghar O, Alam U,
et al. Corneal confocal microscopy detects early nerve regeneration in dia-
betic neuropathy after simultaneous pancreas and kidney transplantation.
Diabetes 2013;62:254e60.
[79] Albers JW, Brown MB, Sima AA, Greene DA. Nerve conduction measures in
mild diabetic neuropathy in the Early Diabetes Intervention Trial: the effects
of age, sex, type of diabetes, disease duration, and anthropometric factors.
Tolrestat Study Group for the Early Diabetes Intervention Trial. Neurology
1996;46:85e91.
[80] Sveen KA, Karime B, Jorum E, Mellgren SI, Fagerland MW, Monnier VM, et al.
Small- and large-fiber neuropathy after 40 years of type 1 diabetes: associ-
ations with glycemic control and advanced protein glycation: the Oslo Study.
Diabetes Care 2013;36:3712e7.
[81] Malik RA, Veves A, Tesfaye S, Smith G, Cameron N, Zochodne D, et al. Small
fibre neuropathy: role in the diagnosis of diabetic sensorimotor poly-
neuropathy. Diabetes Metab Res Rev 2011;27:678e84.
[82] Quattrini C, Tavakoli M, Jeziorska M, Kallinikos P, Tesfaye S, Finnigan J, et al.
Surrogate markers of small fiber damage in human diabetic neuropathy.
Diabetes 2007;56:2148e54.
[83] Edwards K, Pritchard N, Vagenas D, Russell A, Malik RA, Efron N. Utility of
corneal confocal microscopy for assessing mild diabetic neuropathy: baseline
findings of the LANDMark study. Clin Exp Optom 2012;95:348e54.
[84] Perry JR, Bril V. Complications of sural nerve biopsy in diabetic versus non-
diabetic patients. Can J Neurol Sci 1994;21:34e7.
[85] Tavakoli M, Quattrini C, Abbott C, Kallinikos P, Marshall A, Finnigan J, et al.
Corneal confocal microscopy: a novel noninvasive test to diagnose and
stratify the severity of human diabetic neuropathy. Diabetes Care 2010;33:
1792e7.
[86] Dyck PJ, Norell JE, Tritschler H, Schuette K, Samigullin R, Ziegler D, et al.
Challenges in design of multicenter trials: end points assessed longitudinally
for change and monotonicity. Diabetes Care 2007;30:2619e25.
[87] Dehghani C, Pritchard N, Edwards K, Vagenas D, Russell AW, Malik RA, et al.
Natural history of corneal nerve morphology in mild neuropathy associated
with type 1 diabetes: development of a potential measure of diabetic pe-
ripheral neuropathy. Investig Ophthalmol Vis Sci 2014;55:7982e90.
[88] Mehra S, Tavakoli M, Kallinikos PA, Efron N, Boulton AJ, Augustine T, et al.
Corneal confocal microscopy detects early nerve regeneration after pancreas
transplantation in patients with type 1 diabetes. Diabetes Care 2007;30:
2608e12.
[89] Ziegler D, Papanas N, Zhivov A, Allgeier S, Winter K, Ziegler I, et al. Early
detection of nerve fiber loss by corneal confocal microscopy and skin biopsy
in recently diagnosed type 2 diabetes. Diabetes 2014;63:2454e63.
[90] Dehghani C, Pritchard N, Edwards K, Vagenas D, Russell AW, Malik RA, et al.
Morphometric stability of the corneal subbasal nerve plexus in healthy in-
dividuals: a 3-year longitudinal study using corneal confocal microscopy.
Investig Ophthalmol Vis Sci 2014;55:3195e9.
[91] Hu L, Chen J, Zhang L, Sun X, Huang J, Xie W, et al. Effects of long-term soft
contact lenses on tear menisci and corneal nerve density. Eye Contact Lens
2016;42:196e201.
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
[92] Niederer RL, Perumal D, Sherwin T, McGhee CN. Age-related differences in
the normal human cornea: a laser scanning in vivo confocal microscopy
study. Br J Ophthalmol 2007;91:1165e9.
[93] Parissi M, Karanis G, Randjelovic S, Germundsson J, Poletti E, Ruggeri A, et al.
Standardized baseline human corneal subbasal nerve density for clinical
investigations with laser-scanning in vivo confocal microscopy. Investig
Ophthalmol Vis Sci 2013;54:7091e102.
[94] Cruzat A, Qazi Y, Hamrah P. In vivo confocal microscopy of corneal nerves in
health and disease. Ocul Surf 2017;15:15e47.
[95] Petropoulos IN, Alam U, Fadavi H, Asghar O, Green P, Ponirakis G, et al.
Corneal nerve loss detected with corneal confocal microscopy is symmetrical
and related to the severity of diabetic polyneuropathy. Diabetes Care
2013;36:3646e51.
[96] Pritchard N, Edwards K, Dehghani C, Fadavi H, Jeziorska M, Marshall A, et al.
Longitudinal assessment of neuropathy in type 1 diabetes using novel
ophthalmic markers (LANDMark): study design and baseline characteristics.
Diabetes Res Clin Pract 2014;104:248e56.
[97] Jiang MS, Yuan Y, Gu ZX, Zhuang SL. Corneal confocal microscopy for
assessment of diabetic peripheral neuropathy: a meta-analysis. Br J Oph-
thalmol 2016;100:9e14.
[98] Leppin K, Behrendt AK, Reichard M, Stachs O, Guthoff RF, Baltrusch S, et al.
Diabetes mellitus leads to accumulation of dendritic cells and nerve fiber
damage of the subbasal nerve plexus in the cornea. Investig Ophthalmol Vis
Sci 2014;55:3603e15.
[99] Edwards K, Pritchard N, Poole C, Dehghani C, Al Rashah K, Russell A, et al.
Development of a novel technique to measure corneal nerve migration rate.
Cornea 2016;35:700e5.
[100] Tavakoli M, Kallinikos P, Iqbal A, Herbert A, Fadavi H, Efron N, et al. Corneal
confocal microscopy detects improvement in corneal nerve morphology
with an improvement in risk factors for diabetic neuropathy. Diabet Med
2011;28:1261e7.
[101] Smith AG, Russell J, Feldman EL, Goldstein J, Peltier A, Smith S, et al. Lifestyle
intervention for pre-diabetic neuropathy. Diabetes Care 2006;29:1294e9.
[102] Shaheen BS, Bakir M, Jain S. Corneal nerves in health and disease. Surv
Ophthalmol 2014;59:263e85.
[103] Hsu HY, Modi D. Etiologies, quantitative hypoesthesia, and clinical outcomes
of neurotrophic keratopathy. Eye Contact Lens 2015;41:314e7.
[104] Shih KC, Lam KSL, Tong L. A systematic review on the impact of diabetes
mellitus on the ocular surface. Nutr Diabetes 2017;7. e251.
[105] Yeh S, Song XJ, Farley W, Li DQ, Stern ME, Pflugfelder SC. Apoptosis of ocular
surface cells in experimentally induced dry eye. Investig Ophthalmol Vis Sci
2003;44:124e9.
[106] Gekka M, Miyata K, Nagai Y, Nemoto S, Sameshima T, Tanabe T, et al. Corneal
epithelial barrier function in diabetic patients. Cornea 2004;23:35e7.
[107] Chang SW, Hsu HC, Hu FR, Chen MS. Corneal autofluorescence and epithelial
barrier function in diabetic patients. Ophthalmic Res 1995;27:74e9.
[108] Alper M. The anesthetic eye: an investigation of changes in the anterior
ocular segment of the monkey caused by interrupting the trigeminal nerve
at various levels along its course. Trans Am Ophthalmol Soc 1975;73:313.
[109] Mackie I. Role of the corneal nerves in destructive disease of the cornea.
Trans Ophthalmol Soc U. K 1978;98:343.
[110] Beuerman RW, Schimmelpfennig B. Sensory denervation of the rabbit cornea
affects epithelial properties. Exp Neurol 1980;69:196e201.
[111] Yin J, Huang J, Chen C, Gao N, Wang F, Yu FS. Corneal complications in
streptozocin-induced type I diabetic rats. Investig Ophthalmol Vis Sci
2011;52:6589e96.
[112] Kaji Y, Usui T, Oshika T, Matsubara M, Yamashita H, Araie M, et al. Advanced
glycation end products in diabetic corneas. Investig Ophthalmol Vis Sci
2000;41:362e8.
[113] Xu KP, Li Y, Ljubimov AV, Yu FS. High glucose suppresses epidermal growth
factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and
attenuates corneal epithelial wound healing. Diabetes 2009;58:1077e85.
[114] Kim SJ, Toma HS. Ophthalmic antibiotics and antimicrobial resistance a
randomized, controlled study of patients undergoing intravitreal injections.
Ophthalmology 2011;118:1358e63.
[115] Nakamura M, Kawahara M, Nakata K, Nishida T. Restoration of corneal
epithelial barrier function and wound healing by substance P and IGF-1 in
rats with capsaicin-induced neurotrophic keratopathy. Investig Ophthalmol
Vis Sci 2003;44:2937e40.
[116] Ljubimov AV. Diabetic complications in the cornea. Vis Res 2017 Apr 28.
https://doi.org/10.1016/j.visres.2017.03.002. pii: S0042e6989(17)30047-
0 [Epub ahead of print].
[117] Lambiase A, Micera A, Sacchetti M, Cortes M, Mantelli F, Bonini S. Alterations
of tear neuromediators in dry eye disease. Arch Ophthalmol 2011;129:
981e6.
[118] Yamada M, Ogata M, Kawai M, Mashima Y, Nishida T. Substance P in human
tears. Cornea 2003;22:S48e54.
[119] Felipe CD, Gonzalez GG, Gallar J, Belmonte C. Quantification and immuno-
cytochemical characteristics of trigeminal ganglion neurons projecting to the
cornea: effect of corneal wounding. Eur J Pain 1999;3:31e9.
[120] Stern ME, Pflugfelder SC. Inflammation in dry eye. Ocul Surf 2004;2:124e30.
[121] Nakamura M, Chikama T, Nishida T. Synergistic effect with Phe-Gly-Leu-
Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1
on epithelial wound healing of rabbit cornea. Br J Pharmacol 1999;127:
489e97.
55
[122] Nishida T, Nakamura M, Ofuji K, Reid TW, Mannis MJ, Murphy CJ. Synergistic
effects of substance P with insulin-like growth factor-1 on epithelial
migration of the cornea. J Cell Physiol 1996;169:159e66.
[123] Nakamura M, Ofuji K, Chikama T, Nishida T. Combined effects of substance P
and insulin-like growth factor-1 on corneal epithelial wound closure of
rabbit in vivo. Curr Eye Res 1997;16:275e8.
[124] Nakamura M, Chikama T, Nishida T. Up-regulation of integrin alpha 5
expression by combination of substance P and insulin-like growth factor-1 in
rabbit corneal epithelial cells. Biochem Biophys Res Commun 1998;246:
777e82.
[125] Reid TW, Murphy CJ, Iwahashi CK, Foster BA, Mannis MJ. Stimulation of
epithelial cell growth by the neuropeptide substance P. J Cell Biochem
1993;52:476e85.
[126] Garcia-Hirschfeld J, Lopez-Briones LG, Belmonte C. Neurotrophic influences
on corneal epithelial cells. Exp Eye Res 1994;59:597e605.
[127] Kunt T, Forst T, Schmidt S, Pfutzner A, Schneider S, Harzer O, et al. Serum
levels of substance P are decreased in patients with type 1 diabetes. Exp Clin
Endocrinol Diabetes 2000;108:164e7.
[128] Stapleton F, Chao C, Jalbert I, Mandathara PS, Kolanu S, Willcox MDP, et al.
Relationships between central corneal nerve parameters and tear neuro-
peptides. Investig Ophthalmol Vis Sci 2016;57.
[129] Markoulli M, Lum E, You JJ, Duong C, Tolentino J, Kim J. Corneal nerve
morphology and tear film substance p in diabetes. Investig Ophthalmol Vis
Sci 2016;57:6153.
[130] Yamada M, Ogata M, Kawai M, Mashima Y. Decreased substance P concen-
trations in tears from patients with corneal hypesthesia. Am J Ophthalmol
2000;129:671e2.
[131] Yamada M, Ogata M, Kawai M, Mochizuki H, Mashima Y. Topical diclofenac
sodium decreases the substance P content of tears. Arch Ophthalmol
2002;120:51e4.
[132] Cashman JN. The mechanisms of action of NSAIDs in analgesia. Drugs
1996;52(Suppl 5):13e23.
[133] Mohamed-Noriega K, Butron-Valdez K, Vazquez-Galvan J, Mohamed-
Noriega J, Cavazos-Adame H, Mohamed-Hamsho J. Corneal melting after
collagen cross-linking for keratoconus in a thin cornea of a diabetic patient
treated with topical nepafenac: a case report with a literature review. Case
Rep Ophthalmol 2016;7:119e24.
[134] Marfurt CF, Echtenkamp SF. The effect of diabetes on neuropeptide content
in the rat cornea and iris. Investig Ophthalmol Vis Sci 1995;36:1100e6.
[135] Bonini S, Lambiase A, Rama P, Caprioglio G, Aloe L. Topical treatment with
nerve growth factor for neurotrophic keratitis. Ophthalmology 2000;107:
1347e51. discussion 51e52.
[136] Lambiase A, Bonini S, Micera A, Rama P, Bonini S, Aloe L. Expression of nerve
growth factor receptors on the ocular surface in healthy subjects and during
manifestation of inflammatory diseases. Investig Ophthalmol Vis Sci
1998;39:1272e5.
[137] Lambiase A, Micera A, Pellegrini G, Merlo D, Rama P, De Luca M, et al. In vitro
evidence of nerve growth factor effects on human conjunctival epithelial cell
differentiation and mucin gene expression. Investig Ophthalmol Vis Sci
2009;50:4622e30.
[138] Rocha EM, Cunha DA, Carneiro EM, Boschero AC, Saad MJ, Velloso LA.
Identification of insulin in the tear film and insulin receptor and IGF-1 re-
ceptor on the human ocular surface. Investig Ophthalmol Vis Sci 2002;43:
963e7.
[139] Cunha DA, de Alves MC, Stoppiglia LF, Jorge AG, Modulo CM, Carneiro EM,
et al. Extra-pancreatic insulin production in RAt lachrymal gland after
streptozotocin-induced islet beta-cells destruction. Biochim Biophys Acta
2007;1770:1128e35.
[140] Ding J, Liu Y, Sullivan DA. Effects of insulin and high glucose on human
meibomian gland epithelial cells. Investig Ophthalmol Vis Sci 2015;56:
7814e20.
[141] Alves Mde C, Carvalheira JB, Modulo CM, Rocha EM. Tear film and ocular
surface changes in diabetes mellitus. Arq Bras Oftalmol 2008;71:96e103.
[142] Cruzat A, Pavan-Langston D, Hamrah P. In vivo confocal microscopy of
corneal nerves: analysis and clinical correlation. Semin Ophthalmol 2010;25:
171e7.
[143] Chen DK, Frizzi KE, Guernsey LS, Ladt K, Mizisin AP, Calcutt NA. Repeated
monitoring of corneal nerves by confocal microscopy as an index of pe-
ripheral neuropathy in type-1 diabetic rodents and the effects of topical
insulin. J Peripher Nerv Syst 2013;18:306e15.
[144] Zagon IS, Klocek MS, Sassani JW, McLaughlin PJ. Use of topical insulin to
normalize corneal epithelial healing in diabetes mellitus. Arch Ophthalmol
2007;125:1082e8.
[145] Zagon IS, Sassani JW, McLaughlin PJ. Insulin treatment ameliorates impaired
corneal reepithelialization in diabetic rats. Diabetes 2006;55:1141e7.
[146] Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Naltrexone and insulin are
independently effective but not additive in accelerating corneal epithelial
healing in type I diabetic rats. Exp Eye Res 2009;89:686e92.
[147] Wu YC, Buckner BR, Zhu M, Cavanagh HD, Robertson DM. Elevated IGFBP3
levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal
epithelium. Ocul Surf 2012;10:100e7.
[148] Gasset AR, Braverman LE, Fleming MC, Arky RA, Alter BR. Tear glucose
detection of hyperglycemia. Am J Ophthalmol 1968;65:414e20.
[149] Sen DK, Sarin GS. Tear glucose levels in normal people and in diabetic pa-
tients. Br J Ophthalmol 1980;64:693e5.
56 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
[150] Peng B, Lu J, Balijepalli AS, Major TC, Cohan BE, Meyerhoff ME. Evaluation of
enzyme-based tear glucose electrochemical sensors over a wide range of
blood glucose concentrations. Biosens Bioelectron 2013;49:204e9.
[151] Zhao Z, Liu J, Shi B, He S, Yao X, Willcox MD. Advanced glycation end product
modified proteins in tears of diabetic patients. Mol Vis 2010;16:1576e84.
[152] Grus FH, Sabuncuo P, Dick HB, Augustin AJ, Pfeiffer N. Changes in the tear
proteins of diabetic patients. BMC Ophthalmol 2002;2:4.
[153] Stolwijk TR, Kuizenga A, van Haeringen NJ, Kijlstra A, Oosterhuis JA, van
Best JA. Analysis of tear fluid proteins in insulin-dependent diabetes melli-
tus. Acta Ophthalmol Copenh 1994;72:357e62.
[154] de Luca C, Olefsky JM. Inflammation and insulin resistance. FEBS Lett
2008;582:97e105.
[155] Costagliola C, Romano V, De Tollis M, Aceto F, dell'Omo R, Romano MR, et al.
TNF-alpha levels in tears: a novel biomarker to assess the degree of diabetic
retinopathy. Mediat Inflamm 2013;2013:629529.
[156] Gill SE, Parks WC. Metalloproteinases and their inhibitors: regulators of
wound healing. Int J Biochem Cell Biol 2008;40:1334e47.
[157] Nagase H, Woessner Jr JF. Matrix metalloproteinases. J Biol Chem 1999;274:
21491e4.
[158] Berman M, Dohlman CH, Gnadinger M, Davison P. Characterization of col-
lagenolytic activity in the ulcerating cornea. Exp Eye Res 1971;11:255e7.
[159] Garrana RM, Zieske JD, Assouline M, Gipson IK. Matrix metalloproteinases in
epithelia from human recurrent corneal erosion. Investig Ophthalmol Vis Sci
1999;40:1266e70.
[160] Schultz GS, Wysocki A. Interactions between extracellular matrix and
growth factors in wound healing. Wound Repair Regen 2009;17:153e62.
[161] Kaiserman I, Kaiserman N, Nakar S, Vinker S. Dry eye in diabetic patients. Am
J Ophthalmol 2005;139:498e503.
[162] Lv H, Li A, Zhang X, Xu M, Qiao Y, Zhang J, et al. Meta-analysis and review on
the changes of tear function and corneal sensitivity in diabetic patients. Acta
Ophthalmol 2014;92:e96e104.
[163] Seifart U, Strempel I. The dry eye and diabetes mellitus. Ophthalmologe
1994;91:235e9.
[164] Creuzot-Garcher C, Lafontaine PO, Gualino O, D'Athis P, Petit JM, Bron A.
Study of ocular surface involvement in diabetic patients. J Fr Ophtalmol
2005;28:583e8.
[165] Goebbels M. Tear secretion and tear film function in insulin dependent di-
abetics. Br J Ophthalmol 2000;84:19e21.
[166] Sullivan DA, Edwards JA, Wickham LA, Pena JD, Gao J, Ono M, et al. Identi-
fication and endocrine control of sex steroid binding sites in the lacrimal
gland. Curr Eye Res 1996;15:279e91.
[167] Shetty R, Saeed T, Rashed H, Adeghate E, Singh J. Effect of diabetes mellitus
on acinar morphology, peroxidase concentration, and release in isolated rat
lacrimal glands. Curr Eye Res 2009;34:905e11.
[168] Inoue K, Kato S, Ohara C, Numaga J, Amano S, Oshika T. Ocular and systemic
factors relevant to diabetic keratoepitheliopathy. Cornea 2001;20:798e801.
[169] Sagdik HM, Ugurbas SH, Can M, Tetikoglu M, Ugurbas E, Ugurbas SC, et al.
Tear film osmolarity in patients with diabetes mellitus. Ophthalmic Res
2013;50:1e5.
[170] DeMill DL, Hussain M, Pop-Busui R, Shtein RM. Ocular surface disease in
patients with diabetic peripheral neuropathy. Br J Ophthalmol 2016;100:
924e8.
[171] Beckman KA. Characterization of dry eye disease in diabetic patients versus
nondiabetic patients. Cornea 2014;33:851e4.
[172] Leibowitz HM, Krueger DE, Maunder LR, Milton RC, Kini MM, Kahn HA, et al.
The Framingham Eye Study monograph: an ophthalmological and epide-
miological study of cataract, glaucoma, diabetic retinopathy, macular
degeneration, and visual acuity in a general population of 2631 adults, 1973-
1975. Surv Ophthalmol 1980;24:335e610.
[173] Ederer F, Hiller R, Taylor HR. Senile lens changes and diabetes in two pop-
ulation studies. Am J Ophthalmol 1981;91:381e95.
[174] Haddad NM, Sun JK, Abujaber S, Schlossman DK, Silva PS. Cataract surgery
and its complications in diabetic patients. Semin Ophthalmol 2014;29:
329e37.
[175] Pershing S, Morrison DE, Hernandez-Boussard T. Cataract surgery compli-
cations and revisit rates among three states. Am J Ophthalmol 2016;171:
130e8.
[176] Ozdemir M, Buyukbese MA, Cetinkaya A, Ozdemir G. Risk factors for ocular
surface disorders in patients with diabetes mellitus. Diabetes Res Clin Pract
2003;59:195e9.
[177] Tsaousis KT, Panagiotou DZ, Kostopoulou E, Vlatsios V, Stampouli D. Corneal
oedema after phacoemulsification in the early postoperative period: a
qualitative comparative case-control study between diabetics and non-di-
abetics. Ann Med Surg Lond 2016;5:67e71.
[178] Hugod M, Storr-Paulsen A, Norregaard JC, Nicolini J, Larsen AB, Thulesen J.
Corneal endothelial cell changes associated with cataract surgery in patients
with type 2 diabetes mellitus. Cornea 2011;30:749e53.
[179] Mathew PT, David S, Thomas N. Endothelial cell loss and central corneal
thickness in patients with and without diabetes after manual small incision
cataract surgery. Cornea 2011;30:424e8.
[180] Kattan HM, Flynn Jr HW, Pflugfelder SC, Robertson C, Forster RK. Nosocomial
endophthalmitis survey. Current incidence of infection after intraocular
surgery. Ophthalmology 1991;98:227e38.
[181] Phillips 2nd WB, Tasman WS. Postoperative endophthalmitis in association
with diabetes mellitus. Ophthalmology 1994;101:508e18.
[182] Javitt JC, Street DA, Tielsch JM, Wang Q, Kolb MM, Schien O, et al. National
outcomes of cataract extraction. Retinal detachment and endophthalmitis
after outpatient cataract surgery. Cataract Patient Outcomes Research Team.
Ophthalmology 1994;101:100e5. discussion 6.
[183] Menikoff JA, Speaker MG, Marmor M, Raskin EM. A case-control study of risk
factors for postoperative endophthalmitis. Ophthalmology 1991;98:1761e8.
[184] Misra SL, Goh YW, Patel DV, Riley AF, McGhee CN. Corneal microstructural
changes in nerve fiber, endothelial and epithelial density after cataract
surgery in patients with diabetes mellitus. Cornea 2015;34:177e81.
[185] Jiang D, Xiao X, Fu T, Mashaghi A, Liu Q, Hong J. Transient tear film
dysfunction after cataract surgery in diabetic patients. PloS One 2016;11,
e0146752.
[186] Gemensky-Metzler AJ, Sheahan JE, Rajala-Schultz PJ, Wilkie DA, Harrington J.
Retrospective study of the prevalence of keratoconjunctivitis sicca in dia-
betic and nondiabetic dogs after phacoemulsification. Vet Ophthalmol
2015;18:472e80.
[187] Chikamoto N, Chikama T, Yamada N, Nishida T, Ishimitsu T, Kamiya A. Effi-
cacy of substance P and insulin-like growth factor-1 peptides for preventing
postsurgical superficial punctate keratopathy in diabetic patients. Jpn J
Ophthalmol 2009;53:464e9.
[188] Schulze SD, Sekundo W, Kroll P. Autologous serum for the treatment of
corneal epithelial abrasions in diabetic patients undergoing vitrectomy. Am J
Ophthalmol 2006;142:207e11.
[189] Fujishima H, Tsubota K. Improvement of corneal fluorescein staining in post
cataract surgery of diabetic patients by an oral aldose reductase inhibitor,
ONO-2235. Br J Ophthalmol 2002;86:860e3.
[190] Nakahara M, Miyata K, Otani S, Miyai T, Nejima R, Yamagami S, et al.
A randomised, placebo controlled clinical trial of the aldose reductase in-
hibitor CT-112 as management of corneal epithelial disorders in diabetic
patients. Br J Ophthalmol 2005;89:266e8.
[191] United States Food and Drug Administration. When is LASIK not for me?
http://www.fda.gov/medicaldevices/productsandmedicalprocedures/
surgeryandlifesupport/lasik/ucm061366.htm2014.
[192] Fraunfelder FW, Rich LF. Laser-assisted in situ keratomileusis complications
in diabetes mellitus. Cornea 2002;21:246e8.
[193] Halkiadakis I, Belfair N, Gimbel HV. Laser in situ keratomileusis in patients
with diabetes. J Cataract Refract Surg 2005;31:1895e8.
[194] Cobo-Soriano R, Beltran J, Baviera J. LASIK outcomes in patients with un-
derlying systemic contraindications: a preliminary study. Ophthalmology
2006;113(1118):e1e8.
[195] Spadea L, Paroli MP. Laser refractive surgery in diabetic patients: a review of
the literature. Clin Ophthalmol 2012;6:1775e83.
[196] Ono T, Yuki K, Ozeki N, Shiba D, Tsubota K. Ocular surface complications after
trabeculectomy: incidence, risk factors, time course and prognosis. Oph-
thalmologica 2013;230:93e9.
[197] Friberg TR, Ohji M, Scherer JJ, Tano Y. Frequency of epithelial debridement
during diabetic vitrectomy. Am J Ophthalmol 2003;135:553e4.
[198] Shimada H, Nakashizuka H, Hattori T, Mori R, Mizutani Y, Yuzawa M. Inci-
dence of endophthalmitis after 20- and 25-gauge vitrectomy causes and
prevention. Ophthalmology 2008;115:2215e20.
[199] Dogru M, Kaderli B, Gelisken O, Yucel A, Avci R, Goto E, et al. Ocular surface
changes with applanation contact lens and coupling fluid use after argon
laser photocoagulation in noninsulin-dependent diabetes mellitus. Am J
Ophthalmol 2004;138:381e8.
[200] Lu L, Reinach PS, Kao WW. Corneal epithelial wound healing. Exp Biol Med
Maywood 2001;226:653e64.
[201] Veys J, Efron N, Boulton A. A survey of contact lens wear among diabetic
patients in the United Kingdom. Cont Lens Anterior Eye 1997;20:S27e33.
[202] O'Donnell C, Efron N. Contact lens wear and diabetes mellitus. Cont Lens
Anterior Eye 1998;21:19e26.
[203] Eichenbaum JW, Feldstein M, Podos SM. Extended-wear aphakic soft contact
lenses and corneal ulcers. Br J Ophthalmol 1982;66:663e6.
[204] Spoor TC, Hartel WC, Wynn P, Spoor DK. Complications of continuous-wear
soft contact lenses in a nonreferral population. Arch Ophthalmol 1984;102:
1312e3.
[205] O'Donnell C, Efron N, Boulton AJ. A prospective study of contact lens wear in
diabetes mellitus. Ophthalmic Physiol Opt 2001;21:127e38.
[206] March W, Long B, Hofmann W, Keys D, McKenney C. Safety of contact lenses
in patients with diabetes. Diabetes Technol Ther 2004;6:49e52.
[207] O'Donnell C, Efron N. Corneal hydration control in contact lens wearers with
diabetes mellitus. Optom Vis Sci 2006;83:22e6.
[208] Leem HS, Lee KJ, Shin KC. Central corneal thickness and corneal endothelial
cell changes caused by contact lens use in diabetic patients. Yonsei Med J
2011;52:322e5.
[209] March WF, Mueller A, Herbrechtsmeier P. Clinical trial of a noninvasive
contact lens glucose sensor. Diabetes Technol Ther 2004;6:782e9.
[210] Phan CM, Subbaraman L, Jones LW. The use of contact lenses as biosensors.
Optom Vis Sci 2016;93:419e25.
[211] Ascaso FJ, Huerva V. Noninvasive continuous monitoring of tear glucose
using glucose-sensing contact lenses. Optom Vis Sci 2016;93:426e34.
[212] Farandos NM, Yetisen AK, Monteiro MJ, Lowe CR, Yun SH. Contact lens
sensors in ocular diagnostics. Adv Healthc Mater 2015;4:792e810.
[213] Azar DT, Spurrmichaud SJ, Tisdale AS, Gipson IK. Decreased penetration of
anchoring fibrils into the diabetic stroma - a morphometric analysis. Arch
Ophthalmol 1989;107:1520e3.
 M. Markoulli et al. / The Ocular Surface 16 (2018) 45e57
[214] Dursun D, Kim MC, Solomon A, Pflugfelder SC. Treatment of recalcitrant
recurrent corneal erosions with inhibitors of matrix metalloproteinase-9,
doxycycline and corticosteroids. Am J Ophthalmol 2001;132:8e13.
[215] Akpek EK, Merchant A, Pinar V, Foster CS. Ocular rosacea: patient charac-
teristics and follow-up. Ophthalmology 1997;104:1863e7.
[216] Bonini S, Rama P, Olzi D, Lambiase A. Neurotrophic keratitis. Eye Lond
2003;17:989e95.
[217] Cho BJ, Lee GJ, Ha SY, Seo YH, Tchah H. Co-infection of the human cornea
with Stenotrophomonas maltophilia and Aspergillus fumigatus. Cornea
2002;21:628e31.
[218] Holifield K, Lazzaro DR. Case report: spontaneous Stenotrophomonas mal-
tophilia keratitis in a diabetic patient. Eye Contact Lens 2011;37:326e7.
[219] Jin H, Parker WT, Law NW, Clarke CL, Gisseman JD, Pflugfelder SC, et al.
Evolving risk factors and antibiotic sensitivity patterns for microbial keratitis
at a large county hospital. Br J Ophthalmol 2017;101(11):1483e7.
[220] Brown CT, Davis-Richardson AG, Giongo A, Gano KA, Crabb DB, Mukherjee N,
et al. Gut microbiome metagenomics analysis suggests a functional model
for the development of autoimmunity for type 1 diabetes. PloS One 2011;6,
e25792.
[221] Giongo A, Gano KA, Crabb DB, Mukherjee N, Novelo LL, Casella G, et al. To-
ward defining the autoimmune microbiome for type 1 diabetes. ISME J
2011;5:82e91.
[222] Larsen N, Vogensen FK, van den Berg FW, Nielsen DS, Andreasen AS,
Pedersen BK, et al. Gut microbiota in human adults with type 2 diabetes
57
differs from non-diabetic adults. PloS One 2010;5, e9085.
[223] Yang C, Fei Y, Qin Y, Luo D, Yang S, Kou X, et al. Bacterial flora changes in
conjunctiva of rats with streptozotocin-induced type i diabetes. PloS One
2015;10, e0133021.
[224] Coburn PS, Wiskur BJ, Christy E, Callegan MC. The diabetic ocular environ-
ment facilitates the development of endogenous bacterial endophthalmitis.
Investig Ophthalmol Vis Sci 2012;53:7426e31.
[225] Moreno NP, Moreno RD, Sousa LB. Aerobic bacterial microbiota of the con-
junctiva in diabetic patients with normal and altered glycated hemoglobin
levels in two regions in Brazil. Arq Bras Oftalmol 2014;77:351e4.
[226] Bilen H, Ates O, Astam N, Uslu H, Akcay G, Baykal O. Conjunctival flora in
patients with type 1 or type 2 diabetes mellitus. Adv Ther 2007;24:1028e35.
[227] Karimsab D, Razak SK. Study of aerobic bacterial conjunctival flora in pa-
tients with diabetes mellitus. Nepal J Ophthalmol 2013;5:28e32.
[228] Fernandez-Rubio ME, Rebolledo-Lara L, Martinez-Garcia M, Alarcon-
Tomas M, Cortes-Valdes C. The conjunctival bacterial pattern of diabetics
undergoing cataract surgery. Eye Lond 2010;24:825e34.
[229] Nahar N, Anwar S, Miah MRA. Conjunctival bacterial flora in diabetic pa-
tients. Ibrahim Med Coll J 2013;7:4.
[230] Adam M, Balci M, Bayhan HA, Inkaya AC, Uyar M, Gurdal C. Conjunctival flora
in diabetic and nondiabetic individuals. Turk J Ophthalmol 2015;45:193e6.
[231] Martins EN, Alvarenga LS, Hofling-Lima AL, Freitas D, Zorat-Yu MC, Farah ME,
et al. Aerobic bacterial conjunctival flora in diabetic patients. Cornea
2004;23:136e42.