Cornea

Diabetes Mellitus Leads to Accumulation of Dendritic Cells

and Nerve Fiber Damage of the Subbasal Nerve Plexus in
the Cornea
Katja Leppin,1 Ann-Kathrin Behrendt,1 Maria Reichard,2 Oliver Stachs,2 Rudolf F. Guthoff,2
Simone Baltrusch,3 Johanna C. Eule,4 and Brigitte Vollmar1
1Institute for Experimental Surgery, University of Rostock, Rostock, Germany
2
Department of Ophthalmology, University of Rostock, Rostock, Germany
3
Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Rostock, Germany
4
Small Animal Clinic, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany

Correspondence: Brigitte Vollmar, In-
stitute for Experimental Surgery, Uni-
versity of Rostock, Schillingallee 69a,
18057 Rostock, Germany;
brigitte.vollmar@med.uni-rostock.de.
Submitted: March 5, 2014
Accepted: April 16, 2014
Citation: Leppin K, Behrendt A-K,
Reichard M, et al. Diabetes mellitus
leads to accumulation of dendritic
cells and nerve fiber damage of the
subbasal nerve plexus in the cornea.
Invest Ophthalmol Vis Sci.
2014;55:3603–3615. DOI:10.1167/
iovs.14-14307
PURPOSE. To evaluate whether nerve fibers of the subbasal nerve plexus (SNP) and dendritic
cells (DCs) are in association with each other leading to neuropathy in the diabetic cornea.
METHODS. BALB/c mice were injected with streptozotocin (STZ) for 5 days for induction of
diabetes mellitus (DM) or with vehicle solution (control). B6.VLepob/ob (ob/ob) mice served
as an obese and glucose-intolerant DM type 2 (DM II) model and lean B6.VLepob/þ (ob/þ)
mice as respective controls. Using in vivo corneal confocal microscopy (CCM), nerve fibers
and DCs were quantified over a period of 9 weeks and additionally analyzed by in vitro
immunofluorescence whole-mount staining.
RESULTS. In STZ-diabetic mice, CCM revealed an increase of DC density (DCD) in contrast to
controls, whereas nerve fiber density (NFD) was decreased with duration of DM. In ob/ob
mice, DCD was 3-fold higher than in both ob/þ mice and STZ-diabetic mice. Whole-mount
staining displayed CD11c(þ) and major histocompatibility complex (MHC) class II(þ) mature
DCs in colocalization with class III b-tubulin(þ) nerve fibers in the cornea.
CONCLUSIONS. Hyperglycemia leads to corneal DC infiltration, and obesity aggravates this
immune response. The direct contact between DCs and the SNP can be assumed to be a
trigger of nerve fiber damage and thus a contributing factor to polyneuropathy in diabetic
corneas.
Keywords: diabetes mellitus, dendritic cells, subbasal nerve plexus

iabetes mellitus (DM) is a common metabolic disease
D characterized by an increased blood glucose (BG) level due
to a relative or absolute deficit of insulin and is classified into
DM I and DM II.1 The insulin-dependent type, DM I, is caused
by an autoimmune destruction of the b-cells in the pancreas,
whereas the insulin-independent type, DM II, is characterized
by insulin resistance. International analyses show a doubling of
the diabetic population over the last three decades.1 The
increased BG level leads to various symptoms based on
pathologic changes in organs and tissues of the whole body,
including eyes, kidneys, heart, blood vessels, and nerves.2
However, the underlying mechanisms are not yet fully
understood.
The most common type of neuropathy is diabetic neurop-
athy,3 defined as change in morphology and function as well as
the loss of nerve fibers in different tissues. To determine
diabetic neuropathy, only invasive methods like skin biopsy
exist. The cornea displays the highest nerve fiber density (NFD)
in the human body, comprising innervating nerves with Ad and
C fibers4 that originate from the ophthalmic division of the
trigeminal nerve. These fibers are damaged in the earliest stage
of DM, leading to peripheral neuropathy.5 Corneal confocal
microscopy (CCM) represents an alternative and highly
attractive method to assess corneal pathology due to its ability
to display detailed information on the different corneal cell
layers, including the visualization of nerve fibers of the corneal
subbasal nerve plexus (SNP). Degeneration or regeneration of
nerve fibers results in changes in corneal NFD.6 Diabetes
mellitus leads to the loss of nerve fibers, thus resulting in a
lower level of NFD7–9 that can be displayed by CCM.10
Besides being able to examine the corneal SNP, CCM has also
the ability to detect immunologic cells like the highly reflective
dendritic cells (DCs). These bone marrow–derived cells can be
found in the noninflamed cornea with a high density in the
periphery in contrast to the center, where only a few sessile
DCs appear.11 Due to their function to recognize, process, and
present antigens, they are also called professional antigen-
presenting cells (APCs), reflecting a kind of guard against
foreign influences on the cornea. Dendritic cells exhibit a
distinct morphology, showing specific cell processes growing
especially to the apical surface of the corneal epithelium.12 As
DCs are found to be increased in patients with no or mild
diabetic neuropathy but decreased with moderate and severe
diabetic neuropathy, it has been postulated that they might be
involved in the initial stage of diabetic nerve destruction.13 In
addition, a strong correlation between an increase of DCs and a
decrease of nerve fibers in inflamed corneas has been shown.14
Within this context, the term neuroimmunologic synapse was

Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3604

FIGURE 1. (A) Schematic drawing of in vivo CCM using the combination of the Heidelberg Retina Tomograph II and the in-house–developed
Rostock Cornea Module. (B) In vivo CCM image of a DC (white arrow) and nerve fibers of the SNP as well as stromal nerves (yellow arrows) of a
normoglycemic BALB/c mouse. Scale bar: 50 lm. (C) Schematic drawing of the nine images, captured in a meandering pattern, for in vivo CCM
analysis of the corneal center. (D) Corneal flattening of whole mounts by use of four to six radial incisions toward the center.

coined to refer to interconnections of the immune and nervous
systems.15 In previous studies, an interplay between the
immune and nervous systems has been demonstrated in skin16
and gut.17 To add to our current understanding about the role
of DCs and nerve fibers in DM, we hypothesize that corneal
polyneuropathy is closely linked to an infiltration of DCs. For
this purpose, we provide a comprehensive longitudinal
analysis of corneal DCs and nerve fibers in experimental
mouse models of both streptozotocin (STZ)-diabetic mice and
ob/ob mice.
MATERIALS AND METHODS
Animals
Female 8- to 12-week-old B6.VLepob/ob and BALB/c mice were
purchased from Charles River Laboratories (Sulzfeld, Ger-
many). Animals were kept on water and standard laboratory
chow ad libitum. All experiments were approved by the local
animal welfare committee and were performed in accordance
with the German legislation and the principles of laboratory
animal care, as well as with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
Induction of Diabetes; Blood Glucose and Weight
Measurements
For induction of DM, BALB/c mice (n ¼ 7) received an
intraperitoneal (i.p.) injection of 50 mg/kg per body weight
STZ (Sigma-Aldrich, Steinheim, Germany) dissolved in 0.05 M
sodium citrate buffer on 5 days. BALB/c mice, treated with the
vehicle solution, served as controls (n ¼ 7). Ob/ob mice (n ¼ 5)
exhibit obesity and hyperglycemia.18 Lean and normoglycemic
ob/þ mice (n ¼ 5) were used as controls. The onset and
progression of diabetes were monitored by BG (mmol/L)
assessment using the BG meter Contour (Bayer Vital, Leverku-
sen, Germany). All animals underwent regular weight analysis.
Corneal Confocal Microscopy
By means of in vivo CCM, nerve fibers of the SNP and DCs were
quantified biweekly over a period of 9 weeks using the
combination of Heidelberg Retina Tomograph II (Heidelberg
Engineering, Heidelberg, Germany) and the in-house–devel-
oped Rostock Cornea Module19 (University of Rostock,
Germany) adapted to mouse imaging.20 Mice were anesthe-
tized with 75 mg/kg per body weight ketamine (bela-pharm,
Vechta, Germany) and 5 mg/kg per body weight xylazine i.p.
(Bayer Health Care, Leverkusen, Germany) and fixed in a

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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3605

FIGURE 2. Average BG levels of STZ- and sodium citrate–treated BALB/c mice (n ¼ 7) (A) as well as ob/ob and ob/þ mice (n ¼ 5) (B). Quantitative
analysis of DCD (number/mm2) (C) and NFD (mm/mm2) (E) in the corneal epithelium of healthy mice and mice with STZ-induced DM over an
observation period of 9 weeks. Sodium citrate–treated BALB/c mice (n ¼ 7) served as normoglycemic controls (sham), and STZ-treated BALB/c mice
(n ¼ 7) represent hyperglycemic DM I mice (STZ). Quantitative analysis of DCD (number/mm2) (D) and NFD (mm/mm2) (F) in the corneal
epithelium of healthy mice and mice with DM II over an observation period of 9 weeks. B6.VLepob/þ mice (n ¼ 5) served as normoglycemic controls
(ob/þ), and B6.VLepob/ob mice (n ¼ 5) represent hyperglycemic DM II mice (ob/ob). Means 6 SEM; *P < 0.05 vs. sham at the respective time points;
#P < 0.05 vs. 1 week.

MouseFix animal holder (Steven GmbH, Ochtrup, Germany).
The body temperature was maintained at 378C with a heating
pad. The corneas were wetted with gel (Vidisic; Bausch &
Lomb/Dr. Mann Pharma, Berlin, Germany) to prevent desicca-
tion. The curved cornea was then lightly touched with a
TomoCap (Heidelberg Engineering GmbH) (Fig. 1A) that was
also filled with gel, and prescanned for differentiation between
the center and the periphery. In contrast to a previous work,21
in this study planar images revealing clear and sharp
morphology throughout the whole area were interpreted as
located ‘‘inside the center’’ and served for subsequent analysis
(Fig. 1B). Upon movement of the microscope as images
revealed a loss of focus due to the convexity of the cornea
toward the periphery, the images were interpreted as located
‘‘outside the center’’ and were not considered for further
analysis. Additionally, the nerve fiber vortex of the SNP located
in the center of the cornea served as a landmark, being
characteristic of the corneal center. Within one of the nine

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 Corneal Changes in Diabetic Mice
FIGURE 3. Regression analysis between BG (mmol/L) and DCD (number/mm2) of sodium citrate–treated sham mice (n ¼ 7) (A), STZ-treated mice (n
¼ 7) (C), ob/þ mice (n ¼ 5) (B), and ob/ob mice (n ¼ 5) (D). R, regression coefficient.
images taken of each animal’s eye, captured in a meandering
pattern (Fig. 1C), the vortex was routinely observed, which
further underscored the exclusive analysis of the corneal
center. However, the vortex was found located at the border of
the central regions and not necessarily located in the most
central image. Since nine different pictures were captured, the
entire analyzed region contained tissue in very close proximity
to the vortex and represents the central cornea. Digital images
(0.3 3 0.3 mm, 384 3 384 pixels) were taken using a 0.3-mm2
field-of-view lens. Using the sequence mode, three images per
second were captured. Finally, offline analysis of these images
was performed in a masked manner. Dendritic cell density
(DCD) and NFD were assessed by the use of ImageJ (available
in the public domain at http://rsb.info.nih.gov/ij/) NeuronJ as
plug-in for ImageJ, Analysis 3.1 (Soft Imaging System, Münster,
Germany).10 Dendritic cell density is defined as the total
number of counted DCs per square millimeter (number/mm2),
and NFD includes the total accumulative length of all nerve
fibers and branches of the SNP (mm/mm2).
Corneal Whole-Mount Staining
Before enucleation, mice were killed and subsequently the
corneas were processed as described elsewhere.22 In brief, the
separated globes were fixed for 20 minutes at room
temperature (RT) in 4% paraformaldehyde (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA), 0.2% picric acid in
0.1 M phosphate-buffered saline (PBS; Life Technologies
GmbH, Darmstadt, Germany). Afterward the anterior segment
was excised and placed back in the fixative solution for 20
minutes, followed by the extraction of the lens and a last
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IOVS j June 2014 j Vol. 55 j No. 6 j 3606
incubation in the fixative solution for 20 minutes. After the
corneas were freed from the iris, four to six radial incisions
toward the center of the cornea were performed with an
ophthalmic scalpel (pfm medical ag, Köln, Germany) to flatten
the tissue (Fig. 1D). The preparations were stored in 0.1 M PBS
with 30% sucrose (Sigma-Aldrich Chemie GmbH Taufkirchen,
München, Germany) for 24 hours. Corneas were incubated at
378C by gentle agitation in 0.1% EDTA (Sigma-Aldrich, Inc., St.
Louis, MO, USA) and 0.01% hyaluronidase (type IV-S, product
no. H4272; Sigma-Aldrich) in 0.1 M PBS, pH 5.3. Tissues were
rinsed for 90 minutes in PBS with 0.3% Triton X-100 (PBS-TX;
Roth, Karlsruhe, Germany) and were blocked at RT for 2 hours
in PBS-TX containing 1% bovine serum albumin (BSA; Santa
Cruz Biotechnology, Inc.). To identify mature DCs, we used
CD11c (purified anti-mouse CD11c, clone N418, 1:200;
BioLegend, San Diego, CA, USA) combined with major
histocompatibility complex (MHC) class II (I-A/I-E) (purified
anti-mouse MHC class II, clone M5/114.15.2, 1:200; eBio-
science, Inc., San Diego, CA, USA). Neuronal class III b-tubulin
antibody (TUJ1) (clone TUJ1 1-15-79, 1:1000; Covance,
Princeton, NJ, USA) was used as a pan-neuronal surface marker
for corneal nerve fibers. Corneas were rinsed for 90 minutes in
PBS-TX followed by the application of DyLight 488-conjugated
anti-hamster (Armenian) IgG (1:200; BioLegend), Alexa Fluor
555-conjugated anti-rat (1:400; Life Technologies GmbH), and
Alexa Fluor 633-conjugated anti-rabbit (1:400; Life Technolo-
gies GmbH) for 2 hours. After rinsing of the tissue for 90
minutes in PBS-TX, corneas were stained with 4 0 ,6-diamidino-
2-phenylindole (1:1000, AppliChem GmbH, Darmstadt, Ger-
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FIGURE 4. Regression analysis between DCD (number/mm2) and NFD (mm/mm2) of sodium citrate–treated sham mice (n ¼ 7) (A), STZ-treated
mice (n ¼ 7) (C), ob/þ mice (n ¼ 5) (B), and ob/ob mice (n ¼ 5) (D).

FIGURE 5. Localization of DCs and nerve fibers within the corneal epithelium and anterior stroma assessed by performing a z-stack through the
corneal whole mount of a normoglycemic BALB/c mouse. DCs express CD11c (A) and MHC class II (B). In general, they are localized between the
epithelium and anterior stroma. The cell bodies reside beneath the basal cell layer, and the processes of the DCs are directed toward the corneal
surface. Class III b-tubulin(þ) (TUJ1) nerve fiber bundles of the stroma (C) sprout into single nerve fibers forming the SNP between the anterior
stroma and the epithelium. Associated small fibers resulting from the SNP are arranged among epithelial cells. Scale bars: 30 lm.

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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3608

FIGURE 6. Distribution of CD11c(þ) and MHC class II(þ) DCs in corneal whole mounts. Images were taken with a 103 objective lens. All groups
revealed a decreasing gradient of DCs from the periphery to the center. In contrast to sodium citrate–treated BALB/c and B6.VLepob/þ mice (A, C),
STZ-treated BALB/c and B6.VLepob/ob mice display a higher density of DCs (B, D). Scale bars: 500 lm.

many) as nuclear staining and mounted in fluorescent
mounting medium (Dako, Jena, Germany).
Confocal Imaging of Corneal Whole Mounts
Images from processed corneas were captured with a 60-fold
oil immersion objective lens of an Olympus Fluoview 10i
confocal microscope (Olympus, Hamburg, Germany). To assess
the three-dimensionality with precise information on the SNP
and DCs including their processes, z-stacks were performed.
Slices were captured in steps of 0.5 lm and reconstructed to
one image. Images were taken from the center of each cornea
to allow comparability between CCM and confocal microscopy
of corneal whole mounts. Offline analysis of the histologic
specimen was performed in a masked manner.
Statistical Analysis
The SPSS version 20.0. (IBM Corp., Armonk, NY, USA) General
Linear Model (GLM) repeated measures ANOVA was used for

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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3609

FIGURE 7. In vivo CCM and confocal imaging of corneal whole mounts in normoglycemic BALB/c mice at week 9. (A) CCM of nerve fibers. Scale
bar: 50 lm. (B–E) Confocal imaging of corneal whole mounts with a single CD11c(þ) (B, C, green) and MHC class II(þ) (B, D, red) DC and class III
b-tubulin(þ) (TUJ1) nerve fibers (B, E, yellow). DAPI (blue) as nuclear staining. Scale bars: 30 lm.

statistical analysis of the data to test the effect of both the
between-subject factor (treatment) and the within-subject
factor (time). By means of SigmaStat 3.5 software (SigmaStat;
Jandel Corporation, San Rafael, CA, USA), differences between
groups were calculated with one-way repeated measures
ANOVA with an appropriate post hoc test, and linear
regression was performed for analyzing correlations between
BG, DCD, and NFD. A P value of <0.05 was considered
statistically significant.
RESULTS
Blood Glucose Response
Two weeks after the first STZ application, mice showed an
increase in BG level of 10.8 mmol/L and an average blood BG
level of 13.5 mmol/L during the remainder of the experiment
(Fig. 2A).
Ob/ob mice revealed an average BG level of 15.9 mmol/L
from the first detection of BG until week 4 and showed
decreased BG levels (10.6 and 11.8 mmol/L) at the last two
time points (Fig. 2B).
CCM-Based Analysis of the Cornea in STZ-Diabetic
Mice
The parallel arrangement of single nerve fibers of the SNP and
between residing DCs is exemplified in Figure 1B. After
treatment with STZ, BALB/c mice over the period of 1 to 9
weeks showed a gradual increase of corneal DCD in contrast to
the constant level of DCD in control mice. At weeks 7 and 9,
DCD displayed a significant rise (Fig. 2C) when compared to
both the corresponding values for control animals (Fig. 2C) and
within-group values at week 1, with an approximately 3-fold
increase at week 9 (17 6 2 / mm2). In addition, the increase of
DCD in STZ-diabetic mice showed a significant positive

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FIGURE 8. In vivo CCM and confocal imaging of corneal whole mounts in hyperglycemic BALB/c mice at week 9. (A) CCM of DCs (arrows) and
nerve fibers. Scale bar: 50 lm. (B–E) Confocal imaging of corneal whole mounts with three CD11cþ ([B, C], green) and MHC class II(þ) ([B, D], red)
DCs and class III b-tubulin(þ) (TUJ1) nerve fibers ([B, E], yellow). DAPI (blue) as nuclear staining. Scale bar: 30 lm.

correlation with increasing BG levels (Fig. 3C) in contrast to
sham mice (Fig. 3A). The NFD in the cornea of STZ-diabetic
mice significantly decreased at weeks 5, 7, and 9, in contrast to
constant levels in sham mice (Fig. 2E). Regression analysis of
STZ-diabetic mice showed a significant negative correlation
between DCD and NFD (Fig. 4C) but not in sham mice (Fig.
4A). The GLM repeated measures ANOVA revealed that for BG
and NFD, the time effect and the interaction between time and
treatment were significant (P < 0.05). For DCD, the time effect
was also significant (P < 0.05).
mice (23 6 5 / mm2) (Fig. 2D). The values for DCD in ob/ob
mice showed a significant negative correlation with BG levels
(Fig. 3D) in contrast to ob/þ mice (Fig. 3B). Nerve fiber density
in ob/ob mice was lower than in control mice (Fig. 2F).
Regression analysis on ob/ob and ob/þ mice showed no
correlation between DCD and NFD (Figs. 4D, 4B). The GLM
repeated measures ANOVA revealed that for NFD the time
effect was significant (P < 0.05).
Corneal Whole-Mount Staining in Mice

CCM-Based Analysis of the Cornea in ob/ob Mice
Both ob/þ and ob/ob mice exhibited different levels of all
measured parameters during the entire period when compared
to BALB/c mice. Ob/ob mice showed a nearly constant high
level of DCD (65 6 6 / mm2) (Fig. 2D), which was statistically
different over the entire observation period in contrast to ob/þ
Whole-mount staining procedure enables an accurate presen-
tation of different structures in the cornea. CD11c(þ) and MHC
class II(þ) DCs reside in the basal layer of the corneal
epithelium and represent mature cells. Their processes
originating from cell bodies follow different directions leading
to the surface of the cornea (Figs. 5A, 5B). The SNP is located
between the epithelium and the anterior stroma, and class III b-

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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3611

FIGURE 9. In vivo CCM and confocal imaging of corneal whole mounts in normoglycemic B6.VLepob/þ mice at week 9. (A) CCM of DCs (arrows)
and nerve fibers. Scale bar: 50 lm. (B–E) Confocal imaging of corneal whole mounts with three CD11c(þ) ([B, C], green) and MHC class II(þ) ([B,
D], red) DCs and class III b-tubulin(þ) (TUJ1) nerve fibers ([B, E], yellow). DAPI (blue) as nuclear staining. Scale bar: 30 lm.

tubulin(þ) single nerve fibers sprout within the superficial
epithelial layers to the surface of the cornea (Fig. 5C).
Corneal Whole-Mount Staining in Both DM Models
Control BALB/c and ob/þ mice at week 9 showed a gradient of
corneal DCD with a decrease from the periphery toward the
center (Figs. 6A, 6C). Streptozotocin-diabetic and ob/ob mice
at week 9 also presented a gradient of DCD from the periphery
to the center, however with much higher numbers of mature
DCs (Figs. 6B, 6D).
All corneal whole mounts of each treatment group
displayed DCs, which are positive for both CD11c and MHC
class II. Control BALB/c mice at week 9 showed many class III
b-tubulin(þ) nerve fibers forming the typical parallel structure
of the SNP (Figs. 7B, 7E) and sparsely dispersed CD11c(þ) and
MHC class II(þ) DCs (Figs. 7B–D). In agreement with the
quantitative analysis of CCM images, corneal whole mounts of
STZ-diabetic mice at week 9 revealed a higher density of DCs
and a lower density of nerve fibers (Figs. 8B–E). In contrast to
ob/þ mice (Figs. 9B–D), ob/ob mice showed a much higher
level of DCD (Figs. 10B–D). Higher magnification of corneal
whole mounts, as exemplified in Figure 11, reveals that DCs
often colocalize with nerve fibers and seem to establish close
contacts.
DISCUSSION
Methodological Considerations
In contrast to invasive skin biopsy, CCM represents a
noninvasive method for repetitive in vivo investigations,
allowing immediate identification of morphologic characteris-
tics such as changes in corneal nerve fibers and DCs. For
evaluation of corneal neuropathy, the study focused on the SNP
and not on stromal nerve fibers. However, one cannot exclude
that possibility that in individual images, stromal nerve fibers

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 Corneal Changes in Diabetic Mice IOVS j June 2014 j Vol. 55 j No. 6 j 3612

FIGURE 10. In vivo CCM and confocal imaging of corneal whole mounts in hyperglycemic B6.VLepob/ob mice at week 9. (A) CCM of DCs (arrows)
and nerve fibers. Scale bar: 50 lm. (B–E) Confocal imaging of corneal whole mounts with several CD11c(þ) ([B, C], green) and MHC class II(þ) ([B,
D], red) DCs and class III b-tubulin(þ) (TUJ1) nerve fibers ([B, E], yellow). DAPI (blue) as nuclear staining. Scale bars: 30 lm.

were detected as well (Fig. 1B, yellow arrows). Nevertheless,
for offline quantification of the in vivo CCM images, only single
nerve fibers of the SNP were quantified. In general, the
visualization of whole nerve fiber bundles, such as those
present in the stromal level, does not allow identification of the
loss of single nerve fibers and thus does not allow the exact
quantification of a reduction of NFD. Analysis of single nerve
fibers of the SNP might have an additional advantage in that the
loss of an individual nerve fiber might precede whole-bundle
degeneration. Therefore, in contrast to stromal nerve fiber
bundles, alteration of the SNP might serve as an earlier
indicator for diabetic polyneuropathy. To ensure optimal
reproducibility of the distribution of subbasal nerve fibers
and DCs, nine images per cornea were quantified in this study.
The question whether diabetic neuropathy occurs from the
center or from the periphery cannot be answered by the
present study because the peripheral area was not investigated.
Nevertheless, we suppose that corneal neuropathy would start
from the periphery and not within the center of the cornea
due to the fact that corneal neuropathy, among others, might
be initiated by paracrine signals reaching the cornea via the
limbal vascular plexus located in the very periphery, forming
the border between the conjunctiva and the avascular cornea.
The in vitro analysis of corneal whole mounts allowed
discrimination of CD11c(þ) DCs and their immature or mature
status through the use of MHC class II, supporting further
interpretation of CCM images. With the concomitant use of the
neuronal class III b-tubulin(þ) antibody as pan-neuronal
marker, a comprehensive analysis of both DCs and nerves
under different BG levels was possible.
Induction of DM can be achieved through a single high-dose
or multiple low-dose injections of STZ. The application of high-
dose STZ leads to a severe necrosis of pancreatic b-cells, in
contrast to the multiple low-dose application of STZ that leads
to specific b-cell death. 23 BALB/c mice underwent an

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 Corneal Changes in Diabetic Mice
FIGURE 11. Higher magnification of a confocal image of a corneal
whole mount, displaying the close position between a mature DC and
nerve fibers of the SNP. CD11c(þ) (green), MHC class II(þ) (red) DC,
and class III b-tubulin(þ) (TUJ1) nerve fibers (yellow). DAPI (blue) as
nuclear staining. Scale bar: 20 lm.
application of STZ in multiple low doses over 5 days and as
lean hyperglycemic animals represent a model of DM I.24–26
Due to the leptin deficiency, B6.VLepob/ob mice display a
spontaneous DM II model exhibiting impaired glucose
tolerance, hyperglycemia, hyperinsulinemia, and obesity.27
This mouse strain is commonly used as a model for a
diabetes-like syndrome.25,27,28 It has to be noted that ob/ob
mice display only a transient hyperglycemia between weeks 8
and 12, compensated later by increasing insulin levels.29
Nerve Fibers of the SNP in the Diabetic Cornea
Several studies using CCM have reported changes in morphol-
ogy of corneal nerve fibers during the progress of DM and
described a decrease in NFD.9,10,30,31 The present study could
confirm these observations in DM I by the significant decrease
of NFD at weeks 5, 7, and 9 in STZ-treated mice. The NFD of
ob/ob mice remained approximately constant over time with a
lower level compared to that in ob/þ mice. The absence of
significant differences between the two groups relates to the
tendency toward higher NFD in ob/ob mice from week 5 on.
Notably, the higher NFD values inversely correlate with lower
BG values, whereas both parameters remain constant in ob/þ
mice. Another study reported the development of peripheral
neuropathy in ob/ob mice.18 However, that study used 4-week-
old animals that showed high BG (>13.8 mmol/L).18 In
contrast, our study used 8- to 12-week-old animals, which
had lower BG at later time points, most likely due to
hyperinsulinemia occurring at this age.
DCs in the Diabetic Cornea
In line with Knickelbein et al.,12 DCs were found beneath cells
of the basal epithelium and displayed several long processes
with direction toward the corneal surface. In addition, the
current data confirm those of Knickelbein et al.12 and Lee et
al.,32 in that the DCD revealed a decreasing gradient from the
periphery toward the center of the cornea under normal
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IOVS j June 2014 j Vol. 55 j No. 6 j 3613
conditions, but extend the current knowledge by demonstrat-
ing that diabetic mice have much higher DCD than found in
nondiabetic controls. To our knowledge this is the first report
that STZ-induced DM is associated with a constant increase of
DCs over 9 weeks, while ob/ob mice show a constantly high
level of DCD, most probably due to enhanced inflammatory
and altered immunologic status of these mice.33
The increase of DCs upon diabetes induction might be
interpreted as a cellular response to inflammation, as DM is
known to be associated with systemic inflammation.34,35 This
view is supported by the fact that inflammatory stimuli like
electric cautery to the ocular surface or application of
lipopolysaccharide and tumor necrosis factor-a induce corneal
DC infiltration and maturation32,36 and are closely linked to a
decrease of subbasal nerve fibers in patients with infectious
keratitis.14 Accordingly, Popper and coworkers (Popper M, et
al. IOVS 2005;46:ARVO E-Abstract 879) described diabetic
corneal neuropathy in association with an increase of very
highly reflective cells in patients with DM II. Tavakoli et al.13
also showed these features in the cornea of DM I and DM II
patients with an additional correlation to the loss of corneal
nerve fibers.
In addition to DCs, Langerhans cells (LCs), which similarly
function as professional APCs, and macrophages reside in the
cornea.36,37 Mayer et al.38 demonstrated that corneal Langerinþ
LCs coexpress MHC class II and CD11c characterizing the DC
lineage. Furthermore, these cells are exclusively located in the
corneal epithelium and display a decreasing gradient from the
periphery to the center.38 During inflammation, the cornea
shows an influx of LCs expressing costimulatory markers
similar to DCs,36 suggesting a likewise behavior of these cells
under diabetic conditions. Moreover, CD11c() and CD11b(þ)
macrophages could be identified in the cornea, mainly located
within the posterior stroma37,38 where they show an
association to nerve fiber bundles.39 This morphologic
proximity of the two cell types might also play a role in
corneal diabetic polyneuropathy and needs further investiga-
tion.
Hamrah et al.36 demonstrated that DCs in the uninflamed
epithelium of the corneal center are MHC class II negative.
Under inflammatory conditions, however, they described a
subset of DCs expressing MHC class II. This observation is
partly in contrast to our findings in corneal whole mounts,
demonstrating that all DCs, independently of diabetic or
nondiabetic conditions, were CD11c(þ) and MHC class II(þ).
This might indicate a permanently activated status of corneal
DCs, confirming them as highly sensitive cells against foreign
antigens.
Due to the significant negative correlation between DCD
and nerve fibers in STZ-induced DM, it might be concluded
that the increase of DCs could play an initial role in the
manifestation of diabetic corneal neuropathy. The close
position of DCs and single nerve fibers of the SNP suggests
communication between the two cell structures. In other
organ systems like the skin, a colocalization of LCs with
epidermal nerves was observed.40 Furthermore, in lung41 and
in the gut,42 an interaction between DCs and nerve fibers was
described. A morphologic association between neurites and
mast cells and the additional presence of vesicles at the points
of contact were also shown in a nerve–mast cell coculture.43
These findings support our assumption that a delicate
interaction between nerve fibers and DCs could also exist in
the diabetic cornea.
However, corneal nerve fiber loss and DC infiltration might
also be different phenomena that occur accidentally upon
hyperglycemia at the same time but independently of each
other. There could be confounding factors, for example,
vascularization that develops after denervation resulting in an
 Corneal Changes in Diabetic Mice
influx of DCs. While Ferrari et al.44 reported on corneal
neovascularization after denervation upon trigeminal stereo-
tactic electrolysis with a concomitant increase of CD45(þ)
inflammatory cells in the cornea,44 we could not observe
corneal neovascularization as a potential attractor of DC
infiltration. However, due to the ability of MHC class II(þ)
DCs to form membrane nanotubes, mounting a possible
transfer system of different molecules in healthy and inflamed
murine corneas,45,46 these cell processes might reach the
nerve fibers of the SNP and contribute to corneal polyneurop-
athy.
In summary, the current data underline the possible
association between an increase of DCs and a decrease of
nerve fibers in the cornea. As DM is an inflammatory disease,
interaction of the two cell types could lead to neurogenic
inflammation, resulting in the release of neuropeptides and
thus contributing to a bidirectional interaction47 of corneal
DCs and nerve fibers.
Acknowledgments
The authors thank Carl F. Marfurt, PhD, for his support in whole-
mount staining (Department of Anatomy and Cell Biology, Indiana
University School of Medicine-Northwest, Gary, Indiana), Heike
Weiss, PhD, for her helpful advice on confocal microscopy of
corneal whole mounts (Institute of Medical Biochemistry and
Molecular Biology, University of Rostock), and Günther Kundt,
PhD (Institute for Biostatistics and Informatics in Medicine and
Aging Research, University of Rostock), for his excellent help and
advice in the statistical revision of the data.
Supported by a grant from the Federal Ministry of Education and
Research (894193; REMEDIS [Höhere Lebensqualität durch neu-
artige Mikroimplantate]). The authors alone are responsible for the
content and writing of the paper.
Disclosure: K. Leppin, None; A.-K. Behrendt, None; M. Reich-
ard, None; O. Stachs, None; R.F. Guthoff, None; S. Baltrusch,
None; J.C. Eule, None; B. Vollmar, None
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