Protective Effect of D--Hydroxybutyrate on Corneal
Epithelia in Dry Eye Conditions through Suppression
of Apoptosis
Shigeru Nakamura,1 Michiko Shibuya,1 Yasukazu Saito,1 Hideo Nakashima,1
Fumio Saito,1 Akihiro Higuchi,2 and Kazuo Tsubota2
PURPOSE. To investigate the effect of D--hydroxybutyrate
(HBA) on ocular surface epithelial disorders induced by tear
fluid deficiency, the potency of HBA and serum, the efficacy of
which has been well documented in clinical application, were
compared.
METHODS. Rat corneal epithelial erosion was induced by expo-
sure of rat eyes to continuous low-humidity airflow, which
accelerated the tear evaporation. During desiccation, one eye
of each rat was treated with HBA (20, 40, or 80 mM) or rat
serum (5%, 20%, or 100%), and in the other eye a drop of
phosphate-buffered saline (PBS) was instilled as the control.
Histopathologic examination and quantification of the epithe-
lial defect area were performed. The apoptosis in the epithelia
was determined by chromatin condensation using the Hoechst
33342 fluorescein probe.
RESULTS. In PBS-treated eyes, thinning in the cell layer was seen
on the periphery of the initial wound after 6 hours, and it
progressed to defects after 12 hours. In the 80-mM HBA and
20% serum applications, the pathologic change in the epithelia
was moderate, and the structure was maintained in an almost
normal state in the 100% serum application. Significant de-
creases in the defect areas were observed in the 5%, 20%, and
100% serum and 40- and 80-mM HBA treatment groups com-
pared with the PBS-treated eyes (n 12). A significant sup-
pression of chromatin condensation was observed with HBA
and serum treatment.
CONCLUSIONS. These results suggest the potential clinical appli-
cation of HBA for ocular surface epithelial disorders to main-
tain epithelial cell viability in patients with dry eye. (Invest
Ophthalmol Vis Sci. 2003;44:4682 4688) DOI:10.1167/
iovs.03-0198
T o date, a variety of clinical approaches have been used to
cure abnormalities in tear fluid status in dry eye conditions.
For improvement of the decreased volume and instability of
aqueous fluid on the ocular surface, frequent application of
preservative-free artificial tears or punctal occlusion has been
performed in conventional management. In addition, tear re-
placement therapy with autologous serum has been attempted
to correct the insufficient composition of tear fluid along with
From 1Ophtecs Corporation, Hyogo, Japan; and the 2Department
of Ophthalmology, Tokyo Dental College, Chiba, Japan.
Submitted for publication February 26, 2003; revised June 2, 2003;
accepted June 14, 2003.
Disclosure: S. Nakamura, Ophtecs (E); M. Shibuya, Ophtecs (E);
Y. Saito, Ophtecs (E); H. Nakashima, Ophtecs (E); F. Saito, Ophtecs
(E); A. Higuchi, None; K. Tsubota, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked advertise-
ment in accordance with 18 U.S.C. 1734 solely to indicate this fact.
Corresponding author: Shigeru Nakamura, Research Center, Oph-
tecs Corporation, 156-5, Kamiyoshidai, Toyooka, Hyogo 668-0831,
Japan; shigeru@mxa.nkansai.ne.jp.
4682
aqueous fluid, on the basis that the growth factor, cytokine,
vitamin, and nutrition content in serum resembles aqueous
tears.1
Fox et al.2 found that the use of a topical application of
diluted autologous serum improved symptoms in patients with
keratoconjunctivitis sicca and reduced the resistance to ther-
apy with commercially available artificial tears. Autologous
serum application has been reported to be effective in the
treatment of severe dry eye states associated with ocular pem-
phigoid, Stevens-Johnson syndrome, and chronic graft-versus-
host disease,3,4 and in persistent epithelial defect.5,6 Recently,
investigators in a placebo-controlled study conducted in pa-
tients with bilateral severe dry eye reported a trend toward an
improvement in symptoms, including cellular changes.7 These
successful clinical results showed practically that tear replace-
ment therapy using autologous serum is an effective clinical
management for dry eye and suggest that the key concept in
effective ophthalmic formulations is related to providing a
proper environment for the ocular surface cells.1,8
HBA is produced primarily in the liver by the degradation of
a long fatty acid and exists abundantly in the plasma and
peripheral tissues. The levels in human plasma and tissues are
maintained below 0.1 mM in the normal state.9 The role of HBA
as an oxidative fuel and lipogenic precursor has been well
recognized for some time.10 Because HBA has good penetra-
tion and rapidly diffuses in the peripheral tissues, the thera-
peutic benefits of exogenously applied HBA have been docu-
mented under stressful conditions, such as hemorrhagic
shock,11,12 extensive burns,13 and cerebral hypoxia, anoxia,
and ischemia.14 In these states, HBA ameliorates tissue damage,
protein catabolism, and metabolic dysfunction. Furthermore,
HBA has been shown to decrease cell death in human neuronal
cell cultures in Alzheimers and Parkinsons disease models.15
This evidence shows the possible therapeutic efficacy of HBA
in a variety of stress-induced conditions.16
However, the effects of HBA on ocular surface epithelial
disorders have not been investigated. In the present study, we
used a rat model of dry eye to determine whether topically
applied HBA could heal corneal epithelial erosion caused by
ocular surface desiccation. In addition, to assess the therapeu-
tic benefit of HBA in ophthalmic formulations, we compared
the potency of HBA with serum, the efficacy of which has been
well documented in clinical application. We found that HBA, as
well as serum, ameliorates the appearance of corneal epithelial
erosion through suppression of apoptosis.
MATERIALS AND METHODS
Animals
Male 8-week-old Sprague-Dawley rats (n 6 12 in each experiment)
were purchased from Tokyo Laboratory Animal Science Co., Ltd. (To-
kyo, Japan). They were quarantined and acclimatized before the ex-
periments for a 1-week period under standard conditions: room tem-
perature 23 2C, relative humidity 60% 10%, an alternating
Investigative Ophthalmology & Visual Science, November 2003, Vol. 44, No. 11
Copyright Association for Research in Vision and Ophthalmology
IOVS, November 2003, Vol. 44, No. 11
12-hour light dark cycle (8 AM to 8 PM), with water and food available
ad libitum. All procedures were performed according to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Ophthalmic Solutions
Sodium HBA (Fig. 1) was synthesized by Ophtecs Corporation, Ltd.
(Hyogo, Japan), and was found to be more than 99% pure when tested
by HPLC. Rat serum was the pooled serum, collected in a sterile
manner from six rats. The endogenous concentration of HBA was
determined as 37 M by using HBA dehydrogenase coupled to a
bioluminescence assay.17 The serum was kept at 4C and shielded from
light until use. Ophthalmic solutions of 20, 40, and 80 mM of HBA
(wt/vol) and 5%, 20%, and 100% of serum (vol/vol) were formulated in
PBS, and osmolarities were adjusted with NaCl from 290 to 300 mOsm.
Rat Dry Eye Model
Corneal Epithelial Scraping. The rats were anesthetized by
intramuscular injection of an anesthesia cocktail containing ketamine
and xylazine. After deep anesthesia was achieved, the central region of
the corneal epithelium (0.4 mm2) was scraped mechanically with an
ophthalmic surgical blade.
Desiccation Procedure. Just after scraping, the rats were
placed in a desiccation room, with room temperature of 23 2C,
relative humidity of 28% 2%, and constant air flow (2 4 m/sec), and
maintained for 12 hours.
Topical Eye Drop Application. During desiccation, one eye
of each rat was treated with HBA or serum eye drops, and the other eye
was given a drop of PBS as the control. Ten microliters of eye drops
was administered every hour for 12 hours.
Quantitative Determination of Corneal Epithelial Ero-
sion. The damaged areas were photographed immediately after and 2,
6, and 12 hours after scraping, by applying a fluorescein solution under
cobalt blue light. The stained area (epithelial erosion) was digitized
with an optical scanner and quantified with image-analysis software
(NIH Image, ver. 1.53; available by ftp at zippy.nimh.nih.gov/ or at
http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, Na-
tional Institutes of Health, Bethesda, MD).
Histopathologic Study. The rats were killed with an overdose
of a mixture of ketamine and xylazine and the eyeballs removed and
fixed in Davidsons solution (37.5% ethanol, 12.5% acetic acid, and 25%
formaldehyde [37% solution]). The corneal specimens were embedded
in paraffin, cross-sectioned, and stained with hematoxylin and eosin.
Chromatin Condensation Evaluation in Corneal Epi-
thelia. The level of apoptosis in the corneal epithelia was quantified
by modification of a previously described method.18 Whole corneal
epithelium was scraped with an ophthalmic surgical blade, placed in a
microtube, and washed twice with PBS. The cells were incubated in
the dark with 10 g/mL Hoechst 33342 (Molecular Probes, Inc.,
Eugene, OR) in PBS for 30 minutes (500 L/tube). The cells and
Hoechst solution were placed in a 48-well plate. The plates were read
at an excitation of 360 nm and emission of 450 nm (Cytofluor 4000;
PerSeptive Biosystems, Inc., Framingham, MA). The cells were col-
lected again and extracted in lysis buffer (50 mM HEPES, 1 mM
dithiothreitol [DTT], 0.1 mM EDTA, 0.1% 3-([3-cholamidopropyl]di-
methylammonio-2-hydroxy-1-propanesulfonate [CHAPS], and 0.1 mM
phenylmethylsulfonyl fluoride [PMSF] [pH 7.4]) and the lysates were
then centrifuged at 15,000g for 10 minutes. The supernatants were
extracted and the protein concentrations measured by a micro bicin-
FIGURE 1. Chemical structure of sodium D--hydroxybutyrate.
Effect of D--Hydroxybutyrate on Corneal Epithelia 4683
choninic acid (BCA) method, using the provided protein assay reagent
(Micro BCA Protein Assay Reagent; Pierce Biotechnology, Rockford,
IL), according to the manufacturers instructions. The fluorescence
intensity of Hoechst 33342 per protein was calculated and expressed
as the percentage of nontreated eyes.
In experiments to distinguish the apoptotic and necrotic cell pop-
ulations, the cells after Hoechst staining were again placed in a micro-
tube, washed twice with PBS, and incubated with 0.005% neutral red
(Nacalai Tesque, Inc., Kyoto, Japan) in Medium 199 (Nissui Pharma-
ceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine
serum (Invitrogen-Gibco, Grand Island, NY) for 3 hours (500 L/tube).
The dye solution was carefully discarded, and the cells were washed
twice in PBS. To elute the dye, the stained cells were treated with a
solution of 1% acetic acid and 50% ethanol (200 L/tube). After the
neutral red crystals were thoroughly mixed to dissolve them entirely,
the neutral red solution was placed in a 48-well plate and rapidly read
at an excitation of 535 nm and emission of 600 nm. The index of
Hoechst 33342/neutral red (Ho/NR) was calculated and compared
with the ratio in nontreated eyes.
Tear Fluid Volume Measurement. We attempted a modified
phenol red thread test19 on the rats eyes to measure the residue tear
fluid volume. In brief, a finely cut Schirmer tear test strip (1 15 mm;
Alcon Laboratories, Inc., Fort Worth, TX) was placed on the temporal
side of the lower eyelid margin for 10 seconds. The length of the
moistened area from the edge was measured to an accuracy of 0.5 mm.
Statistical Analysis
Analysis of the significance of differences was performed on computer
by the paired or unpaired Students t-test between two groups (Stat-
View IV; Abacus Concepts, Berkeley, CA). Differences were accepted
as significant at P 0.05.
RESULTS
Tear Fluid Volume Measurement
First, to confirm that our desiccation condition of exposure to
a continuous 28% 2% humidity airflow at 2 to 4 m/sec
induces a deficiency of tear fluid on the ocular surface, we
attempted a modified phenol red thread test19 on the rats eyes
to measure the residue tear fluid volume. During the ocular
surface desiccation procedure, a significant decrease in tear
fluid was observed compared with the standard condition (Fig.
2). These results indicate that tear fluid deficiency was induced
by exposure to continuous low-humidity airflow due to accel-
eration of tear evaporation from the ocular surface. Thus, we
used this procedure to induce the ocular surface disorder.
Induction of Corneal Erosion
After maintaining the rats in a desiccated condition for at least
5 days, we observed corneal epithelial disorder, superficial
punctate keratopathy, and erosion in most of the eyes. How-
ever, variations in the frequency of appearance, degree of
disorder and time of appearance were noteworthy in this
condition (Nakamura S, unpublished data, 2000). Thus, to
enable us to assess the effect of pharmacological treatments
accurately, we attempted to induce uniform corneal epithelial
disorder by scraping a small area of the corneal epithelium as
a trigger.
After 2 hours, in both the standard and desiccated condi-
tions, the epithelial defect remained in the same area as at the
initial level. In the normal condition, the corneal epithelial
defect started to decrease after 6 hours (Fig. 3A) and had
almost disappeared within 12 hours (Fig. 3C). During the
desiccation of the ocular surface, aggravation of the defective
area was clearly observed. Six hours after the desiccation was
ended, a lightly fluorescein-stained area appeared surrounding
4684 Nakamura et al. IOVS, November 2003, Vol. 44, No. 11

role in the degeneration of the corneal epithelium that leads to

erosion.

Effect of HBA and Serum on the Occurrence of

Corneal Epithelial Erosion
Based on the tear replacement therapy using autologous serum
for severe dry eye,1 we used rat serum as a positive control in

FIGURE 2. Effect of ocular surface desiccation on tear volume. The

modified phenol red thread test was performed 2, 6, or 12 hours after
the standard condition (60% humidity) or ocular surface desiccation
(30% humidity with airflow). Data are the mean SD of 10 measure-
ments. *P 0.05 versus standard condition (unpaired Students t-test).

the initial epithelial defect (Fig. 3B) and progressed to a dis-

tinct, extensively stained spot after 12 hours (Fig. 3D). Quan-
titative analysis of the fluorescein-stained area showed a signif-
icant increase in stained area after 6 and 12 hours of treatment
compared with that before treatment (Fig. 3E). In the his-
topathologic examination, a slight corneal epithelial migration
to the wounded area was observed after 6 hours, and the
defective area was completely covered with multilayer, regen-
erated epithelium after 12 hours in the standard condition
(Figs. 4A, 4C). However, after 6 hours of desiccation, thinning
of the cell layer accompanied by extensive exfoliation of the
corneal epithelia was seen on the periphery of the initial
wound (Fig. 4B), and then these degenerations progressed to
defects after 12 hours (Fig. 4D). These observations suggest
that desiccation of the ocular surface initially induces exfolia-
tion of the corneal epithelium and results in erosion when
these epithelia have entirely detached.

Apoptosis in Corneal Epithelium

Induction of apoptosis on the ocular surface in dry eye has
been documented in the conjunctiva20 and corneal epithe-
lium,21 although the mechanism involved is still unclear. To
confirm apoptosis in the desiccated corneal epithelium, we
used Hoechst 33342 staining to examined the state of chroma-
tin, one of the characteristic features of apoptosis, regardless of
the apoptotic pathways involved.22,23 In addition, to differen-
tiate apoptosis from necrosis, we used an index of chromatin
condensation (Ho; Hoechst 33342 fluorescence)/cell viability
(NR; membrane integrity detection by neutral red assay), as
previously described.18 We assessed the apoptosis after 6 FIGURE 3. Fluorescein staining of corneal epithelial erosion induced
hours of ocular surface desiccation on the cornea, because by ocular surface desiccation. The damaged areas were photographed
thinning of the corneal epithelia, an indication of erosion, was by applying fluorescein solution under cobalt blue light 2, 6, and 12
hours after scraping. Six hours of treatment under standard conditions
apparently observed. Significant increases in Hoechst 33342 (A) and with ocular surface desiccation (B), and 12 hours of treatment
fluorescence (Fig. 5A) and the Ho/NR ratio (Fig. 5B) were under standard conditions (C) and with ocular surface desiccation (D).
observed in the desiccation treatment in contrast to the stan- Quantitative analysis of the fluorescein-stained area (E). Values repre-
dard condition treatment. These results indicate that the pro- sent mean SD of eight measurements. ***P 0.005 versus standard
gression of apoptosis in the corneal epithelia plays a critical condition (unpaired Students t-test).
IOVS, November 2003, Vol. 44, No. 11 Effect of D--Hydroxybutyrate on Corneal Epithelia 4685

due to a protective potential against epithelial cell degenera-

tion.

Effect of HBA and Serum on Corneal

Epithelial Apoptosis
To investigate whether the effects of HBA and serum arises
from the suppression of apoptosis, we performed a Hoechst
33342 assay on 80 mM HBA and 100% serum applied to the
cornea after 6 hours of desiccation. Consistent with the former
results, significant decreases in apoptosis, fluorescence inten-
sity, and Ho/NR ratio were observed when 80 mM HBA and
100% serum were applied to the cornea in contrast to PBS
treatment. The effect of 100% serum was more apparent than
that of 80 mM HBA (Fig. 8).

FIGURE 4. Microphotograph of rat corneal epithelial erosion. Rats

eyes were fixed and the cross-sectioned corneas were stained with
hematoxylin and eosin after 6 and 12 hours of ocular surface desicca-
tion. After 6 hours of treatment under standard conditions (A) and with
ocular surface desiccation (B) and after 12 hours of treatment under
standard conditions (C) and with ocular surface desiccation (D). Mag-
nification, 100.

our rat dry eye model. To assess the effect of HBA and serum
on the appearance of corneal epithelial erosion, we selected a
time point after 12 hours of desiccation, because the appear-
ance of erosion was identical up to this point. As shown in
Figure 6, both HBA and serum dose-dependently decreased the
fluorescein-stained areas. Significant decreases in the fluores-
cein-stained areas were observed in all the serum-treated
groups (5%; P 0.05, 20%; P 0.01, 100%; P 0.005, Fig. 6A)
compared with the PBS group. In the HBA-treated group, a
significant decrease was observed at 40 and 80 mM HBA (40
mM; P 0.05, 80 mM; P 0.005 versus PBS, Fig. 6B). A
maximum effect was observed with 80 mM HBA (65% of PBS)
and 100% serum (49% of PBS).
Because corneal epithelial degeneration evidently occurs 6
hours after ocular surface desiccation treatment, we used this
time point for histopathologic examination. Representative
patterns of histopathologic changes in the corneal epithelium
after 6 hours are shown in Figure 7. The changes in the features
of the epithelial cell layer were consistent with the quantitative
analysis data after 12 hours of treatment. In PBS, thinning in the
cell layer accompanied by detachment of the corneal epithelia
was observed surrounding the periphery of the wound (Fig. FIGURE 5. Involvement of apoptosis in cornea epithelial erosion dur-
7A). The thinning of the cell layer was moderate in the 20% ing desiccation of the ocular surface. Rats were treated under standard
conditions or ocular surface desiccation for 6 hours. Apoptosis was
serum (Fig. 7B) and 80 mM HBA (Fig. 7C) applications, and the determined by a Hoechst 33342 fluorescein probe. The Ho/NR index
structure of the epithelial cell layer was maintained in an was calculated to differentiate apoptosis and necrosis. Fluorescence
almost normal state in the 100% serum application (Fig. 7D). intensity (A), Ho/NR ratio (B). Data are the mean SD of 10 corneas.
These results indicate that the suppressive effects of HBA and *P 0.05, ***P 0.005 versus standard conditions (unpaired Students
serum on the appearance of corneal epithelial erosion were t-test).
4686 Nakamura et al.
FIGURE 6. Effect of topically applied HBA and rat serum on corneal
epithelial erosion induced by ocular surface desiccation. Eye drops
(PBS and HBA or rat serum) were applied to both eyes. Drops were
applied at 1-hour intervals during ocular surface desiccation. After 12
hours, quantitative analysis of the fluorescein-stained area of the cor-
neal epithelium was performed on each eye. Serum application (A),
HBA application (B). Data represent the mean SD of 12 experiments.
*P 0.05, **P 0.01, ***P 0.005, versus PBS treatment (paired
Students t-test).
DISCUSSION
In this study, we used a novel rat dry eye model to evaluate the
effect of HBA on ocular surface epithelial cell disorder. We
demonstrated that HBA, as well as serum, ameliorates the
appearance of corneal epithelial erosion due to ocular surface
desiccation accompanied by suppression of apoptosis.
In our rat model, we found that a mechanically created
partial corneal epithelial defect was aggravated to extensive
epithelial erosion during exposure to continuous low-humidity
airflow on rat eyes, which accelerated the tear evaporation rate
and enhanced ocular surface desiccation. Several animal mod-
els have been reported to mimic the clinical features caused by
tear fluid deficiency on the ocular surface. Increased kerato-
conjunctivitis sicca, rose bengal staining on the conjunctiva,
and/or increased corneal epithelial permeability have been
established in monkeys,24 dogs,2528 rabbits,29,30 rats,31 and
IOVS, November 2003, Vol. 44, No. 11
mice.32 However, to our knowledge, tear fluid depletion-in-
duced corneal epithelial erosion, a serious pattern in ocular
surface epithelial disorder, has not been reported. Pharmaco-
logic blockade of the cholinergic muscarinic receptors or sur-
gical excision of the lacrimal glands to decrease tear secretion
is known to be a useful technique to desiccate the ocular
surface in animal models.24,30 However, these techniques have
some disadvantages in assessing the critical function of ocular
surface disorder, because it is difficult to exclude the complex
influence of surgical insult or pharmacologic alteration after
neural stimulation in the lacrimal gland. In contrast to these
established models, however, the treatment technique we
used to desiccate the ocular surfacenamely, exposing rat
eyes to continuous low-humidity air flowis minimally inva-
sive and provokes no pharmacological alteration in neural
stimulation of the lacrimal gland. Moreover, our results of
serum application in our rat model are in agreement with the
successful clinical results of applying autologous serum to the
ocular surface of individuals afflicted with dry eye.1,2,7 These
findings, taken together, show that this model could be appli-
cable in the investigation of useful drugs for dry eye.
Consistent with previous reports,20,21 our study provides
evidence of a strong relationship between the pathogenesis of
corneal epithelial disorder and induction of apoptosis on the
ocular surface of dry eye. However, the mechanism involved in
drastically aggravated corneal epithelial erosion accompanied
by an increase in apoptotic epithelial cell death was not clear
from this experiment. It is well accepted that the obvious role
of tear fluid is to provide essential factors, such as growth
factors, vitamins, and nutrition to maintain homeostasis of the
ocular surface epithelium or to promote differentiation, migra-
tion, and proliferation during the epithelial wound-healing pro-
cess.33,34 The lack of a tear fluid component may compromise
ocular surface integrity or epithelial reconstruction. During the
corneal epithelial wound-healing process, cytokines triggering
epithelial cell apoptosis by IL-1, IL-6, and TNF have been
expressed on epithelial cells or tear fluid.3538 In our model, it
is presumed that cytokines may be present on the ocular
surface at increased levels, because the tear fluid condensed
due to accelerated tear evaporation rates. It could be specu-
lated, therefore, that a synergic effect between enhanced apo-
ptotic signals and insufficient supplementation of essential
factors to the corneal epithelia may have been involved in the
pathogenesis of corneal epithelial erosion.
FIGURE 7. Micrographs of a representative pattern of rat corneal epi-
thelial erosion treated topically with HBA or serum. After 6 hours of
ocular surface desiccation, rats eyes were fixed, and the cross-sec-
tioned corneas were stained with hematoxylin and eosin. PBS (A), 20%
serum (B), 80 mM HBA (C), 100% serum (D). Magnification, 100.
IOVS, November 2003, Vol. 44, No. 11
FIGURE 8. Suppressive effect of HBA and serum on apoptosis of cor-
neal epithelial cells during desiccation of the ocular surface. The
treatment protocol is described in the legend to Figure 7. After 6 hours,
the eyes were removed and chromatin condensation in the cornea
epithelium was determined by Hoechst 33342 fluorescein probe. The
Ho/NR index was calculated, to differentiate apoptosis from necrosis.
Fluorescence intensity (A), Ho/NR ratio (B). Data are the mean SD
of six corneas. *P 0.05, **P 0.01 versus PBS treatment (paired
Students t-test).
We demonstrated that both serum and HBA significantly
prevented the appearance of corneal epithelial cell degenera-
tion and suppressed apoptotic cell death due to ocular surface
desiccation. Based on the data that the endogenous concentra-
tion of HBA in rat serum used in this study was negligible
compared with the effective dosage, the protective effect of
serum may be mediated by other serum components that are
essential in maintaining ocular surface homeostasis.1 Recently,
Higuchi et al. established a number of serum components that
contribute to the suppression of cultured human conjunctival
Effect of D--Hydroxybutyrate on Corneal Epithelia 4687
cell apoptosis induced by serum deprivation (Higuchi A, et al.
IOVS 2002;43:ARVO E-Abstract 3171). They reported that
apoptosis was suppressed by several components of serum,
such as growth factors, vitamins, and serum proteins, and that
these antiapoptotic effects act synergically. There may be some
overlapping pivotal event between HBA and serum compo-
nents responsible for the improvement of ocular surface epi-
thelial disorder, although the exact mechanism involved re-
mains unclear.
A recent study reported that HBA causes a significant re-
duction in the mitochondrial nicotinamide adenine dinucle-
otide (NAD) couple, which was reflected by an increase in
mitochondrial membrane potential, in the working perfused
rat heart.39 In another study, HBA showed a neuroprotective
effect against mitochondrial dysfunction induced by mitochon-
dria reduced nicotinamide adenine dinucleotide dehydroge-
nase (NADH) multienzyme complex inhibitor, which de-
creases cell respiration and mitochondrial proton pumping and
increases free radical production, in cultured mesencephalic
neurons.15, We suspect that a possible mechanism involved in
the antiapoptotic effect of HBA is the suppression of multiple
types of apoptosis signals induced by ocular surface desicca-
tion, by preventing proapoptotic factor release by protecting
the mitochondrial membrane potential. However, further
study is needed to clarify the critical events involved in the
suppressive effects of HBA on apoptosis.
In this study, topically applied HBA showed no adverse
effects on the rat ocular surface. In addition, an intravenous
dose toxicity test repeated at 4 weeks in dogs and rats showed
no observable adverse effects on both systemic and ocular
tissues at a dosage of a 400 mg (3.17 mM)/kg per day (data not
shown). Taking these data together, we have shown a protec-
tive effect of HBA against corneal epithelia disorder in a tear-
deficiency model, suggesting the potential usefulness of HBA in
the clinical treatment of ocular surface epithelial disorders in
patients with dry eye. However, we have not established the
effect on mild types of dry eye, such as keratoconjunctivitis
sicca, superficial punctate keratopathy, and rose bengal stain-
ing on the conjunctiva. Further investigations are now under
way to clarify the effects on chronic symptoms of dry eye and
the critical mechanisms involved.
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