Hypercalcemia Leads to Delayed Corneal Wound Healing in Ovariectomized Rats

Abstract

Hypercalcemia is often observed in postmenopausal women as well as in patients with primary hyperparathyroidism or malignant tumors. In this study, we investigated the relationship between calcium ion (Ca²⁺) levels in lacrimal fluid and the rate of corneal wound healing in hypercalcemia using ovariectomized (OVX) rat debrided corneal epithelium. We also determined the effects of Ca²⁺ levels on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T). The calcium content in bones of OVX rats decreased after ovariectomy. Moreover, the Ca²⁺ content in the blood of OVX rats was increased 1 month after ovariectomy, and decreased. The Ca²⁺ content in the lacrimal fluid of OVX rats was also increased after ovariectomy, and then decreased similarly as in blood. Corneal wound healing in OVX rats was delayed in comparison with Sham rats (control rats), and a close relationship was observed between the Ca²⁺ levels in lacrimal fluid and the rate of corneal wound healing in Sham and OVX rats (y=−0.7863x+8.785, R=0.78, n=25). In addition, an enhancement in Ca²⁺ levels caused a decrease in the viability in HCE-T cells. It is possible that enhanced Ca²⁺ levels in lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in a delay in corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage of patients with hypercalcemia.

Calcium (Ca) is the most abundant mineral in the human body¹⁾ with about 99% of the body’s entire supply residing in bones, and the remaining 1% in blood, extracellular fluids, and other tissues.^1,2) A reduction in serum calcium ion (Ca²⁺) concentration stimulates specific hormonal balancing mechanisms that influence Ca transport in the intestine, bone and kidney to maintain homeostasis.³⁾ Estrogen is a hormone that participates in the regulation of Ca contents in the body, and postmenopausal women experience a drop in estrogen secretion. Bone resorption is regulated by estrogen, and a decrease in the estrogen level leads to a decrease vitamin D. Since vitamin D enhances Ca²⁺ absorption from the small intestine,^4–7) postmenopausal women are at greater risk of osteoporosis, hypercalcemia or hypocalcemia due to a chronic reduction in their circulating estrogen level.^4–7) Osteoporosis is a condition of bone fragility characterized by decreased bone mass and a microarchitectural deterioration of bone tissues with a subsequent increase in the risk of fractures.⁸⁾ Osteoporosis develops due to increased bone resorption and low calcium ion (Ca²⁺) absorption. In addition, the decrease in estrogen secretion following menopause is related to the prevalence of several disorders within the area innervated by the trigeminal nerve.^9,10) The ocular surface is an integrated unit comprising the corneal and conjunctival epithelia, meibomian glands, main and accessory lachrymal glands, and trigeminal neurons. It has been reported that a decrease in estrogen level results in dry eye in animal models.^9,10) Hypercalcemia is also observed in the some postmenopausal women due to the reduced function of Ca²⁺ regulation.⁸⁾ Hypercalcemia may lead to increased Ca²⁺ contents in the lacrimal fluid, and affect the cornea. Despite these findings, there are, to our knowledge, no reports of the effect of Ca²⁺ levels in the lacrimal fluid on the corneas of postmenopausal women.

In order to elucidate the precise relationship between hypercalcemia and the cornea, the selection of the experimental animal is very important. The ovariectomized (OVX) rat is an accepted female animal model to mimic postmenopausal osteoporosis.¹¹⁾ The OVX procedure promotes bone loss. Following bilateral ovariectomy, serum Ca²⁺ levels rise and bone mass decreases.^11,12) In addition, OVX rats show a decrease in the secretion of lacrimal fluid; the pathogenetic role for estrogen in dry eye involves the upregulation of matrix metalloproteinase 2 (MMP-2) expression.⁹⁾ Some changes in the biological characteristics of OVX rats may correspond to those of human osteoporosis. In this study, we investigated the effect of Ca²⁺ levels in lacrimal fluid on the rate of corneal wound healing in OVX rat debrided corneal epithelium. We also determined the effects of Ca²⁺ content on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T).

MATERIALS AND METHODS

Animals and Reagents

Female Wistar rats, 5 weeks of age, were ovariectomized (OVX rat) or sham operated (Sham). The rats were housed under standard conditions (12 h/d fluorescent light (07:00–19:00), 25°C room temperature), and allowed free access to a commercial diet (CE-2, Clea Japan Inc., Tokyo, Japan) and purified water (PW). All procedures were performed in accordance with the Kinki University Faculty of Pharmacy Committee Guidelines for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. All other chemicals used were of the highest purity commercially available.

Measurement of Calcium Content

Femur, blood and lacrimal fluid from rats at 10, 14 and 18 weeks of age (1, 2, 3 months after ovariectomy) were removed. The femur was boiled for 2 h, and then dried at room temperature. After that, it was calcined in a muffle furnace KDF S-80 (Sansyo Co., Ltd., Osaka, Japan) to 550°C for 48 h. Calcined femur was dissolved in 1% nitric acid, and the sample solution was filtered through a 0.45 µm membrane filter. The Ca²⁺ concentration of the calcined femur was measured using an inductively coupled plasma-atomic emission spectrometer ICPS-7500 (ICP-AES, Shimadzu Corp., Kyoto, Japan). The blood and lacrimal fluid were centrifuged at 10000 rpm for 30 min at 4°C, and the Ca²⁺ concentrations were determined. The Ca²⁺ concentrations in the blood and lacrimal fluid were determined by the methyl xylenol blue colorimetric method using a Ca test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and CL-770 (Shimadzu Corp.).

Image Analysis of Corneal Wound Healing in Rats

Rats were anesthetized with isoflurane, and a patch of corneal epithelium was removed with a BD Micro-Sharp™ (blade 3.5 mm, 30°, Becton Dickinson, Fukushima, Japan) as described previously.^13–16) The areas of debrided corneal epithelium in Sham and OVX rats 1-3 month after operation (10-18 weeks of age) were as follows: 1-month-old Sham rat, 12.45±0.43 mm²; 2-month-old Sham rat, 12.37±0.62 mm²; 3-month-old Sham rat, 11.99±0.67 mm²; 1-month-old OVX rat, 12.03±0.42 mm²; 2-month-old OVX rat, 12.07±0.61 mm²; 3-month-old OVX rat, 12.34±0.46 mm² (mean±standard error (S.E.) for 5 independent rat corneas). The debrided corneal epithelium was dyed by the instillation of a solution containing 1% fluorescein (Alcon Japan, Tokyo, Japan) and 0.4% Benoxil (Santen Pharmaceutical Co., Ltd., Osaka, Japan). Changes in the corneal wounds were monitored under a TRC-50X fundus camera (Topcon, Tokyo, Japan) equipped with a digital camera (EOS Kiss Digital N, Canon Inc., Tokyo, Japan), and the images obtained were analyzed with Image J. The amounts of corneal wound healing (%) were calculated according to Eq. 1^13–16):   

(1)

The rates of corneal wound healing, represented by the corneal wound healing rate constant (k_H, h⁻¹) over the period 0-36 h after corneal epithelial abrasion were calculated according to Eq. 2^13–16):   

(2)

where t is time (0–36 h) after corneal abrasion, and H_∞ and H_t are the percentages of corneal wound healing (%) at time ∞ and t, respectively.

Cell Culture and Treatment

An immortalized human corneal epithelial cell line (HCE-T) was used, and cultured as described previously.^17,18) In this study, Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12, normal medium, Ca²⁺ content 4.2 mg/dL, Gibco, Tokyo, Japan) and DMEM/F12 without Ca²⁺ (experimental medium) were used. The Ca²⁺ contents in the experimental medium were achieved by the addition of CaCl₂ (Ca²⁺ content, 4–12 mg/dL), and the Ca²⁺ contents in the medium were detected following the concentration in blood and lacrimal fluid (Figs. 2B, C). HCE-T cells were cultured in these media containing 5% (v/v) heat-inactivated fetal bovine serum (FBS, Ca²⁺ content, 0.8 mg/dL, Gibco) for 2 weeks prior to the start of the cell adhesion and proliferation experiments.

Measurement of Cell Adhesion

Cell adhesion of HCE-T cells was calculated by TetraColor One according to the manufacturer’s instructions. HCE-T cells (1×10⁴ cells) were seeded in 96-well microplates (IWAKI, Chiba, Japan), and incubated under humidified air containing 5% CO₂ at 37°C for 12 h. After that, TetraColor One (Seikagaku Co., Tokyo, Japan) was added, and the absorbance (Abs) at 490 nm was measured. Cell adhesion (%) is represented as the ratio of the Abs of control HCE-T cells cultured in normal medium (DMEM/F12).¹⁸⁾

Measurement of Cell Proliferation

HCE-T cell cultures one day after seeding (1×10⁴ cells) in 96-well microplates were incubated under humidified air containing 5% CO₂ at 37°C for 24 h, after which TetraColor One (Seikagaku Co.) was added, and the Abs at 490 nm was measured. The proliferation was not observed in the culture using DMEM/F12 medium without FBS for the 0–24 h incubation. Therefore, the Abs data reflecting the cell proliferation were evaluated according to Eq. 3:   

(3)

The Abs_{with FBS} and Abs_{without FBS} were the Abs values of HCE-T cells incubated in medium with and without FBS, respectively. Cell proliferation (%) is defined as the ratio of the Abs compared with control HCE-T cells cultured in normal medium (DMEM/F12).¹⁸⁾

Measurement of Cell Viability

HCE-T cell cultures one day after seeding (1×10⁴ cells) in 96-well microplates were incubated in various media without FBS under humidified air containing 5% CO₂ at 37°C for 24 h. cell viability was calculated using TetraColor One. Cell viability (%) is defined as the ratio compared with the Abs of control HCE-T cells cultured in normal medium (DMEM/F12).¹⁹⁾

Statistical Analysis

All values are presented as mean±standard deviation (S.D.) or S.E. Unpaired Student’s t-test was used to evaluate statistical differences, and multiple groups were evaluated by one-way ANOVA followed by Dunnett’s multiple comparison. The p values less than 0.05 were considered significant.

RESULTS

Body Weight and Calcium Contents in Bone, Blood and Lacrimal Fluid of OVX Rats

Figure 1 shows the changes in food intake (A), water consumption (B) and body weight (C) of Sham and OVX rats from 1–3 months after ovariectomy (10–18 weeks of age). Although the food intake and water consumption of OVX rats were similar to those of Sham rats, the body weight of OVX rats was higher than that of Sham rats. Figure 2 shows the changes in the calcium contents of bone (A), blood (B) and lacrimal fluid (C) of Sham and OVX rats. The Ca²⁺ content in the bones of OVX rats decreased following ovariectomy with the level 3 month after ovariectomy approximately 65% that of Sham rats. On the other hand, the Ca²⁺ contents in blood and lacrimal fluid of OVX rats peaked 1 month after ovariectomy, and subsequently decreased. The Ca²⁺ content in the blood of OVX rats 1–3 months after ovariectomy were significantly higher than in Sham rats, and the Ca²⁺ contents in lacrimal fluid of OVX rats 1 and 2 months after ovariectomy were also significantly higher than in Sham rats.

Fig. 1. Food Intake (A), Water Consumption (B) and Body Weight (C) of Sham and OVX Rats 1–3 Months after Surgery

Open columns, Sham rats; closed columns, OVX rats. The data are presented as the means±S.E. of 5 independent rats. * p<0.05 vs. Sham rats for each category.

Fig. 2. Calcium Contents in the Bone (A), Blood (B) and Lacrimal Fluid (C) of Sham and OVX Rats 1–3 Months after Surgery

The Ca content in the bone (femur) was measured using the ICP-AES. The Ca²⁺ concentrations in the blood and lacrimal fluid were determined using a Ca test kit. Open columns, Sham rats; closed columns, OVX rats. The data are presented as the means±S.E. of 5 independent rats. * p<0.05 vs. Sham rats for each category.

Changes in Corneal Wound Healing in OVX Rats

Figures 3, 4 show images following corneal epithelial abrasion (Fig. 3) as documented by a TRC-50X equipped with a digital camera, and the rates of corneal wound healing (Fig. 4) of Sham and OVX rat eyes 1–3 months after surgery. The corneal wounds of Sham rats 1–3 months after surgery were approximately 50% healed at 12 h, approximately 83% healed at 24 h, and almost entirely healed 36 h after corneal epithelial abrasion. Although the corneal healing rates of OVX rats 3 months after ovariectomy were similar to those of Sham rats, the corneal healing rates 1 and 2 months after ovariectomy were significantly slower than in Sham rats, and rate constants (k_H) for OVX rats 1 and 2 months after ovariectomy 18.3% and 73.3% those of Sham rats, respectively (Table 1). In addition, a close relationship was observed between the Ca²⁺ content in lacrimal fluid and k_H in Sham and OVX rats (y=－0.7863x+8.785, R=0.78, n=25).

Fig. 3. Corneal Images of Sham and OVX Rats 1–3 Months after Surgery

The corneal epithelium was removed with a BD Micro-Sharp™, and the resulting corneal wounds were dyed with 1% fluorescein solution. The wounds were monitored using a TRC-50X equipped with a digital camera. The area inside the dashed lines shows the corneal wound.

Fig. 4. Corneal Wound Healing in Sham and OVX Rats 1 (A), 2 (B), and 3 (C) Months after Surgery

The corneal epithelium was removed with a BD Micro-Sharp™, and the resulting corneal wounds were dyed with 1% fluorescein solution. The wounds were monitored using a TRC-50X equipped with a digital camera, and analyzed with Image J software. The degree of corneal wound healing (%) was calculated according to Eq. 1 in Materials and Methods. Open columns, Sham rats; closed columns, OVX rats. The data are presented as the means±S.E. of 5 independent rats. * p<0.05 vs. Sham rats for each category.

Table 1. Rates of Corneal Wound Healing in Sham and OVX Rats 1–3 Months after Surgery

	Corneal wound healing rate (kH, ×10−2/h)
	1 month	2 months	3 months
Sham rats	5.25±0.56**	5.28±0.43**	5.15±0.39**
OVX rats	0.96±0.22*	3.87±0.63*,**	4.96±0.31**

The rates (k_H) were calculated according to Eq. 2 in Materials and Methods. The data are presented as means±S.E. of 5 independent rat corneas. * p<0.05 vs. Sham rats for each category. ** p<0.05 vs. OVX rats 1 month after surgery.

Effect of Ca²⁺ Levels on the Adhesion, Proliferation and Viability of HCE-T Cells

Figure 5 shows the changes in adhesion (A), proliferation (B) and viability (C) of HCE-T cells cultured in medium containing 4–12 mg/dL Ca²⁺. Cell adhesion and proliferation were similar at all Ca²⁺ concentrations. On the other hand, cell viability was significantly decreased by incubation in medium containing 8–12 mg/dL Ca²⁺ with the viabilities of HCE-T cells incubated in medium containing 8, 10, and 12 mg/dL Ca²⁺ being 75%, 60% and 59% that of cells incubated in normal medium (DMEM/F12).

Fig. 5. Effect of High Ca²⁺ Levels on the Adhesion (A), Proliferation (B), and Viability (C) of HCE-T Cells

The Ca²⁺ levels in the free-medium were achieved by the addition of CaCl₂ (experimental medium, Ca²⁺ content, 4–12 mg/dL). Cell adhesion, proliferation and viability were measured by TetraColor One, and the data in cell proliferation was evaluated according to Eq. 3. Control (open columns), HCE-T cells cultured in normal medium (DMEM/F12, Ca content, 4.2 mg/dL). The data are presented as means±S.E. of 10 experiments. * p<0.05 vs. Control HCE-T cells for each category.

DISCUSSION

In this study, we found that high Ca²⁺ levels in lacrimal fluid cause a delay in the rate of corneal wound healing in the debrided corneal epithelium of OVX rats.

It is known that serum Ca²⁺ levels rise and that bone mass decreases in rats following bilateral ovariectomy.^11,12) We found that the Ca²⁺ content in bone of OVX rats decreased following ovariectomy, and that Ca²⁺ contents in the blood of OVX rats were elevated 1 month after ovariectomy, and subsequently decreased (Fig. 2). This decrease may be caused by other hormones regulating Ca²⁺ levels in the body, such as calcitonin and parathyroid hormone. We also investigated the effect of hypercalcemia on the Ca²⁺ contents of lacrimal fluid in OVX rats. The Ca²⁺ contents in lacrimal fluid of OVX rats increased at first after ovariectomy, and then decreased subsequently similar to the findings in blood (Fig. 2). These results show that the OVX rats used in this study showed bone loss and hypercalcemia, and that the hypercalcemia may lead to enhanced Ca²⁺ levels in lacrimal fluid.

The cornea does not contain blood vessels, and receives nutrients via diffusion from the lacrimal fluid through the outside surface and the aqueous humour through the inside surface. In particular, corneal epithelial cells receive nutrients from the lacrimal fluid. Therefore, we investigated the effect of Ca²⁺ levels in lacrimal fluid on corneal wound healing using OVX rats 1–3 month after ovariectomy. Corneal wound healing in OVX rats was slower than in Sham rats, and a close relationship was observed between the Ca²⁺ contents in lacrimal fluid and the rate constants (k_H) for corneal wound healing. Moreover, the k_H values for OVX rats 1, 2, and 3 months after ovariectomy were approximately 18.3, 73.3, and 96.3% those of Sham rats, respectively (Table 1, Fig. 4), and the volume of lacrimal fluid in OVX rats 3 month after ovariectomy (4.81±1.30 µm) was lower than in OVX rats 1 month after ovariectomy (5.61±1.52 µm, means±S.E., n=5; the volume of lacrimal fluid was measured by the Schirmer test²⁰⁾). These results show that there is not the relationship between the volume and Ca²⁺ levels of lacrimal fluid in OVX. In addition, it is suggest that the enhanced Ca²⁺ levels in lacrimal fluid may be related to a slower rate of corneal wound healing.

The maintenance of corneal epithelial cell mass can be viewed as the result of three separate, independent phenomena²¹⁾: X, the proliferation of basal epithelial cells; Y, the contribution to the cell mass of the centripetal movement of peripheral cells; and Z, epithelial cell loss from the surface.²²⁾ Corneal epithelial maintenance thus can be defined by the equation: X+Y=Z, which simply states that if the corneal epithelium is to be maintained, cell loss must be balanced by cell replacement. The corneal wound healing process is divided into three sequential and partially overlapping steps: epithelial cell loss from the surface (Z) reduces and eventually covers the wound surface (Y), while cell proliferation (X) provides cells to rebuild the tissue and tissue remodeling to restore the stratified epithelium.^23–29) Therefore, a decrease in centripetal movement, proliferation or viability of corneal epithelial cells acts to delay corneal wound healing, and the cell adhesion, growth and viability were related the centripetal movement of cell, cell proliferation, cell loss from the surface, respectively. Based on these findings, we evaluated the effect of Ca²⁺ levels on the centripetal movement, proliferation and viability of corneal epithelial cells using HCE-T cells. The Ca²⁺ contents in the medium were selected based on the concentrations in blood and lacrimal fluid of the rats used in this study. Cell adhesion and proliferation were similar for cells incubated in medium containing any Ca²⁺ level in the range of 4–12 mg/dL. On the other hand, cell viability was significantly decreased by incubation in medium containing 8–12 mg/dL Ca²⁺. Sharpe et al. reported that an increase in the extracellular Ca²⁺ level affects the intracellular Ca²⁺ concentration,³⁰⁾ and Berridge et al. reported the high Ca²⁺ levels can lead to cell death.³¹⁾ Taken together, these findings suggest that the viability of corneal epithelium cells is decreased under high Ca²⁺ conditions (over 8 mg/dL) as are observed in the lacrimal fluid of OVX rats, and that a decrease in the viability of corneal epithelial cells may be the cause of delay in corneal wound healing in OXV rats.

Further studies are needed to elucidate the relationship between corneal wound healing and the Ca²⁺ content in lacrimal fluid. In addition, it is important to clarify the mechanism of the decrease in viability of corneal epithelial cells under high Ca²⁺ conditions. It was known that the enhancement of Ca²⁺ levels in the lens, such as ophthalmic organization without blood vessel like cornea, induced the activation of calpain, a Ca²⁺-dependent protease, and caused the degradation of lens proteins, resulting in an opaque lens.^32,33) Therefore, we are now investigating the changes in intracellular Ca²⁺ concentration in corneal epithelial cells under high Ca²⁺ conditions.

In the present study, we investigated the rate of corneal wound healing in the debrided corneal epithelium of OVX rats. The Ca²⁺ content in the lacrimal fluid was shown to increase under conditions of hypercalcemia, and the rate of corneal wound healing in OVX rats was slower than in the Sham rats used as controls. In addition, a close relationship was observed between the rate of corneal wound healing and the Ca²⁺ content in the lacrimal fluid of OVX rats. Further, we show that an enhancement in the Ca²⁺ level in the culture medium causes a decrease in the viability of HCE-T cells. It is possible that the enhanced Ca²⁺ levels in the lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in the slower rate of corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage in patients with hypercalcemia.

Conflict of Interest

The authors declare no conflict of interest.

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