Korean J Ophthalmol.  2015 Aug;29(4):270-279. 10.3341/kjo.2015.29.4.270.

In Vivo Effects of Preservative-free and Preserved Prostaglandin Analogs: Mouse Ocular Surface Study

• Kim JH
• Kim EJ
• Kim YH
• Kim YI
• Lee SH
• Jung JC
• Lee KW
• Park YJ

• KMID: 2363772
• DOI: http://doi.org/10.3341/kjo.2015.29.4.270

Abstract

PURPOSE
Chronic use of topical hypotensive agents induces several side effects caused by preservatives. The purpose of this study was to evaluate the effects of prostaglandin analogs with varying concentrations of benzalkonium chloride (BAC), preservative-free (PF), and alternative preservatives on mouse corneal tissue.
METHODS
Thirty-five, 8- to 10-week-old female C57BL/6 mice (five mice for each group) were used for this study. To the control group, we applied normal saline, and to each drug-treated group we applied 0.02% BAC, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), travoprost 0.004% (with 0.001% polyquad) or tafluprost 0.0015% with/without 0.001% BAC, once a day (9 p.m.) for 4 weeks. Corneal fluorescein staining was evaluated in all groups. After harvest, the corneal tissues were embedded in paraffin and then Hematoxylin-Eosin stain was performed for histopathological examination. Immunofluorescence staining was done against TNF-alpha, IL-6, HLA DR, pJNK, and pAkt.
RESULTS
In corneal fluorescein staining, severe punctate epithelial keratitis was seen in the groups of 0.02% BAC, 0.02% BAC containing bimatoprost 0.01% and latanoprost 0.005%. The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were observed in the groups of BAC-containing eye drops. Moreover, the groups treated with BAC-containing eye drops have high inflammatory markers, significantly decreased cell viability-related signal, pAkt, and higher apoptosis-inducing signal, pJNK, than the control group. On the other hand, travoprost 0.004% and PF tafluprost 0.0015% have less cellular morphologic changes, lower inflammation, and higher cellular viability than BAC-containing formulations.
CONCLUSIONS
Corneal damage, increased inflammation and apoptosis and low cell viability were observed in BAC-containing groups. PF or alternatively preserved glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

Keyword

Benzalkonium compounds; Cornea; Pharmaceutical preservatives; Synthetic prostaglandins

MeSH Terms

Animals
Cell Survival
Conjunctiva/drug effects/*pathology
Disease Models, Animal
Epithelium, Corneal/drug effects/*pathology
Female
Glaucoma/*drug therapy/pathology
Mice
Mice, Inbred C57BL
Microscopy, Fluorescence
Ophthalmic Solutions
Preservatives, Pharmaceutical
Prostaglandins, Synthetic/*administration & dosage
Ophthalmic Solutions
Preservatives, Pharmaceutical
Prostaglandins, Synthetic

Figure

• 

  Fig. 1 Corneal fluorescein staining of seven C57BL/6 mice groups. (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015%(BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free). Severe punctuate epithelial keratitis was seen in the groups of 0.02% BAC (B), 0.02% BAC contained bimatoprost 0.01% (C) and latanoprost 0.005% (D).

• 

  Fig. 2 Histopathologic examination by H&E stain for each group. (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free). The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were shown in the groups of BAC contained eye drops (B,C,D,F). However, travoprost 0.004% (with 0.001% polyquad) (E) and preservative-free tafluprost 0.0015% (G) groups were similar morphology with control group (A).

• 

  Fig. 3 Immunofluorescence staining of TNF-α positive cells in the corneal section (A-G) and the density of TNF-α fluorescence (H). (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free, PF). Immunofluorescence staining of TNF-α was expressed more in preservative contained eye drops groups than control group. But, travoprost 0.004% (with 0.001% polyquad) and preservative-free tafluprost 0.0015% groups had significantly lower TNF-α positive cells than 0.02% BAC group. And BAC contained tafluprost 0.0015% has more TNF-α positive cells than preservative-free one. *,**p < 0.05, #p < 0.02.

• 

  Fig. 4 Immunofluorescence staining of IL-6 positive cells in the corneal section (A-G) and the density of IL-6 fluorescence (H). (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free, PF). Immunofluorescence staining of IL-6 was highly expressed in 0.02% BAC contained eye drops groups than control group. But, travoprost 0.004% (with 0.001% polyquad), 0.001% BAC contained tafluprost 0.0015% and preservative-free tafluprost 0.0015% groups had significantly lower IL-6 positive cells than 0.02% BAC group. And BAC contained tafluprost 0.0015% has more IL-6 positive cells than preservative-free one. *p < 0.006, **,#p < 0.005.

• 

  Fig. 5 Immunofluorescence staining of HLA DR positive cells in the corneal section (A-G) and the density of HLA DR fluorescence (H). (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free, PF). Immunofluorescence staining of HLA DR was highly expressed in 0.02% BAC contained eye drops groups than control group. But, travoprost 0.004% (with 0.001% polyquad), 0.001% BAC contained tafluprost 0.0015% and preservative-free tafluprost 0.0015% groups had significantly lower HLA DR positive cells than 0.02% BAC group. *p < 0.02, **p < 0.01.

• 

  Fig. 6 Immunofluorescence staining of pJNK positive cells in the corneal section (A-G) and the density of pJNK fluorescence (H). (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free, PF). Immunofluorescence staining of pJNK was highly expressed in 0.02% BAC contained eye drops groups than control group. But, travoprost 0.004% (with 0.001% polyquad), 0.001% BAC contained tafluprost 0.0015% and preservative-free tafluprost 0.0015% groups had significantly lower pJNK positive cells than 0.02% BAC group. *p < 0.03, **p < 0.05.

• 

  Fig. 7 Immunofluorescence staining of pAkt positive cells in the corneal section (A-G) and the density of pAkt fluorescence (H). (A) Control, (B) 0.02% benzalkonium chloride (BAC), (C) bimatoprost 0.01% (BAC 0.02%), (D) latanoprost 0.005% (BAC 0.02%), (E) travoprost 0.004% (with 0.001% polyquad), (F) tafluprost 0.0015% (BAC 0.001%), and (G) tafluprost 0.0015% (preservative-free, PF). Immunofluorescence staining of pAkt shows significantly lower expression in 0.02 % BAC and latanoprost 0.005 % (BAC 0.02 %) than control group. PF tafluprost 0.0015 % group had significantly higher expression than control group. Moreover, PF tafluprost 0.0015 % group had significantly higher expression than 0.02 % BAC and preserved tafluprost 0.0015 % group. *,**p < 0.05, #p < 0.01.

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