Suprachoroidal injection of ketorolac tromethamine does not cause retinal damage.

Rabbit right eyes were injected with 3 or 6 mg ketorolac tromethamine into the suprachoroidal space. Electroretinography results demonstrated no abnormal changes in rod cell response, maximum rod cell or cone cell mixing reaction, oscillation potential, cone cell response, waveform, amplitude, and potential of 30 Hz scintillation response in right eyes before injection, and at 1, 2, and 4 weeks after injection. There was no difference between left (control) and right eyes. Under light microscopy, the histomorphology of cells in each retinal layer was normal at 4 weeks following 6 mg ketorolac tromethamine administration. These results indicate that a single suprachoroidal injection of 3 or 6 mg ketorolac tromethamine into rabbits was safe. Suprachoroidal space injection appears to be safe.

Research Highlights

Previous studies have reported vitreous space injection of ketorolac tromethamine to observe neurotoxicity on the retina. However, suprachoroidal injection of ketorolac tromethamine has not been reported.

A single suprachoroidal injection of 6 mg ketorolac tromethamine in rabbits showed no significant influence on electroretinograms and retinal morphological structures.

Results suggested that suprachoroidal injection of 6 mg ketorolac tromethamine in rabbits is safe.

INTRODUCTION

Ketorolac tromethamine is an active nonsteroidal anti-inflammatory drug and non-narcotic analgesic with cyclooxygenase inhibitory activity. Its use does not lead to dependency[123456]. It can be administered intramuscularly, intravenously, orally, and as eye drops as the water-soluble Trometamol salt. Ketorolac tromethamine has been widely used for the treatment of ocular inflammation, such as allergic conjunctivitis and keratitis, or as a prophylaxis to prevent post-procedure pain and cystoid macular edema[78910111213141516171819]. There has been outstanding performance of ketorolac tromethamine in antiangiogenesis and in anti-proliferation in recent years[2021]. The mechanism of action is thought to be primarily through inhibition of cyclooxygenase[22]. The most common adverse reaction of topical nonsteroidal anti-inflammatory drugs such as ketorolac tromethamine and diclofenac is a burning and stinging sensation. Others include delayed corneal epithelial healing and conjunctival hyperemia. Serious complications such as corneal ulcer and corneal perforation have been individually reported in a few postoperative patients[23]. However, these symptoms were due to the preservative in the preparations.

The effects of medical treatments for posterior segment disease are less than satisfactory[24]. Systemic medications usually use large doses of drugs to enhance the effective drug concentration in eyes, and thus the drug inevitably has inherent side effects and contraindications are hard to be overcome[25]. Topical therapy such as retrobulbar injection achieves therapeutic drug levels too slowly, and intravitreal injection can easily lead to retina toxicity and vitreous opacity. Another option is to inject drugs into the suprachoroidal space, which is a potential cavity gap limited anteriorly in the region of the scleral spur and posteriorly by the transscleral connections of the short posterior ciliary vessels to the choroid[26]. Possible advantages of this route of drug delivery are ease of drug absorption, fast achievement of high intraocular drug concentrations after a single dose, and long duration of drug presence because of abundant vessels of the deep-seated choroid. There are many reports of suprachoroidal cavity drug administration to treat ocular perforating injury, endophthalmitis, and to repair retinal detachments[272829303132]. There are also disadvantages such as suprachoroidal hemorrhage and choroidal detachment. To the best of our knowledge, the safety of suprachoroidal injection of ketorolac tromethamine has never been reported.

Thus, this study was designed to determine if ketorolac tromethamine is nontoxic to the retina when injected into the suprachoroidal space of rabbits, and to assess the safety of the drug administration route.

RESULTS

Quantitative analysis of experimental animals

A total of 12 rabbits were equally and randomly divided into 3 mg and 6 mg treatment groups. Rabbits in the 3 mg (0.05 mL) group were injected with ketorolac tromethamine in the right eye, while the dose in the 6 mg group was 6 mg (0.05 mL). The left eye was injected with 0.05 mL saline (0.9% sodium chloride solution) as a control.

Ketorolac tromethamine did not affect the appearance of rabbit retinas

All animals tolerated the injections well with no external signs of inflammation. Slit lamp biomicroscopy at baseline and 1, 2, and 4 weeks after injection revealed no intraocular inflammation or cataract formation. Indirect ophthalmoscopy of ketorolac tromethamine-injected eyes showed no signs of hemorrhage, whitening, retinal pigment epithelium loss, or optic nerve pallor.

Electroretinogram waveform appeared normal after ketorolac tromethamine injection

Representative scotopic and photopic electroretinogram waveforms are shown in Figure 1. All groups of eyes showed similar waveform patterns with clearly distinguishable a-waves and b-waves. No waveform depression was observed in eyes injected with ketorolac tromethamine.

Figure 1

Electroretinogram of rabbit right eye (experimental eye) in the 6 mg treatment group.

Unit of Y-axis is mV. (A) Rod cell response; (B) cone cell response; (C) maximum rod cell-cone cell mixing reaction; (D) 30 Hz scintillation response; (E) oscillation potential.

There was no evidence that wave amplitudes produced by the standard electroretinogram differed significantly for eyes treated with 3 mg or 6 mg ketorolac tromethamine compared with saline-treated eyes (P > 0.05, Tables 1–3).

Table 1

Data from the electroretinogram of rabbit left and right eyes after right eyes were injected with 3 mg ketorolac tromethamine

Table 3

Difference of control eyes and right eyes treated with 3 mg or 6 mg of ketorolac tromethamine (P value)

Ketorolac tromethamine did not affect retinal appearance

After 4 weeks, histopathological specimens were examined by light microscopy (hematoxylin-eosin staining). There were no significant differences between the histological findings of eyes injected with ketorolac tromethamine and saline as shown in Figure 2A for one rabbit injected with 6 mg, and Figure 2B for a control eye. All retinal layers, including the ganglion cell layer, inner nuclear layer, outer nuclear layer, and photoreceptor layer exhibited normal structures. The photoreceptor layer was arranged into a microgroove shape and was homogeneously stained. The cell nuclei of the other layers appeared normal.

Figure 2

Light microscopy of the retina at 4 weeks after injection of 6 mg of ketorolac tromethamine (A) or saline (B) (hematoxylin-eosin staining, × 400).

Ganglion cells, inner plexiform layer, inner nuclear layer, outer nuclear layer, and photoreceptor layer exhibited normal structure after injection with 6 mg of ketorolac tromethamine.

GCL: Ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; PRL: photoreceptor cell layer.

DISCUSSION

Corticosteroids are potent anti-inflammatory medications that are widely used in treating fundus oculi disease.

However, the side effects and the risk-benefit ratio preclude their use in some patients. A growing body of scientific evidence supports the use of nonsteroidal anti-inflammatory drugs in improving the treatment of diseases such as cystoid macular edema, diabetic macular edema, neovascularization, uveitis, and even age-related macular degeneration[3334353637]. In this report, we examined the safety of one such nonsteroidal anti-inflammatory drug, ketorolac tromethamine. The combination of electroretinogram and histopathological analyses demonstrated that a suprachoroidal dose as high as 6 mg of ketorolac tromethamine was well tolerated and safe in the rabbit retina.

Komarowska et al [38] reported that repeated intravitreal injections of commercial ketorolac preparations (3 mg, with preservative) at 2-week intervals induced local histological damage but was not sufficient to affect the electroretinogram data. A previous study showed that doses as high as 4 mg of ketorolac tromethamine injected into adult human eyes was nontoxic tromethamine[39]. However, eyes injected intravitreally with 6 mg of ketorolac tromethamine demonstrated a 10% amplitude decrease at 4 weeks and a 20–30% decrease at 8 weeks. Based on our results, it can be assumed that administration of 3 and 6 mg ketorolac tromethamine may not damage the retina. The differing results of our study may be due to the different routes of administration. Possible advantages of suprachoroidal injection are ease of drug absorption and fast achievement of high intraocular drug concentrations after a single dose because of abundant vessels of the deep-seated choroid in this region. This injection route is obviously better tolerated than intravitreal injection.

There are increasingly more reports of administration of drugs into the suprachoroidal space. Olsen et al [40] demonstrated the safety and feasibility of such administration experimentally, and proved that it was an effective method to deliver targeted drugs into the posterior segment. Wang et al [41] reported that suprachoroidal injection of ketorolac tromethamine could achieve an effective drug level in the retina/choroid. The mean maximum concentration of ketorolac tromethamine in the retina/choroid was 56.71 ± 22.64 μg/g (at 0.5 hours) after suprachoroidal injection of 250 μg ketorolac tromethamine. Our findings clarify and further expand upon these results.

In conclusion, ketorolac tromethamine, a nonsteroidal anti-inflammatory drug, is nontoxic to the retina when injected into the suprachoroidal space of rabbits. The results are promising. However, this study has limitations because of the low number of eyes injected and the limited number of doses used.

MATERIALS AND METHODS

Design

A randomized, controlled animal experiment.

Time and setting

Experiments were performed in Capital Medical University, China between February and September 2011.

Materials

Animals

Twelve 6-month-old, healthy, specific pathogen free, male adult albino rabbits weighing 2.0 kg each were housed in separate cages and maintained in a controlled environment for these experiments. Animals were provided by the Animal Care Department of the Capital Medical University, license No. SCXK (Jing) 2007-0001. All experimental use of animals complied with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[42].

Drug

The ketorolac tromethamine chemical formula is C15H13NO3·C4H11NO3, and the chemical structural formula is as follows:

It was acquired in powder form (Ketorolac tris salt; Weidu Medical Technology Chemical Co., Ltd., Jinan, China) and dissolved in saline (0.9% sodium chloride solution) under sterile conditions.

Methods

Ketorolac tromethamine administration in the suprachoroidal space

Animals were first anesthetized with an intramuscular injection of 50 mg/kg of ketamine hydrochloride and 20 mg/kg of xylazine hydrochloride. Before all suprachoroidal space injections, the eyes were treated with several drops of 0.5% proparacaine hydrochloride (Alcon, Inc., Brussels, Belgium), and tropicamidephenylephrine ophthalmic solution (Santen Pharmaceutical Co., Ltd., Suzhou, China) was applied to dilate the pupils. Then, a conjunctival peritomy was made on the superonasal quadrant, exposing clear access to the sclera. A parallel 2 mm scleral tunnel incision was made with a blade about 4 mm away from the inferior limbus to expose bare choroid. Before injection, 0.1 mL of aqueous humor was drained to minimize the leakage and to reduce the intraocular pressure. Viscoelastic materials (Bausch & Lomb Surgical, Inc., Beijing, China) were injected into the suprachoroidal space to facilitate the drug's delivery. A volume of 0.05 mL was then slowly injected by a 0.1 mL-syringe with a special needle (32 G, 0.22 mm × 5 mm, Beijing Brightway Medical Instruments Co., Ltd., Beijing, China). According to previously reported procedures[3839], the right eyes of each animal in the 3 mg treatment group and 6 mg treatment group were given a single injection into the suprachoroidal space, with 3 mg/0.05 mL, and 6 mg/0.05 mL of ketorolac tromethamine. Left eyes were given a single injection into the suprachoroidal space with 0.05 mL of saline, as a control.

Electroretinography of retina functions in rabbits

All animals underwent electroretinography exams prior to injections and exams were repeated at 1, 2, and 4 weeks after drug delivery. Standard dark-adapted and light-adapted electroretinography was performed after anesthesia as discussed above, and pupillary dilation was induced by topical tropicamide. Electroretinography responses were recorded in the dark-adapted animals after 1 hour of dark equilibration. The recording electrode (Eye Institute of Tongren Hospital, Beijing, China) was positioned on the cornea. The reference electrode (Eye Institute of Tongren Hospital) was inserted into the lip and the ground electrode was placed subcutaneously in the ipsilateral leg. All procedures were carried out under dim red light at the end of the adaptation period. Full-field stimulation was used in the experiments. The flash stimuli were 0.01 cd·s/m2 for rod stimulation, 3.0 cd·s/m2 for all other standard responses, and 30 cd·s/m2 for light adaptation and background luminance. Electroretinography analysis was based on the amplitude of a-wave, b-wave, and the total amplitudes of oscillatory potentials (Ops), as well as the average amplitude of 30 Hz flicker. According to the ISCEV Standard[43], results were designated as Dark-adapted 0.01 electroretinography (rod response, Rod-R), Dark-adapted 3.0 electroretinography (standard combined response, S-combined), Dark-adapted 3.0 oscillatory potentials (oscillatory potentials, Ops), Light-adapted 3.0 electroretinography (photopic response, phot.R), and Light-adapted 3.0 flicker electroretinography (30 Hz), respectively. The a-wave reflects the function of the inner segment of the photoreceptors, and the b-wave reflects the activity of the Muller cells and the bipolar cells. Electroretinography responses of the experimental eyes (right eye) were compared with electroretinography responses of the control eyes (left eye) in each wave study.

Light microscopic observation of rabbit retinal tissue morphology

After the electroretinography test, at 4 weeks, the rabbits were euthanatized with an intravenous injection of air. The eyes were immediately enucleated and fixed in a solution of glacial acetic acid, formaldehyde, and ethanol, provided by the Peking University People's Hospital for light microscopy methods (Zeiss, Oberkochen, Germany). After embedding, the eyes were sliced into 7 μm thick coronal sections, and stained with hematoxylin-eosin. The light microscopy magnification was × 400.

Statistical analysis

The data were analyzed using SPSS 17.0 software (IBM, New York, USA, serial No. 4625180487). The method of general linear model-repeated measures was used to account for the repeated measurements within subjects.

Table 2

Data from the electroretinogram of rabbit left and right eyes after right eyes received 6 mg of ketorolac tromethamine injection