Elimination of hydroxypropyl methylcellulose from the anterior chamber of the rabbit

Jose Fernandez-Vigo, M.D., Miguel F. Refojo, D.Sc. , Marcia Jumblatt, M.S.

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The use of sodium hyaluronate solutions and other viscous or viscoelastic substances has been an impor­tant contribution to anterior segment surgery, espe­cially for the implantation of intraocular lenses. An injectable solution with viscoelastic properties is a viscous solution with elastic elements in its behavior. Thus, a viscoelastic solution may have the elasticity of a jelly, but flows under shear stress. All viscoelastic solutions are viscous, but not all viscous solutions are viscoelastic. Hydroxypropyl methylcellulose (HPMC), as well as sodium hyaluronate solutions, is viscoelastic. The viscosity of a nonviscoelastic substance is relatively constant at all shear rates, but in viscoelastic substances the viscosity decreases under shear stress. Thus, injecting high-viscosity solutions ofHPM C and sodium hyaluronate is easier than injecting a nonviscoelastic substance of similar high viscosity.

These substances protect the corneal endothelium

against surgical trauma and maintain the depth of the anterior chamber during surgery. Sodium hyaluronate, the substance most commonly used in these pro­cedures, is well tolerated by the eye, but its use may result in transitory ocular hypertension1.2,3 apparently because of diminution of aqueous outflow. 4 ,5 Hence, some authors have recommended removing sodium hyaluronate postoperatively. 6, 7

Hydroxypropyl methylcellulose is a physiologically inert polymer used mainly outside the United States for anterior segment surgery.8,9 In the United States, clinical trials of 2% HPMC in extracapsular cataract extraction with IOL implantation were reported re­cently.lO Its ability to protect the endothelium and maintain the depth of the anterior chamber during surgery is similar to that of sodium hyaluronate. 6-13 Although some authors have reported mild transitory hypertension ,8,12 others have not.9, 10

From the Eye Research Institute (Drs. Fernandez-Vigo, Refojo, and Ms. Jumblatt ), and the Department of Ophthalmology, Harvard Medical School (Dr. Refojo), Boston. Dr. Fernandez-Vigo was a Fellow of the Xunta de Galicia (Spain) during the time of this investigation.

Supported by NIH grant EYOO327 and in part by EY05767.

Reprint requests to M.F. Refojo , D. Sc., Eye Research Institute, 20 Staniford Street, Boston, Massachusetts 02114.

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The goals of this study were to determine the clearance from the anterior chamber of an HPMC solution that is used currently in Europe and to compare it with other HPMC solutions of higher viscosity. We also investigated the effect of HPMC on intraocular pressure (lOP) and endothelial cells, using an in vitro technique for toxicity assessment.

MATERIALS AND METHODS

Materials We used 30 eyes of 19 mixed-breed, pigmented

rabbits (male and female) weighing approximately 2.5 kg to 3.0 kg and three different HPMC solutions in balanced salt solution prepared according to the method described by Fechner.14 Solution A, a 2% solution used in Europe for IOL implantation, is made from HPMC (US Pharmacopeia designation 2910) of molecular weight 86,000 daltons with a viscosity of 4,000 centipoises (3,984 centistokes). Solu­tion B is similar to A but is used at 2.5% concentration, which results in a viscosity of 15,000 centipoises (14,922 centistokes). Solution C is a 2% solution made from HPMC (USP designation 2208) of molecular weight 120,000 daltons, which also has a viscosity of 15,000 centipoises. All the solutions were filtered under positive pressure through polycarbonate 8 f..Lm filters.

Surgical Procedure We induced anesthesia by intramuscular injection of

ketamine (100 mg/kg) combined with topical propara­caine HCI 0.5%, introduced a 30-gauge needle at an oblique angle at the limbus, and removed 0.1 ml of anterior aqueous humor. We then injected the same volume of a HPMC solution through the same needle into the anterior chamber. The procedure caused minimal trauma, the rabbits were not visually im­paired, and both eyes of each animal were used.

Two series of experiments were done. In one series of seven rabbits and 14 eyes, after extracting 0.1 ml aqueous humor, the same amount of balanced salt solution, as a control, and Solution A was injected into four right and three right eyes, respectively. In the seven left eyes, 0.1 ml of Solution A was substituted for aqueous humor.

In the second series of eight rabbits and 16 eyes, the eight right eyes received 0.1 ml of Solution B and the eight left eyes the same volume of Solution C. Thus, ten eyes were injected with Solution A, and eight eyes each with Solutions Band C. The controls were four eyes injected with balanced salt solution.

Tonometry was performed using a pneumatonome­ter, calibrated for rabbit eyes, connected to a poly­graph. The lOP values used were the averages of three consecutive determinations as follows: (a) normal eyes with topical anesthesia (proparacaine HCI, 0.5%)

(N = 30); (b) after general anesthesia (N = 30); (c) imme­diately after injection of balanced salt solution (n = 4) or the HPMC solution (Solution A: n = 10; Solutions B and C: n = 8 each); (d) with topical anesthesia at three and six hours (Solution A: n = 8; Solutions Band C: n = 6 each), 9 and 12 hours (n = 4), and 24 hours (n = 2) after injection of balanced salt solution or HPMC solution into the anterior chamber.

The concentration ofHPMC in the anterior chamber was determined after extracting 0.1 ml of fluid, using a 23-gauge needle, with the animals under general anesthesia. However, before the aqueous sample was withdrawn, the aqueous was aspirated and reinjected twice to ensure that a representative sample was obtained. Immediately after removing the intraocular fluid for analysis, the animals were sacrificed by an intravenous overdose of pentobarbital sodium. The samples were removed from the anterior chamber at time 0 (immediately after injection), 6, 12, and 24 hours postinjection. The concentration of HPMC was deter­mined by means of its reaction with diphenylamine 15 modified as follows: 0.1 ml of intraocular fluid was diluted up to 1 ml with distilled water and added to 1 ml of a saturated solution of calcium chloride. The mixture was stirred for 45 seconds. If the sample contained HPMC, a precipitate was formed that was further separated by ten minutes of centrifugation at medium speed in a standard laboratory centrifuge. The clear solution was pipetted from the centrifuge tube, and the precipitate saved. The pipetted solution was put through a 0.45 f..Lm filter that was rinsed by filtering 2 ml of saturated calcium chloride solution through it. The filtrate was discharged. The precipitate 'retained on the filter was redissolved in 5 m.l of freshly prepared diphenylamine reagent (diphenylamine [3.75 g] dis­solved in glacial acetic acid [150 ml] and concentrated HCL [90ml]) and added through the filter into the centrifuge tube containing the HPMC precipitate. The HPMC dissolved in the reagent solution was placed in a stoppered tube in boiling water (30 minutes), cooled in ice (ten minutes), and allowed to stand at room temperature (ten minutes).

A characteristic blue color developed in the reaction mixture. The concentration of HPMC was obtained from the average of three spectrophotometer readings at 635 nm, at 0.5 nm slit-width. The absorbance at 635 nm (0.225 ± 0.017) that was obtained when second­ary aqueous humor of normal rabbits was reacted with the diphenylamine reagent was subtracted from the absorbance obtained for all the aqueous humor samples of the eyes injected with HPMC. Aqueous humor taken at different times from normal rabbit eyes provided results identical to the secondary aqueous. The concentration of HPMC in the anterior chamber was determined using graphs of standard concentra­tions versus absorbance obtained with serial dilutions

192 J CATARACT REFRACT SURG-VOL 15, MARCH 1989

of HPM C for each of the two molecular weights used in these experiments.

In Vitro Rabbit Endothelial Toxicity Determination Rabbit corneal endothelial cells were grown and

subcultured into 24-well multiplates as previously described. 16 After one week, the cell number was determined in four replicate wells. After removal of the medium, circular defects were produced in the re­maining wells by direct application of a filter (type HA, 6 mm diameter) to the apical surface of the culture. The cultures were rinsed in control medium and the fil­ter discs carefully removed. Test medium containing 2.5% HPMC (86,000 daltons), prepared by diluting a 5% HPMC solution with the appropriate control media, was added and the cultures were kept for three days at 37°C in a humidified CO2 incubator. At the end of the incubation period, the media were removed and the cell number was determined in duplicate in test and control cultures. The remaining culture wells were fixed in neutral buffered formalin and stained with full­strength Giemsa as previously described for cultures of corneal epithelium .17 The areas of the remaining unstained central wound areas and the intensities of peripheral cellular staining were compared visually.

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RESULTS Intraocular Pressure

The results of these experiments are given in Fig­ure 1. The lOP of the rabbits changed very little pre­and postanesthesia. Immediately after the exchange of the aqueous humor for an equal amount of balanced salt solution, there was no change. When the aqueous humor was exchanged with Solution A, a slight lOP increase was observed. When Solution B or C was injected, a marked decrease was observed. However, in no case was leakage of HPMC solution observed after the injection. At three hours postinjection, all eyes injected with HPMC solutions had higher-than­normal lOPs, but the eyes injected with balanced salt solution were still hypotensive. Six hours postinjec­tion, the lOP of the HPMC-injected eyes approached normal values, which were reached at nine hours and maintained thereafter at 12 and 24 hours postinjection.

Concentration of HPMC in the Anterior Chamber The results of these experiments are given in Ta­

ble 1. The aqueous humor of two eyes was analyzed at each interval, except for the eyes injected with Solu­tion A, which was analyzed at six hours in four eyes. At time 0, immediately after 0.1 ml of aqueous humor was

-0- BSS -_ .• --- HPMC 2% (HMw), Solution C -.- HPMC 2.5% (LMw), Solution B ~ HPMC 2% (LMw), Solution A

12 24

Time (hours)

Fig. 1. (Fernandez-Vigo) Intraocular pressure (± standard deviation) of eyes in which 0.1 ml of aqueous humor was substituted by (a) balanced salt solution (BSS), (b) Solution A (hydroxypropyl methylcellulose [HPMCl 2%, low molecular weight), (c) Solution B (HPMC 2.5%, low molecular weight), and (d) Solution C (HPMC 2%, high molecular weight). Pr.O. = preoperative; G.A. = general anesthesia; HPMC 0 hours = immediately after injection of test solution into anterior chamber.

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Table 1. Hydroxypropyl methylcellulose in rabbit eyes after exchange of 0.1 ml aqueous humor for 0.1 ml HPMC solution.

Molecular Concentration Viscosity HPMC (% Standard Deviation) in Anterior Chamber (Hours)

Weight (%) (centipoises) 0 6 12 24

86,000 2.0 4,000 1.09S±0.070 0.138±0.OS4* 0.1l0±0.020 0.02S ± 0.020

86,000 2.S IS,OOO 1.091 ± O.OSO 0.34S±0.030 0.21S ± 0.100 0.060 ± 0.014

120,000 2.0 IS,OOO 1.106 ± O.OSO 0.3S0 ± 0.040 0.210±0.IS0 0.02S±0.020

*P<.OO1

substituted by an injection of equal amounts of Solu­tion A, B, or C, the concentration of HPMC in the anterior chamber was, as expected, about half that injected because of dilution in approximately equal aqueous humor volume. The exception was Solution B, which was slightly lower than expected (1.091 ± 0.050 standard deviation versus the expected value of ap­proximately 1.25%). At six hours, there was a marked decrease in the concentration of HPMC remaining in the anterior chamber; it was significantly lower (P<.OOl) in the eyes injected with solution A than in those injected with Solution B or C. The concentration of HPMC in the anterior chamber continued to decrease, and at 24 hours the amount was at the lower limit of detectability of the analytical technique used in these experiments.

In Vitro Endothelial Cell Toxicity of HPMC At a final concentration of 2.5% in nutrient tissue

culture medium, HPMC caused no damage to periph­eral stationary endothelial cells or to cells migrating centripetally into the central defect area. Three days after wounding, both control and experimental cul­tures demonstrated partial coverage of the central defect area, with coverage similar in extent under all conditions. Hemacytometer counts of test and control cultures showed similar cell densities after three days' exposure to HPMC. Control cultures contained 9.3 ± 0.3 X 104 versus 8.9 ± 0.3 X 104 cells per well in experimental cultures (mean ± SEM, n = 4 to 12).

DISCUSSION Hydroxypropyl methylcellulose is an inert polymer

used in Europe8,9 that has proven to be a good protector of the endothelium9 -12 and that maintains the anterior chamber depth during intraocular lens surgery. 11,12,13

Sodium hyaluronate has been reported to have a half-life in the anterior chamber of80 minutes. 19 There are also reports showing the complete elimination of low-molecular-weight sodium hyaluronate from the anterior chamber at 24 hours and of high molecular weight at 72 hours (S. Miyauchi, S. Iwata, Evaluations on the Usefulness of Viscous Agents in Anterior Segment Surgery, Proceedings of the International Society for Eye Research, 1986), but others report between one

and two weeks. 20 This elimination appears to be through Schlemm's canal, but some investigators spec­ulate on an enzymatic mechanism of elimination. 4,5 For chondroitin sulfate, 40 hours has been reported as the time of complete elimination from the anterior cham­ber.l8 Hydroxypropyl methylcellulose has been re­ported to remain in the anterior chamber for 72 hours, but the information that supports these data is incomplete. Also, there are no data in the literature, as far as we know, that determine the difference in retention in the anterior chamber and the lOP effect of HPMC of different concentrations and molecular weights, which are the determinants of the viscosity of the HPMC solutions.

For two of the solutions tested, our results show an lOP decrease after removing the aqueous humor and injecting the same amount of HPMC into the anterior chamber. We believe this was due to the difficulties of exchanging the exact amount of fluid. The exact lOP peak time was not detected; nevertheless, three hours after injection of the three solutions, an lOP increase to between 28 mm Hg and 33 mm Hg was observed. At three hours, the lOP in the eyes injected with HPMC solutions was significantly higher than in the balanced salt solution control group (P<.Ol). This indicates that the transient lOP increase was due to the viscous substance injected into the anterior chamber and not to the protein in the secondary aqueous. By six hours, the lOP approached normal value, which was reached at nine hours and maintained by 12 and 24 hours, in agreement with previous reports. 12

The effect ofHPMC on the lOP seems independent of its molecular weight and concentration, which is clinically important in relation to the use of HPMC solutions of different viscosities according to the sur­geon's needs.

The closeness of the results of the HPMC concentra­tion in the anterior chamber to the expected values immediately upon injection indicates that the sampling error in these experiments was negligible. Despite the viscosity difference, the aqueous humor and the HPMC solutions have approximately the same density (approximately 1.006 glml) and are readily miscible.

The concentration ofHPMC in the anterior chamber diminished gradually, until at 24 hours it was at the lower limit of detectability of the analytical procedure

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used. The half-life of HPMC in the anterior chamber was about three hours for the 4,000-centipoise sample and about four and a half hours for the two solutions of high viscosity (15,000 centipoises). In the early stages, the elimination of the HPMC oflower viscosity (86,000 daltons at 2% concentration) occurred more rapidly than the solutions of higher viscosity.

Although the anterior chamber angle of the rabbit differs from that of humans and the intraocular dy­namics of HPMC in rabbits and humans may be different, this model may predict the elimination of HPMC in humans. Although further research is needed to ascertain the mechanism of elimination of HPMC from the anterior chamber and because it is improbable that there is an enzymatic degradation of HPMC in the eye, the polymer seems to be eliminated from the eye through Schlemm's canal.

The lack of damage to stationary or migratory endothelial cells in culture leads us to conclude that HPMC is relatively nontoxic. A similar assay using corneal epithelial cells has been suggested as an in vitro toxicologic alternative to animal testing. 17 In these experiments, epithelial cells were substituted for en­dothelial cells. This assay was found to be a good indicator of toxicity (unpublished results). Although it is well known that rabbit endothelial cells are more hardy than human endothelium, these cells would show toxicity with use of the assay. Thus, our evaluation does not rule out the possibility of transient inter­ference with the human endothelial function in vivo, but strongly suggests that such an effect is reversible upon elimination ofHPMC from the anterior chamber.

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