Experimental Study on Effect of Hydroxypropyl Methyl Cellulose on Angle Tissue

Abstract: In order to investigate the effect of hydroxypropyl methylcellulose (HPMC) on the angle tissue after entering the anterior chamber, 24 pure white rabbits were randomly divided into two groups: 40 eyes in the experimental group, 2% HPMC was injected into the anterior chamber, and the control group was injected with 2% HPMC in the anterior chamber. Group 8 eyes, one eye as normal control, 7 eyes injected with BSS in the anterior chamber, the experimental group was divided into A and B groups according to normal and increased intraocular pressure. Specimens were collected on the 5th day, 3rd week and 10th week after injection for pathological examination. It was found that at the initial stage of injection, the trabecular meshwork was slightly expanded in the experimental group A, and the cytoplasmic vacuoles of the endothelial cells in the inner wall of the aqueous humor plexus increased. The vacuoles of the endothelial cells in the wall of the aqueous humor cluster increased significantly, but they all returned to normal in the long-term follow-up observation. Experiments have shown that HPMC entering the anterior chamber and remaining can lead to a transient increase in intraocular pressure and thus cause pathological changes in the chamber angle tissue, but the changes are reversible. HPMC itself is non-toxic and destructive to the angle tissue.

Key words:hydroxypropyl methylcellulose; angle tissue; pathological changes

Hydroxypropyl methylcellulose (HPMC) is widely used in clinical ophthalmology, especially in surgery to maintain the anterior chamber for surgical operations, protect corneal tissue, liquid separation and manipulation of tissue, as well as lubricating filling, protective machinery pad and prevent postoperative adhesions. In addition, HPMC is also used in the treatment of dry eye syndrome, eye trauma surgery, PRK, glaucoma and posterior segment surgery. After entering the anterior chamber, HPMC has good tolerance, high safety, no inflammatory effect, and no toxicity to corneal endothelium. And because of its easy-to-obtain and low-cost raw materials, it can be sterilized under high pressure and stored at room temperature, and has good coating properties, etc., it has become a good substitute for Healon and has high clinical application value. Whether the structure has any effect, and what effect has not been reported. In this paper, animal experiments were used to inject 2% HPMC into the anterior chamber of rabbits and observe whether there were pathological changes in the anterior chamber angle tissue in different periods, so as to further evaluate the safety of its intraocular application, so as to provide reference for clinical application.

1.Materials and methods

1.1 Materials

1.1.1 Experimental animals

24 healthy adult pure white rabbits, weighing 2.0-2.5 kg. Male or female.

1.1.2 Main reagents

2% Celoftal (2% HPMC, Al-con, USA)

1.2 Method

1.2.1 Grouping and intraocular pressure measurement

First, the rabbits were randomly divided into 20 experimental groups (40 eyes) and 4 control groups (8 eyes). The experimental group was injected with 2% HPMC, and 7 eyes in the control group were injected with BSS (one eye was not injected as a normal eye) before injection. Chloramphenicol eye drops were administered twice a day, and the initial intraocular pressure was measured with a Schiotz tonometer under 0.5% procaine anesthesia.

1.2.2 Anesthesia

Intramuscular injection of 1 ml/kg with 100 mg/2 ml of ketamine and 100 mg/2 ml of promethazine equal volume mixture can be maintained for 1 hour.

1.2.3 Anterior chamber injection

After anesthesia, anterior chamber puncture was performed under aseptic operation, 0.25 ml of aqueous humor was extracted, 0.25 ml of 2% HPMC was injected into the experimental group, and 0.25 ml of BSS was injected into the control group, and the puncture port was sutured under a microscope with 10-0 non-invasive suture , to ensure watertightness.

1.2.4 IOP measurement and regrouping

The intraocular pressure was measured once respectively at 3, 6, 12, and 24 hours after the injection (the method was the same as before), and the rabbit eyes of the experimental group were grouped again according to the following criteria after the first intraocular pressure measurement: the eyes of the rabbits in the experimental group were grouped higher or lower than the initial eyes. Eyes with intraocular pressure within 3mmHg (1mmHg=0.133kPa) were regarded as normal eyes and included in the experimental group A; Eyes above 3 mmHg were regarded as eyes with decreased intraocular pressure and included in the experimental group C; rabbit eyes that did not meet the above criteria were screened out from the experiment (in the future, only the intraocular pressure was observed without collecting its specimens, the same below). After each intraocular pressure measurement, those who did not meet the criteria were screened out from the experiment in each group according to this standard. As a control, only those with normal intraocular pressure (that is, those with high intraocular pressure or within 3mmHg lower than the initial intraocular pressure) were kept out of the screening experiment.

1.2.5 Second injection and screening

The intraocular pressure was measured 24 hours after the first injection, and the second anterior chamber injection was performed. The injection volume and method were the same as before, and the intraocular pressure was measured once at 3, 6, 12, and 24 hours after the injection. Rabbit eyes that did not meet the standards in each group were screened out. 24 hours after the second injection, finally determine the number of rabbit eyes in each group A.B.C, and then measure the intraocular pressure twice every day until the 5th day.

1.2.6 Fixation in situ

On the 5th day after the first injection, samples were collected from one eye and one normal eye of each of the experimental groups A, B, C and the control group. Anesthetize first (the method is the same as before), connect the No. 5 infusion needle with a 1 ml syringe, draw out 0.25 ml of aqueous humor by anterior chamber puncture under aseptic operation, immediately inject 0.25 ml of 4% glutaraldehyde solution, and clamp the rubber tube of the infusion needle , keep and fix for 60min.

1.2.7 In vivo sampling and secondary fixation

Live samples were collected under a microscope, the bulbar conjunctiva and fascia were cut circularly, and the angle tissue of 1.5 mmX1.0 mmX1.0 mm was cut off with a razor blade and cornea, washed with BSS and immediately put into 4% glutaraldehyde solution , and then the eyeballs were removed, and the accumulated blood was washed clean with BSS and immediately put into 4% glutaraldehyde solution.

1.2.8 Specimen preparation and observation

Electroplated specimens were fixed in glutaraldehyde for 12 hours, washed, fixed, dehydrated, soaked, embedded and other routine treatments were taken to make sagittal slices of the eyeballs, with a thickness of 60 nm, stained with lead and uranium, and then observed with an Opton FM 10CR transmission electron microscope. After the eyeballs were fixed in 4% glutaraldehyde for 48 hours, specimens were taken from the site adjacent to the electron microscope. After routine treatment, sagittal slices of the eyeballs were taken, with a thickness of 4 pum, and stained with HE.

1.2.9 The 2nd and 3rd sampling

On the 3rd and 10th week after the first injection, one eye was randomly selected from each of the experimental groups A, B, C and the control group, and the 2.3rd time was taken, and the specimens were made and observed. The specific operation was the same as before.

2. Results

2.1 Changes in intraocular pressure

The initial intraocular pressure before injection was (2.607+0.372) kPa (n=40) in the experimental group and (2.608+2.527) kPa (n=8) in the control group, P>0.1, the difference was not statistically significant. Before the second injection, compared with the initial intraocular pressure, there were 14 eyes with normal intraocular pressure (group A), 9 eyes with increased intraocular pressure (group B), and no decrease in intraocular pressure (group C). The intraocular pressure was normal. 24 hours after the second injection, 9 rabbit eyes were left in experiment A and group B (one eye of experiment B was screened out due to extremely low intraocular pressure caused by suture rupture) and one eye of control group was due to leakage The intraocular pressure decreased, and the intraocular pressure of the remaining 6 eyes was normal.

The average intraocular pressure at different periods after injection showed that the intraocular pressure could be temporarily increased after injection of HPMC in the anterior chamber, but returned to normal within 2-3 days on average, and the fluctuation of intraocular pressure in the control group was not statistically significant (P>0.05). .

The comparison of the difference between the initial intraocular pressure and the initial intraocular pressure of the experimental group A shows that the difference between the intraocular pressure fluctuation after the injection of the experimental group A and the initial intraocular pressure is not statistically significant.

2.2 Observations

2.2.1 Normal eye

The trabecular sheet is centered on collagen connective tissue, surrounded by elastic fibers, covered with a single layer of endothelial cells and forms a trabecular space, and the proximal tubular tissue is composed of endothelial cells and extracellular space, and there are amorphous substances of different densities in the space. Aqueous plexus and comb ligament can be seen. Similar to other literature reports.

2.2.2 Control group

There was no significant difference between the specimens of each stage and the normal eyes in each eye.

2.2.3 Experimental Group A

In the early stage, the trabecular meshwork was slightly expanded, and the cytoplasmic vacuoles of the endothelial cells in the inner wall of the aqueous humor cluster increased, and no abnormalities were observed in the rest of the specimens.

2.2.4 Experimental Group B

In the early stage, trabecular mesh expansion, collagen fiber proliferation, interstitial fiber proliferation can be seen in the proximal tubular tissue, and medium-to-high-density amorphous substances can be seen in the gap. In the 3rd week after the injection, only mild hyperplasia of collagen fibers was observed in the samples, and the chamber angle tissue returned to normal in the 10th week after the injection.

3 Discussion

Regarding the change of intraocular pressure caused by anterior chamber injection of HPMC, different authors have different opinions. In this experiment, 2% HPMC was injected into the anterior chamber of the rabbit twice, and it was found that the intraocular pressure rose to the peak 3~6 hours after the injection, and returned to normal within 2~3 days. It is well known that high intraocular pressure will lead to a series of pathological changes in trabecular tissue. Although the use of HPMC in human eye surgery can lead to a transient increase in intraocular pressure, it is easy to control and avoid, and there are many reasons for postoperative intraocular pressure increase. Therefore, the interference of intraocular pressure factors must be excluded to discuss the influence of HPMC on the angle tissue. The purpose of observing the intraocular pressure levels in different periods after injection in this experiment is to observe and exclude the influence of high intraocular pressure factors on the experimental results.

Intraocular pressure fluctuations during the sampling and fixation process will also cause changes in the morphology of the trabecular meshwork, so we adopt in-situ fixation and in vivo sampling under approximately constant pressure.

The slight expansion of the trabecular meshwork and the increase of cytoplasmic vacuoles in the endothelial cells of the lumen wall of the aqueous humor plexus seen in the experimental group A at the initial stage of injection are a kind of adaptation of the tissue to the stimulation of the macromolecular substance HPMC entering the anterior chamber. Sexual response, the discharge of aqueous humor depends on the drinking effect of vacuoles in endothelial cells, the expansion of trabecular meshwork and the increase of vacuoles are beneficial to the discharge of aqueous humor and HPMC. By the 3rd and 10th week, the anterior chamber angle tissue had returned to normal, indicating that HPMC itself had no toxic effect on the anterior chamber angle tissue after excluding the influence of high intraocular pressure factors, and the presence of HPMC in the anterior chamber would not cause irreversible changes. Moreover, in this experiment, 2% HPMC was injected into the anterior chamber of the rabbit twice, and its influence on intraocular pressure and chamber angle tissue has exceeded that of HPMC used in clinical operations. The amount of remaining HPMC is much smaller than that in this experiment.

The intraocular pressure of rabbit eyes in experimental group B was once significantly increased at the initial stage of anterior chamber injection, and the observed pathological changes were consistent with the lesions caused by the high intraocular pressure model reported in the literature, and were similar to some changes in glaucoma. It is generally believed that This is a change in tissue response and compliance to elevated intraocular pressure. With the passage of time and the recovery of intraocular pressure, various pathological changes gradually disappeared. It shows that the pathological changes caused by the transient high intraocular pressure caused by injection of HPMC in the anterior chamber are also transient.

To sum up, massive injection of 2% HPMC and retention of the anterior chamber can lead to a transient increase in intraocular pressure and corresponding pathological changes, but the lesions are also transient. 2% HPMC is non-toxic and destructive to the angle tissue, so as long as the intraocular pressure can be well controlled after the operation, it is safe to apply a conventional amount of HPMC in the eye. Of course, the structure of the angle of the human eye is different from that of the rabbit eye, and HPMC enters the anterior chamber The postoperative metabolic process is not completely clear, and there is a risk of increasing intraocular pressure, so it is still advisable to rinse as appropriate after surgery, but if there is difficulty, it is not necessary to force it out, so as not to cause other serious complications.