Comparison of Single versus Multiple Injections of the Protein Saratin for Prolonging Bleb Survival in a Rabbit Model

Abstract

Purpose.: We compared the anti-fibrotic effects of single versus multiple postoperative injections of saratin following glaucoma filtration surgery (GFS) in the rabbit model.

Methods.: The experiment was in two parts. To determine the optimal frequency for postoperative therapy, seven New Zealand White (NZW) rabbits received an injection of saratin under the superior conjunctiva bilaterally, and ocular tissue concentration was determined using Western blot and bicinchoninic acid (BCA) assay. Next, 32 additional NZW rabbits underwent filtration surgery and received either single or multiple-dose saratin treatments. Mitomycin-C (MMC) and balanced saline solution (BSS) treatment acted as positive and negative controls, respectively.

Results.: Rabbits receiving only one perioperative saratin injection had a mean bleb survival time of 29.8 ± 5.3 days, while those receiving multiple (either 3 or 5+) injections of saratin had mean bleb survival times of 26.3 ± 8.1 and 26.4 ± 4.2 days, respectively. Analysis of variance with post-hoc testing showed the single injection group had a statistically favorable effect on bleb survival duration compared to BSS controls and was not significantly different from MMC. The conjunctivas of the saratin-treated rabbits did not show the thinning or avascularity that was seen in the MMC treatment group. Rabbits receiving more than three injections of saratin suffered temporary conjunctival redness and two rabbits had upper eyelid edema.

Conclusions.: A single postoperative injection of saratin was able to prolong the duration of bleb elevation when compared to BSS controls. Additional treatments of saratin seemed to reduce effectiveness and caused short-term eye inflammation.

Introduction

Glaucoma filtration surgery (GFS) is the gold standard when medication and laser surgery have proven insufficient in managing intraocular pressure (IOP). ^1,2 However, postoperative scar tissue formation at the implant site can cause the filter to fail and remains a serious problem. ^3,4 Intraoperative treatment with the antimetabolites mitomycin-C (MMC) and 5-fluorouracil (5-FU) can improve the outcome of GFS, but carry significant risks as well ⁵ ; because they work in a nonselective manner, antimetabolites can compromise the integrity of the filtering bleb, resulting in hypotony, blebitis, endophthalmitis, bleb leakage, and vision loss. ^2,3,5–8  

Saratin, a 12 kilodalton (kD) protein originally isolated from the saliva of the leech Hirudo medicinalis , was studied initially for its ability to prevent platelet aggregation, ^9–12 making it useful in the treatment of intimal hyperplasia ^13,14 and atherosclerotic lesions. ¹⁵  

In a previous study, we demonstrated that Saratin significantly improves the duration of bleb survival in a rabbit model of filtration surgery compared to saline controls, but only when delivered by a topical application with a partial-thickness Weck sponge (in a manner used clinically for MMC application) augmented with subconjunctival injections on postoperative days (POD) 4 and 8, and not when administered only using a partial-thickness Weck sponge direct application technique for 5 minutes during surgery. ¹⁶ Given these data, we hypothesized that subconjunctival injection, rather than sponge application, was necessary to achieve therapeutic levels of the protein within ocular tissues. 

The goals of the current study were first to determine the optimum frequency of administration for subconjunctival injections of saratin, second to compare the duration of bleb survival using different numbers of saratin applications, and third to identify potential side effects of the saratin injections. 

Materials and Methods

We used for this study 39 New Zealand White (NZW) rabbits weighing between 2 and 4 kg. All animal experiments performed adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the University of Florida's Institutional Animal Care and Use Committee. 

Western Blot Analysis

To determine the retention of saratin in ocular tissues, seven NZW rabbits received injections of biotinylated saratin protein at maximum solubility (0.5 mg/0.1 mL) beneath the superior conjunctiva in each eye. Conjunctiva, Tenon's capsule, and scleral tissues were harvested for Western blot assay at 30 minutes; and 1, 2, 4, and 8 hours; and 1, 2, 3, 4, 5, 6, and 7 days post-injection. 

Samples were homogenized in 0.5 mL of buffer and the levels of protein measured by bicinchoninic acid (BCA) assay. Values ranged from approximately 2 to 4 mg/ml. Homogenate samples were added to 2× SDS sample buffer with reducing agent, and a constant amount of protein (50 μg/lane) was added to lanes of 10% polyacrylamide SDA gels (Invitrogen, Grand Island, NY) and electrophoresis was performed until the tracking dye had reached the end of the gel. Proteins then were transferred to nitrocellulose membranes using the Invitrogen I-Blot transfer apparatus and the membranes were incubated overnight at 4°C in Pierce Superblock blocking buffer (Thermo Scientific, Rockford, IL) containing 0.05% of Tween 20. The blots were washed 4 × 5 minutes with 1 × PBS with 0.05% Tween 20 wash buffer and incubated for one hour on a rocker with anti-saratin monoclonal antibody at 5 μg/mL diluted in blocking buffer. The blots then were washed 4 × 5 minutes and incubated for one hour on a rocker with Sigma rabbit anti mouse IgG-AP whole molecule secondary antibody (Sigma-Aldrich, St. Louis, MO) at a concentration of 1:1000 diluted in blocking buffer. Blots were washed 4 × 5 minutes and incubated for 3 minutes in Sigma NBT + BCIP substrate solution (Sigma-Aldrich, St. Louis, MO). 

GFS Treatment Groups

After the optimal injection interval for saratin was determined, 32 NZW rabbits were randomized into the five treatment groups. All 32 rabbits underwent glaucoma filtering operation in their left eyes with one surgeon (MBS) performing all the procedures; the right eyes were not operated on and served as controls. 

Rabbits in the first experimental group (n = 6) received a single 0.1 mL injection of 5 mg saratin under the superior conjunctiva near the bleb immediately following surgery. In the second experimental group (n = 6), rabbits were scheduled to receive a total of three injections of 0.5 mg/0.1 mL saratin, with a single perioperative injection at the end of surgery (similar to group 1) plus two post-surgical injections given on POD 3 and 6. Rabbits in the third experimental group (n = 6) were scheduled to receive a total of eight injections of 0.5 mg/0.1 mL saratin, with the same perioperative injection plus a total of seven postoperative injections, given on POD 3, 6, 9, 12, 15, 18, and 21. 

Those in the negative control group (n = 7) received a 0.1 mL injection of balanced salt solution (BSS) delivered in a similar fashion immediately following surgery and up to four additional injections, given every third day afterwards to match the experimental groups. 

Finally, rabbits in the positive control group (n = 7) received a partial thickness Weck sponge (Alcon Surgical, Fort Worth, TX) application of 0.4 mg/mL MMC at the time of surgery and were not given any postoperative injections. 

Glaucoma Filtering Operation

The rabbits were anesthetized with an intramuscular injection of a combination of ketamine (Ketaject, 50 mg/kg; Phoenix Pharmaceuticals, Inc., Burlingame, CA) and xylazine (Xyla-ject, 10 mg/kg; Phoenix Pharmaceuticals, Inc.). A topical anesthetic, 0.1% proparacaine eye drop (Bausch & Lomb, Tampa, FL) also was administered. The surgical technique for the glaucoma filtration operation was identical to the procedures described in our previous publication using saratin. ¹⁶  

Postoperative Injections and Clinical Evaluations

Following the operation, the eyes were examined three times per week by an examiner masked to the treatment group. The examiner looked for the presence of bleb elevation as well as for any complications that may have resulted from the surgeries or various treatments, including conjunctival injection, edema, anterior chamber shallowing, tube malpositioning, hyphema or subconjunctival hemorrhage, bleb leaks, and corneal or lens opacification. Bleb failure was declared after the masked observer deemed the bleb to be flat in two consecutive examinations. The first of the two dates was recorded as the endpoint. After the clinical examination, rabbits in the saratin and BSS treatment groups also were given injections of 0.1 mL saratin or 0.1 mL BSS at their scheduled times by a separate, nonmasked researcher. 

The rabbits were anesthetized with isoflurane during their postoperative examinations. Topical 0.1% proparacaine anesthetic eye drop was administered for those that required a postoperative injection and a speculum was used to retract the eyelids. The 0.1 mL injections were given near the bleb using a 30-gauge needle on a 1 mL syringe. 

Histology

Eyes were obtained from one rabbit in each of the five groups on POD 12, so bleb tissues from each group could be examined at a uniform time. Eyes were obtained from all the remaining rabbits after the bleb was observed to be flat in two consecutive clinical evaluations by a masked observer. 

The tissues were fixed for 24 hours in a 10% neutral buffered formalin solution. The globes were placed in tissue cassettes, and then processed through graded ethanol and xylene, using a Sakura Tissue-Tek VIP 5 tissue processor (Sakura, Torrance, CA). The tissues were infiltrated with paraffin (Richard-Allan Scientific, Kalamazoo, MI) and embedded on a Tissue Tek III embedding center. Sagittal serial sections of the globes were taken. Once deparaffinized, the sections were stained using either standard Harris hematoxylin and eosin (H&E) or Masson's trichrome. The slides were examined under light microscopy and representative sections were photographed using a Canon EOS T1i digital camera (Canon, Lake Success, NY) attached to an Olympus Vanox microscope (Olympus, Center Valley, PA). 

A semiquantitative comparison of the implant site was conducted on the POD 12 specimens by a single masked observer (JM). Slides stained with Masson's trichrome were used to determine level of fibrosis. Slides stained with Harris hematoxylin and eosin were used to compare the amount of cellularity. 

Statistical Analysis

ANOVA testing was used to determine any significant difference in bleb survival duration among the treatment groups. Two post-hoc tests, the Tukey's Honestly Significantly Different (HSD) test and Fisher's Least Significant Difference (LSD) test, then were used to determine significance between pairs of relevant groups. 

Results

Western Blot Analysis

Western blot assay showed a strong band for the saratin protein for up to 2 days (Fig. 1). On day 3, protein levels began to drop off and were barely detectable on days 4 and 7. These results were confirmed using BCA protein assay to quantify total protein concentrations (Fig. 2). 

Figure 1. 

 

Western blot assay for saratin protein for time points 30 minutes; and 1, 2, 4, and 8 hours (top); and 1, 2, 3, 4, and 7 days (bottom).

Figure 2. 

 

Total protein concentration in ocular tissue samples as determined by BCA protein assay.

Bleb Survival

Rabbits receiving only one injection of saratin had an average time to bleb failure of 29.8 ± 5.3 days. Rabbits in group 2, which received two additional injections of saratin had an average time of 26.3 ± 8.1 days, and those in group 3, which received more than five injections, had an average time of 26.4 ± 4.2 days. The average time to bleb failure for the negative control rabbits receiving BSS injections was 21.0 ± 6.0 days. Rabbits receiving MMC treatments had bleb elevation durations averaging 33.8 ± 6.2 days. The rabbits from each treatment group sacrificed on POD 12 were omitted from data calculations. In addition, one rabbit in the three-injection group (group 2) and one from the BSS control group had medical complications unrelated to the surgery and treatment, and also were omitted from bleb survival calculations. These results are represented in a Kaplan-Meier survival plot (Fig. 3) and summarized using a box-and-whisker plot (Fig. 4). 

Figure 3. 

 

Kaplan-Meier bleb survival plot of eyes treated with one injection of saratin (solid line), 3 injections of saratin (dashed line), more than five injections of saratin (dotted line), as well as positive MMC (grey solid line), and negative BSS (grey dotted line) controls. For each rabbit, bleb failure was declared after the bleb appeared flat in two consecutive masked clinical examinations.

Figure 4. 

 

Box-and-whisker plot of bleb survival for each treatment group. First and third quartiles are delineated by boxes. Whiskers represent maxima and minima.

ANOVA testing revealed a significant difference among the treatment groups (P = 0.018) at a 95% confidence interval (CI). Additional post-hoc testing using Tukey's HSD and Fisher's LSD tests confirmed that the MMC-positive control group had a significantly longer bleb survival duration than the BSS group at a 95% CI. LSD analysis indicated a significant difference in bleb survival duration between the single saratin injection and the BSS groups (P = 0.024) at a 95% CI. No significant improvements over BSS were found for the multiple saratin injection treatment groups (P > 0.1). Finally, there was no statistically significant difference between the MMC group and any of the three saratin treatment groups (P > 0.1). 

Clinical Evaluation

During clinical examinations, bleb avascularity was noted among the MMC rabbits. Rabbits in the saratin treatment group that received more than five injections of the protein all had eye redness, and two had upper eyelid edema in the operated eye (Fig. 5A). The edema was temporary and resolved 10 to 15 days after injections were halted (Figs. 5B, 5C). Rabbits receiving only one injection of saratin did not show any adverse effects, while those receiving three total injections had some eye redness but no eyelid edema. Through consultation with the veterinarians of animal care services at the University of Florida, the maximum number of additional saratin and BSS injections was limited to 5 (POD 0–12) rather than the initially-planned 8 injections (POD 0–21). In addition, a once-daily oral meloxicam treatment was administered to the two rabbits that exhibited eyelid swelling. 

Figure 5. 

 

(A) Temporary tissue swelling and redness observed after sixth saratin injection on POD 15. (B) Eyelid edema in same rabbit on POD 18. (C) Improvement noted 10 days later on POD 28, 13 days after injections discontinued.

Histology

Histologic examination of tissue samples taken on POD 12 showed differences in implant site morphology among the treatment groups (see Table). No capsule was seen in rabbits that received the saratin or MMC treatments (Figs. 6A, 6B). By comparison, the rabbits that received BSS injections consistently formed a fibrotic capsule around the implant (Fig. 6C). Further examination of the 12-day samples showed that eyes treated with MMC had thin, relatively avascular conjunctivas (Fig. 7B). In contrast, saratin- and BSS-treated eyes displayed normal conjunctival morphology with an even distribution of goblet cells (Figs. 7A, 7C). 

Figure 6. 

 

Representative sections of the implant site taken on POD 12. H&E ×20. (A) Saratin after 1 injection. (B) MMC. (C) Balanced saline solution.

Figure 7. 

 

Representative sections of the bleb conjunctiva taken on POD 12. H&E ×100. (A) Saratin. (B) MMC. (C) Balanced saline solution.

Table. 

 

Histologic Changes around Implant Site of POD 12 Rabbits

Treatment Group	Fibrosis (Masson's Trichrome)	Cellularity (H&E)
Saratin (1 injection)	++	++
Saratin (3 injections)	++	+++
Saratin (5 injections)	+	+++
BSS	+++	++
MMC	+/−	+/−

 −, absent; +/−, weakly present; +, ++, +++, present in increasing amounts, as graded by the masked observer (JM).

Histologic comparison of eyes receiving only one injection of saratin to those from the two groups that received multiple injections showed least inflammatory response in the former group. Eyes receiving multiple treatments showed sites of inflammation that increased in area concomitant with the number of treatments, and demonstrated marked inflammation and necrosis in the bulbar conjunctiva (Fig. 8). In conjunction with the increased inflammatory response, mast cells appeared, often in clusters of cells, in the multi-treatment eyes (Fig. 9), but these clusters were not seen in the single injection or BSS samples. 

Figure 8. 

 

Tissues near injection site, close to the medial canthus of a rabbit that received 6 total injections of saratin. Arrows show bulbar conjunctiva with areas of inflammation and necrosis ([A], H&E, ×20). Higher magnification of the area ([B], H&E, ×250).

Figure 9. 

 

Mast cells seen by H&E (A) and toluidine blue (B) in the bulbar conjunctiva away from the implant site of a rabbit receiving 4 total injections of saratin, sacrificed on POD 12 (×20).

Discussion

The prevention of ocular scarring is a relatively unexplored use for the saratin protein. A previous report from this group ¹⁶ concluded that 2 postoperative injections of saratin could prolong bleb survival significantly over saline controls. However, little was known regarding the optimal frequency and duration for these injections to maximize bleb survival, nor the potential for toxic effects following repeated treatment. 

Using Western blot analysis, it was determined that tissue concentrations of saratin remained moderately high for up to three days following injection, after which they dropped significantly. These data indicated that the saratin injections should be given every third day in the two multi-treatment groups (2 and 3) to maintain continuous saratin tissue levels. 

In the second part of this investigation, we re-evaluated the effect of saratin on bleb survival. Post-hoc testing showed that rabbits receiving only one perioperative injection of saratin had a significantly longer duration of bleb elevation than BSS controls. No differences over saline controls could be observed for rabbits receiving multiple saratin injections. 

While the mean bleb survival duration was slightly shorter for the single injection saratin group, it was not statistically significantly different from the MMC group. Histologic examination of the single injection treatment group showed that saratin and MMC had similar effects on reducing capsule formation when compared to the negative BSS controls. Of note, saratin-treated eyes did not have the conjunctival thinning and bleb avascularity that was seen in the MMC rabbits. 

Interestingly, the multiple saratin injection treatment groups had reduced mean bleb survival times (26.3 ± 8.1 and 26.4 ± 4.2 days, respectively) when compared to the single saratin injection group (29.8 ± 5.3 days). In addition, clinical and histologic examination found evidence suggesting that multiple injections of saratin initiated a temporarily heightened inflammatory response within the surrounding ocular tissues, including the upper eyelid. This inflammation seemed to worsen with an increased number of injections, presenting as conjunctival redness after the third injection and in two cases upper eyelid edema after five injections. The swelling did not appear painful to the touch and did not interfere with feeding or any other activities of the rabbit. This side effect did not appear to be permanent; with discontinuation of injections, the swelling improved after a few days and ultimately resolved completely. 

In conclusion, there was no statistical difference in duration of bleb survival between a single perioperative injection of saratin and MMC (0.4 mg/mL given by sponge for 3 minutes). Histologically, saratin, similarly to MMC, delayed capsule formation without causing conjunctival thinning and avascularity. Multiple injections of saratin given postoperatively, if anything, seemed to reduce efficacy and increase inflammatory side effects. Given the observed side effects and possible decreased efficacy associated with multiple treatments, future studies should be designed using only a single, perioperative dose of saratin. Clinically, this is the preferred dosage regimen as it avoids extra postoperative visits and would mimic current MMC use. 

Footnotes

 Supported by a grant from BioVascular, Inc. and in part by an unrestricted departmental grant from Research to Prevent Blindness (RPB).

Footnotes

 Disclosure: J. Min, None; Z.L. Lukowski, None; M.A. Levine, None; C.A. Meyers, None; A.R. Beattie, None; G.S. Schultz, None; D.A. Samuelson, None; M.B. Sherwood, BioVascular, Inc. (F)