We have demonstrated in this study that PI elevates the inflammatory response compared to ACP alone, which manifested as fibrinous anterior chamber reaction and peripheral anterior synechiae (PAS). The selective induction of aqueous humor TGF-β1 and TGF-β3 by PI suggests this procedure may molecularly impact the success of glaucoma surgery by inducing postoperative fibrosis.

Fibrin reaction may result from increased vascular permeability caused by surgical procedures. It typically occurs in the early postoperative period and responds well to steroid therapy, resolving within 1 day to 3 weeks, supporting inflammation as an underlying cause. Hence, PI may increase intraocular inflammation, resulting in fibrinous AC reaction in the affected rabbit eyes.

Similarly, peripheral anterior synechiae (PAS) is commonly associated with AC inflammation. PAS has been observed in a variety of ocular conditions including uveitis, neovascular glaucoma, iridocorneal endothelial syndrome, as well as following trauma and surgeries. Patients typically display symptoms associated with underlying active or prior inflammatory processes. The deposition of inflammatory cells, fibrin and proteins, which is thought to stimulate the formation of adhesions between structures, is central to PAS pathophysiology. Overall, the increased incidents of PAS associated with PI in rabbits corroborate observations of increased inflammation in patients who have had this procedure [19].

We did not find a significant difference between aqueous VEGF-A levels in eyes with PI compared to ACP alone. Even though PI did not increase aqueous VEGF-A, it is still possible for PI to increase VEGF-A in other sites. Local VEGF-A elevation in the iris may contribute to iridal inflammation that potentially promotes fibrinous anterior chamber reaction and PAS. Nonetheless, we expect that the lack of elevation of aqueous VEGF-A will be beneficial in avoiding a wider dissemination of angiogenic influence in the AC and the wound site.

The expression profiles of TGF-β isoforms in the rabbit aqueous humor is striking, with ratios heavily skewed towards TGFβ2 (4 TGF-β1: 800 TGF-β2: 1 TGF-β3). This is similar to that in humans, where TGF-β2 comprised the bulk of the isoforms [21, 22]. TGF-β2 was reported to be elevated in the aqueous humor of glaucoma patients [15]. Together with data correlating aqueous TGF-β2 with high IOP [23], and experimental evidence for the capacity of TGF-β2 to reduce outflow facility when perfused into cultured human anterior segments [24], the implication of aqueous TGF-β2 in the pathogenesis of the ocular hypertension in glaucoma is compelling. Our study revealed that neither ACP nor PI caused significant elevation in aqueous TGF-β2 levels.

On the other hand, aqueous TGF-β1 levels increased in response to ACP on POD 1, but this elevation was only made significant when PI was performed in conjunction with ACP. ACP may trigger TGF-β1 induction as this procedure is associated with increased inflammation. An experimental study on beagle eyes indicated that ACP caused significant increase in inflammatory markers in the canine aqueous humor within 4 hours [25]. This time frame may correspond with the increase in aqueous TGF-β1 shortly following ACP, and suggests that TGF-β1 may be a constituent of the cocktail of molecules induced as part of the inflammatory response to ACP. The extra push rendered by PI to cause significant increase of TGF-β1 above baseline suggests that concomitant iris injury adds to the rise of this cytokine. Interestingly, Tenon’s fibroblasts from pseudoexfoliation (PEX) syndrome/ glaucoma were demonstrated to have heightened sensitivity to TGF-β1 than TGF-β2 in relation to expression and synthesis of profibrotic markers [26]. It may therefore be tempting to speculate that, at least in PEX glaucoma, where aqueous TGF-β2 levels were normal [16] but TGF-β1 was elevated [27], there is the potential for further increase in aqueous TGF-β1 induced by ACP/ PI to raise the risk of unfavorable bleb development after surgery than other glaucomas.

TGF-β3 levels were also induced by PI in the rabbit aqueous humor. Again, the PI-associated significance suggests that the damaged iris is a source of the induced aqueous TGF-β3. However, it is unclear whether the iris is a depot of TGF-β3. Interrogation of the anterior segment by means of immunohistochemical evaluation failed to detect TGF-β3 [15]. Although measurable, mean TGF-β3 concentration detected in the rabbit aqueous humor was in the order of 10 pg/ml. Similarly, TGF-β3 in normal human aqueous was no higher than 10 pg/ml21, or below detectable level in at least one study [22], supporting the likelihood that TGF-β3 is present at physiologically low levels in the aqueous humor. Nevertheless, the induction of TGF-β3 in the aqueous may have interesting implications for wound healing, with direct impact on the damaged iris, and potential indirect effects on the postoperative conjunctiva after glaucoma surgery. A striking quality of the iris is its ability to resist abundant scarring after injury, as suggested by observations of limited scar formation in human iris subjected to surgical iridectomy [28]. While the mechanism for the ability of the iris to avoid a profound scar response remains unclear, the aqueous humor is speculated to be involved [29]. Our data suggest that increased aqueous TGF-β3 may be a candidate for proffering the anti-fibrotic response in the wounded iris. The basis for this conjecture is founded on cumulative experimental and clinical evidence that TGF-β3 has anti-fibrotic activities, particularly in reducing dermal scarring [30, 31]. It is therefore tempting to speculate that elevated levels of TGF-β3 induced by the wounded iris may be a protective strategy by preventing excessive scarring in response to certain traumas, such as surgical iridectomy. By extension, increased aqueous TGF-β3 induced by PI as part of glaucoma surgery may be expected to percolate into the conjunctival bleb and aid in the reduction of postoperative scarring. Yet, recombinant TGF-β3 was shown to stimulate the mouse conjunctival scarring response [12]. Contradictory experimental findings relating to the functions of TGF-β3 are not new, leading to the suggestion that TGF-β3 has complex roles [30, 32], which we predict to be tissue-specific and condition-dependent. It is possible that the distinct molecular environments of the wounded iris and the postoperative conjunctiva may provide opposite regulation of TGF-β3 activities such that divergent wound healing outcomes may ensue. Interestingly, like TGF-β1 [27], increased TGF-β3 was detected in the aqueous humor of PEX glaucoma eyes [21]. The further induction of not one, but these two specific TGF-β isoforms by ACP/ PI in the aqueous humor as part of glaucoma filtration surgery suggests the potential for even greater risk of postoperative scarring in the PEX glaucoma conjunctiva. Indeed, a recent comparative study of the long-term success rates of glaucoma surgeries with PI indicated lower success for PEX glaucoma than for primary open-angle glaucoma eyes [33]. Hence, the necessity of PI in glaucoma surgery, particularly for treatment of PEX glaucoma, deserve additional consideration with respect to the potential impact of TGF-β isoforms on postoperative scarring and long-term surgical success.

In summary, we have demonstrated that PI, as performed in a rabbit model, is associated with increased inflammation. While PI did not increase the amount of aqueous VEGF-A, it caused significant aqueous elevations of TGF-β1 and TGF-β3. The upregulation of TGF-β3 may have implication for reduced iris fibrosis and long-term PI patency, whereas increased aqueous TGF-β1 and TGF-β3 may promote scarring, especially in PEX glaucoma eyes with their apparent unique sensitivities to these isoforms. Although all PI-associated tissue and molecular responses were primarily acute and not sustained in this normal rabbit model, it may contribute to the increased wound healing response in glaucoma eyes undergoing filtration surgery.

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