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引言
猫角膜坏死灶（FCS）是一种独特的角膜疾病，以局灶性基质坏死伴角膜呈深棕色至黑色变色为特征。该病最常见于短头猫品种，可能导致慢性眼部不适，需要手术治疗[1, 2, 3, 4, 5]。尽管其外观具有特征性且已被认识多年，但角膜坏死灶的病因发病机制仍不完全清楚[6]。已提出多种机制，包括慢性角膜刺激、病毒感染、泪膜异常和遗传易感性[1, 2, 3, 4, 5, 6, 7]。
特征性棕黑色色素沉着的来源也已被研究。早期研究提示铁沉积可能参与变色，因为据报道坏死灶中的铁浓度较正常角膜组织显著升高[8]。然而，后续研究得出了相互矛盾的结果。Featherstone等未能证实铁水平升高，但报告在坏死灶内存在黑色素颗粒[9]。同样，Cullen等描述了基质变性和电子致密物质，但未能一致地鉴定出黑色素[10]。最近，有研究探讨了来源于泪膜的卟啉的可能作用；然而，光谱荧光分析未能在猫角膜坏死灶或眼组织中检测到卟啉[11]。
微量元素在生物组织内的氧化应激、炎症和细胞外基质重塑中发挥重要作用[12]。分析技术的进步使得能够详细表征生物样品（包括泪膜）中的元素组成。粒子诱导X射线发射（PIXE）是一种灵敏的多元素分析技术，能够以最少的样品制备检测少量生物样品中的微量元素[13, 14]。PIXE此前已被用于分析人和动物泪液中的微量元素组成[15, 16, 17]。
本研究旨在使用PIXE分析比较猫角膜坏死灶与健康角膜组织的元素组成。我们假设坏死灶组织会表现出独特的元素谱，从而为探讨该病的发病机制提供线索。
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材料与方法
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动物
本研究设计为观察性病例对照研究，纳入在Koret兽医学院收集的被诊断为FCS的猫以及健康对照角膜。该研究经机构动物护理和使用委员会批准（方案PET‐2024‐14‐NE）。在采集样品前已获得畜主书面知情同意，以将客户拥有的猫纳入研究，并将临床数据和手术切除的角膜组织用于研究目的。
最初纳入9只客户拥有的猫（11只眼；2只猫为双侧患病），均被诊断为FCS并接受板层角膜切除术手术治疗。从病历中获取包括年龄、性别和品种在内的信号特征数据，以及眼部病史、既往局部或全身治疗、病变侧别、角膜血管化、病变大小和病变深度。所有患者均由委员会认证的兽医眼科专家进行完整的眼科检查，包括裂隙灯生物显微镜检查（Kowa SL‐17，Kowa Company Ltd.，东京，日本）、回弹式眼压计测量（TonoVet PLUS，Icare Finland Oy）、Schirmer泪液试验测量和荧光素染色。
手术当天早晨停用局部药物，以确保最后一次局部治疗与坏死灶切除之间至少间隔12小时。板层角膜切除术在全身麻醉下使用手术显微镜进行。如先前所述[9]，将坏死灶连同周围约1 mm健康角膜组织整块切除。切除的样品立即放入无菌Eppendorf管中，并在采集后10分钟内于−80°C冷冻。
对照角膜取自4只无眼部病变、因与本研究无关的原因被安乐死的猫。所有对照眼均经裂隙灯检查证实眼科正常。角膜样品在死后采集，并采用相同方案储存。
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PIXE元素分析
样品在处理前解冻，并使用双面碳胶带直接固定在硅片上。随后将固定好的样品放入高真空腔室（约10⁻⁷ Torr）中进行分析。PIXE测量使用1.7 MV串联Pelletron加速器（National Electrostatics Corporation，Middleton，WI，USA）进行，提供约2.014 MeV的质子束，准直至直径约1.5 mm。从辐照样品发射的特征X射线谱使用配备Si₃N₄窗口的FAST SDD硅漂移探测器（Amptek，Bedford，MA，USA）采集。探测器相对于束流法线置于约40°位置（IBM几何构型），并使用12 μm Mylar箔作为滤片。按照实验室方案，样品被辐照至固定的收集电荷量（Q），并使用电子抑制器以尽量减少二次电子效应。能量校准和质量控制使用金属标准品及美国国家标准与技术研究院（NIST，Gaithersburg，MD，USA）的有证标准物质进行。谱图使用GUPIX软件处理以确定净峰面积。由于标本厚度和水合状态存在差异，元素值以相对峰丰度而非绝对浓度报告[17]。
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统计分析
数据分布使用Shapiro–Wilk检验评估。由于主要结局变量（微量元素水平）不服从正态分布（p ≤ 0.049），数据以中位数 ± 均值标准误（SEM）表示，并使用非参数方法分析。 sequestration与对照组织间元素值的比较使用Mann–Whitney秩和检验。统计分析使用SigmaPlot 15.0版（Systat Software Inc.）进行，p值 < 0.05视为具有统计学意义。
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结果
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研究群体
手术切除前， sequestration组9只猫中有7只（78%）接受过局部眼部治疗，包括0.5%土霉素眼膏（Ocíl，VAPP—Veterinária e Agropecuária，S.A.）和抗病毒药物（喷昔洛韦；Fenlips，Perrigo）[18]，每日两次给药，单用或联用。部分猫还接受了全身多西环素（Doxycycline；Doxylin，Teva Pharmaceutical Industries Ltd.）10 mg/kg每日一次。
研究组包括9只猫（11只眼）确诊为角膜 sequestration。患病猫主要为短头品种或易感品种，包括三只斯芬克斯猫（33%）、两只波斯猫（22%）、两只英国短毛猫（22%）、一只未指明品种的短头猫和一只家养短毛猫。就诊时中位年龄为3.0岁（范围：0.5–12岁）。七只猫（78%）为雄性，两只（22%）为雌性。病变大小范围为1至8 mm（中位数：3 mm），估计基质深度范围为20%至70%（中位数：40%）。所有病变均呈深色。11只眼中有8只（72%）存在角膜血管化，而三只眼未见血管长入证据。
对照组包括四只因全身性疾病被安乐死且无眼部表现的家养短毛猫，包括肺癌、车祸后创伤性损伤（n = 2）和动脉血栓栓塞。三只猫为雄性，一只为雌性，年龄范围为4至13岁（中位数：5.5岁）。
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元素分析
在所有分析元素中，仅溴和铁在 sequestration组与对照组之间显示出统计学显著差异（图1和表1），在 sequestration组中检测到而在对照组中缺失。溴在所有对照样品中均未检出，但在 sequestration组中始终存在，中位数 ± SEM峰面积为110 ± 50，对照组为0（p = 0.004；图1A）。同样，铁在对照样品中缺失而在 sequestration组中存在（751 ± 336 vs. 0；p = 0.047；图1B）。钠、钾、氯、镁、磷、钙、硫和锌在 sequestration与对照角膜之间无显著差异（均p > 0.05；图2）。 sequestration组中钠、氯、钙、硫和锌的中位峰面积数值上较高，而钾、镁和磷与对照组相比显示相似或较低的值。
[图：箱线图展示角膜sequestra和健康对照角膜中溴（A）和铁（B）的峰面积]
[图：箱线图展示其他微量元素（钠、镁、磷、硫、锌、钾）的峰面积]
TABLE 1. Peak area values for elemental composition in corneal sequestra and healthy control corneas, as determined by particle‐induced X‐ray emission (PIXE).
| Element | Sequestra | Control | p (Mann–Whitney) |
|---|---|---|---|
| Na | 65 609 ± 3113 (14163–134 362) | 46 183 ± 3346 (29762–58 482) | 0.192 |
| Mg | 5849 ± 360 (2649–14 772) | 6014 ± 107 (5214–6149) | 0.896 |
| P | 139 427 ± 8544 (21117–303 829) | 143 325 ± 13 548 (60053–177 123) | 1.000 |
| S | 443 782 ± 6764 (264616–477 509) | 334 365 ± 10 924 (291231–391 789) | 0.090 |
| Zn | 105 ± 8 (25–316) | 72 ± 6 (45–91) | 0.192 |
| K | 82 572 ± 4324 (10868–157 687) | 123 635 ± 15 407 (57980–181 217) | 0.240 |
| Ca | 26 712 ± 6814 (10380–268 554) | 21 023 ± 1786 (15147–32 193) | 0.192 |
| Fe | 751 ± 336 (0–3333) | 0 (0–0) | 0.047 |
| Cl | 79 056 ± 2473 (6344–101 465) | 62 584 ± 8589 (39552–108 189) | 0.794 |
| Br | 110 ± 50 (39–625) | 0 (0–0) | 0.004 |
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讨论
在本研究中，使用PIXE进行的元素分析揭示了在猫角膜sequestra中存在溴和铁，而在正常猫角膜中未检测到这些元素。这些发现表明，sequestral组织具有独特的生化环境，可能反映了与该疾病发展相关的局部炎症、氧化或退行性过程。
重要的是，在sequestral组织和正常角膜组织之间，钠、钾、氯、镁、磷、钙、硫或锌的水平未观察到统计学显著差异。这一观察表明，猫角膜sequestra并非以经典矿化过程为特征，经典矿化过程通常涉及钙或磷酸盐沉积增加。Featherstone等人此前也报告了类似结论，他们未在sequestral组织内发现显著的钙沉积[9]。总之，这些发现提供了强有力的证据，表明猫角膜sequestra特征性的深色色素沉着和基质变性并非源于常规的营养不良性矿化。
Sequestra内的铁积累可能参与该疾病的病理生理过程。铁因其通过Fenton化学催化活性氧形成的能力而被广泛认识，可生成羟基自由基，能够诱导脂质、蛋白质和核酸的氧化损伤[12]。在角膜内，这种氧化应激可能导致胶原降解、角膜细胞损伤和细胞外基质重塑，这些过程与猫角膜sequestra组织学观察到的基质坏死相一致[10]。除氧化特性外，铁还与眼组织中的色素改变有关。铁可与黑色素结合并影响黑色素氧化途径，可能加剧组织色素沉着。这种相互作用可能部分解释了临床上在sequestra中观察到的特征性棕黑色变色，尽管先前研究关于这些病变内黑色素存在的发现存在矛盾[9, 10]。与我们的发现相反，Featherstone等人使用扫描电子显微镜配合能量色散X射线光谱（SEM–EDX）在正常角膜和sequestra中均检测到低铁水平[9]。这一差异主要归因于采样方法、分析深度和灵敏度的根本差异：SEM–EDX仅评估最外层表面（1–2 μm），检测限约为0.1%（1000 ppm），正常角膜中的表层铁信号可能代表残留泪膜成分或表面伪影，而非真正的基质积累。相反，高能质子PIXE评估切除角膜标本的全层厚度，微量元素灵敏度低至1–10 ppm，捕获整个样本体积的总元素组成，并确认铁积累仅限于sequestral基质，而健康对照角膜中无可检测的铁。
铁介导的氧化应激也是铁死亡的核心机制，铁死亡是最近描述的一种调节性细胞死亡形式，以铁依赖性脂质过氧化为特征[19, 20]。铁死亡已被认为与多种退行性和炎症性疾病有关，并可能在铁积累和氧化应激共存的组织损伤中发挥作用。尽管目前尚不清楚触发铁死亡或角膜基质中黑色素结合所需的绝对浓度阈值，但铁在猫角膜sequestral组织中选择性存在而在正常对照角膜中完全缺失，这提出了一个假说：铁依赖性氧化途径可能促进角膜细胞死亡和基质变性。未来需要进行空间分辨的定量研究，以确定铁是否在特定细胞或细胞外区室中局部浓缩至足以驱动这些推测过程的水平。
溴仅在被隔离组织中检出是另一项有趣的发现。溴最近被确定为人类泪膜的可测量成分，表明其存在于眼表环境中[21]。溴还可作为嗜酸性粒细胞过氧化物酶（EPO）的生理性底物，该酶在炎症组织中嗜酸性粒细胞活化时释放。在过氧化氢存在下，EPO催化溴离子氧化为次溴酸（HOBr），这是一种强氧化剂，能够使细胞外基质蛋白中的酪氨酸残基溴化[22, 23, 24]。该反应导致溴酪氨酸衍生物的形成，后者被广泛认为是嗜酸性粒细胞介导组织损伤的特异性分子标志物[25]。
尽管在猫角膜隔离组织的组织学研究中对嗜酸性粒细胞的报道并不常见，但嗜酸性粒细胞相关的炎症过程可能在疾病的早期阶段或角膜损伤和修复过程中短暂发生。此外，嗜酸性粒细胞性眼表疾病，包括嗜酸性角膜炎和过敏性结膜炎，在猫中已被充分认识[26, 27, 28]，表明嗜酸性粒细胞介导的炎症通路存在于猫眼表环境中。
另一方面，溴的检出可能反映坏死角膜组织内细胞外基质成分的氧化修饰，而非嗜酸性粒细胞性炎症的直接结果。溴化反应可能在炎症性氧化应激过程中发生，并可能促成蛋白质修饰、基质不稳定和基质变性。
综上所述，隔离组织中溴和铁的存在提示氧化生化过程可能在FCS的发病机制中发挥重要作用。这两种元素均与能够修饰蛋白质和细胞外基质成分的氧化反应密切相关，可能促成基质坏死和隔离性病变的进展。
隔离组织中蓄积的微量元素的确切来源仍有待完全阐明，可能涉及潜在的内源性和外源性途径。对于铁，内源性来源可能包括局限性角膜内出血、新生血管的血管渗漏、泪膜铁结合蛋白（如乳铁蛋白），或持续性氧化应激导致的细胞铁蛋白蓄积。还必须考虑长期局部用药的外源性贡献。同样，溴信号可能内源性来源于泪膜中存在的溴化物，或过敏性/慢性炎症反应期间EPO介导的溴化物氧化产生的溴化生物分子，也可能外源性来源于含溴化合物的局部眼用制剂。
角膜内铁沉积是人类眼科学中已被充分认识的现象，最常见表现为局限性上皮铁线，如圆锥角膜中的Fleischer环、Hudson–Stähli线以及滤过泡旁的Ferry线[29]。这些病变通常局限于角膜上皮，被认为源于泪膜动力学、上皮更新和铁蓄积的局部改变，而非组织坏死。类似机制是否在基质坏死发生前的FCS早期阶段发挥作用尚不清楚，值得进一步研究。
本研究存在若干局限性应予说明。首先，样本量相对较小，反映了获取适合元素分析的手术切除隔离组织的困难。其次，尽管PIXE分析可提供高灵敏度的微量元素定量，但它仅能识别元素；不能提供有关化学形式、氧化态或角膜各层及细胞区室内精确空间微定位的信息。第三，本研究的横断面设计无法确定所观察到的元素改变是原发性致病机制还是组织坏死和炎症的继发性结果。
未来整合空间元素成像技术（如micro-PIXE或基于同步辐射的X射线荧光显微镜[30, 31]）以及组织学和分子分析的研究，可能有助于进一步阐明微量元素在猫角膜疾病中的作用。此外，对隔离组织中氧化应激标志物、铁死亡通路和炎症介质的研究可能为基质变性的机制提供进一步见解。
总之，FCS表现出以溴和铁选择性蓄积为特征的特异性元素谱。结合现有的生化和临床证据，这些发现支持氧化性组织重塑在隔离组织形成中的潜在作用。有必要进一步开展整合空间分辨元素分析、免疫组织化学和嗜酸性粒细胞相关标志物的研究，以阐明炎症和过敏通路在这种独特角膜疾病中的作用。
作者贡献
O. Pe'er：概念构思、调查研究、撰写—原稿、方法学、撰写—审阅与编辑、数据管理、形式分析。R. Ofri：经费获取、方法学、数据管理、撰写—原稿、撰写—审阅与编辑。L. Sebbag：撰写—原稿、撰写—审阅与编辑、方法学、数据管理、形式分析。
披露
人工智能声明：作者声明，在本稿件的准备过程中未使用任何生成式人工智能工具。
伦理声明
本研究已获以色列卫生部机构动物护理与使用委员会批准（PET‐2024‐14‐NE）。
利益冲突
作者声明无利益冲突。
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Introduction
Feline corneal sequestrum (FCS) is a distinctive corneal disorder characterized by focal stromal necrosis accompanied by dark brown to black corneal discoloration. The condition is most commonly observed in brachycephalic cat breeds and may lead to chronic ocular discomfort requiring surgical management [1, 2, 3, 4, 5]. Despite its characteristic appearance and long‐standing recognition, the etiopathogenesis of corneal sequestra remains incompletely understood [6]. Several mechanisms have been proposed, including chronic corneal irritation, viral infection, tear film abnormalities, and genetic predisposition [1, 2, 3, 4, 5, 6, 7].
The origin of the characteristic brown‐black pigmentation has also been investigated. Early studies suggested that iron deposition may contribute to the discoloration, as iron concentrations in sequestra were reported to be markedly elevated compared with normal corneal tissue [8]. However, subsequent investigations have produced conflicting results. Featherstone et al. failed to demonstrate increased iron levels but reported the presence of melanin particles within sequestra [9]. Similarly, Cullen et al. described stromal degeneration and electron‐dense material but did not identify melanin consistently [10]. More recently, the possible role of porphyrins originating from the tear film has been investigated; however, spectrofluorometric analyses failed to detect porphyrins within feline corneal sequestra or ocular tissues [11].
Trace elements play important roles in oxidative stress, inflammation, and extracellular matrix remodeling within biological tissues [12]. Advances in analytical techniques have enabled detailed characterization of elemental composition in biological samples, including the tear film. Particle‐induced X‐ray emission (PIXE) is a sensitive multi‐element analytical technique capable of detecting trace elements in small biological samples with minimal preparation [13, 14]. PIXE has previously been used to analyze trace element composition in human and animal tears [15, 16, 17].
The objective of the present study was to compare the elemental composition of feline corneal sequestra with that of healthy corneal tissue using PIXE analysis. We hypothesized that sequestra tissue would exhibit a distinct elemental profile that could provide insight into the pathogenesis of the disease.
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Materials and Methods
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Animals
This study was designed as an observational case–control study including cats diagnosed with FCS and healthy control corneas collected at the Koret School of Veterinary Medicine. The study was approved by the Institutional Animal Care and Use Committee (protocol PET‐2024‐14‐NE). Written informed owner consent was obtained prior to sample collection for inclusion of client‐owned cats in the study and for the use of clinical data and surgically excised corneal tissues for research purposes.
Nine client‐owned cats (11 eyes; two cats with bilateral disease) diagnosed with FCS and treated surgically by lamellar keratectomy were initially included. Signalment data including age, sex, and breed were retrieved from the medical records together with ocular history, previous topical or systemic treatments, lesion laterality, corneal vascularization, lesion size, and lesion depth. All patients underwent a complete ophthalmic examination performed by a board‐certified veterinary ophthalmologist including slit‐lamp biomicroscopy (Kowa SL‐17, Kowa Company Ltd., Tokyo, Japan), rebound tonometry (TonoVet PLUS, Icare Finland Oy), Schirmer tear test measurement, and fluorescein staining.
Topical medications were withheld on the morning of surgery to allow at least 12 h between the last topical treatment and sequestrum excision. Lamellar keratectomy was performed under general anesthesia using an operating microscope. The sequestrum was excised en bloc together with approximately 1 mm of surrounding healthy corneal tissue, as previously described [9]. Excised samples were immediately placed in sterile Eppendorf tubes and frozen at −80°C within 10 min of collection.
Control corneas were obtained from four cats free of ocular pathology that were euthanized for reasons unrelated to this study. All control eyes were confirmed to be ophthalmologically normal by slit‐lamp examination. Corneal samples were collected post‐mortem and stored using the same protocol.
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PIXE Elemental Analysis
Samples were thawed prior to processing and mounted directly on silicon wafers using double‐sided carbon tape. The mounted samples were then introduced into a high‐vacuum chamber (~10−7 Torr) for analysis. PIXE measurements were performed using a 1.7 MV tandem Pelletron accelerator (National Electrostatics Corporation, Middleton, WI, USA) delivering a ~2.014 MeV proton beam collimated to ~1.5 mm in diameter. Characteristic X‐ray spectra emitted from the irradiated samples were acquired using a FAST SDD silicon drift detector (Amptek, Bedford, MA, USA) equipped with a Si₃N₄ window. The detector was positioned at ~40° relative to the beam normal (IBM geometry), and a 12‐μm Mylar foil was used as a filter. Samples were irradiated to a fixed collected charge (Q) according to laboratory protocol, with electron suppressors employed to minimize secondary‐electron effects. Energy calibration and quality control were performed using metallic standards and certified reference materials from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). Spectra were processed using GUPIX software to determine net peak areas. Due to variations in specimen thickness and hydration, elemental values were reported as relative peak abundances rather than absolute concentrations [17].
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Statistical Analysis
Data distribution was assessed using the Shapiro–Wilk test. As the main outcome variable (trace element levels) was not normally distributed (p ≤ 0.049), data are presented as median ± standard error of the mean (SEM) and analyzed using non‐parametric methods. Comparisons between elemental values in sequestra and control tissues were performed using Mann–Whitney rank sum tests. Statistical analysis was performed using SigmaPlot version 15.0 (Systat Software Inc.), with p values < 0.05 considered statistically significant.
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Results
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Study Population
Prior to surgical excision, 7 of 9 cats in the sequestra group (78%) had received topical ocular therapy including 0.5% oxytetracycline ophthalmic ointment (Ocíl, VAPP—Veterinária e Agropecuária, S.A.) and antiviral agents (Penciclovir; Fenlips, Perrigo) [18], administered twice daily either as monotherapy or in combination. A subset of cats also received systemic doxycycline (Doxycycline; Doxylin, Teva Pharmaceutical Industries Ltd.) at 10 mg/kg once daily.
The study group included 9 cats (11 eyes) with confirmed corneal sequestra. Affected cats were predominantly brachycephalic or predisposed breeds, including three Sphynx cats (33%), two Persian cats (22%), two British Shorthair cats (22%), one brachycephalic cat of unspecified breed, and one Domestic Shorthair. The median age at presentation was 3.0 years (range: 0.5–12 years). Seven cats (78%) were male and two (22%) female. Lesion size ranged from 1 to 8 mm (median: 3 mm), and estimated stromal depth ranged from 20% to 70% (median: 40%). All lesions appeared dark in color. Corneal vascularization was present in 8 of 11 eyes (72%), whereas three eyes showed no evidence of vascular ingrowth.
The control group included four Domestic Shorthair cats euthanized for systemic diseases without ophthalmic manifestations, including pulmonary carcinoma, traumatic injuries following vehicular accidents (n = 2), and arterial thromboembolism. Three cats were male and one female, with ages ranging from 4 to 13 years (median: 5.5 years).
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Elemental Analysis
Among all analyzed elements, only bromine and iron demonstrated statistically significant differences between the sequestra and control groups (Figure 1 and Table 1), being detected in the sequestra group but absent in controls. Bromine was undetectable in all control samples but was consistently present in the sequestra group, with a median ± SEM peak area of 110 ± 50 compared with 0 in controls (p = 0.004; Figure 1A). Similarly, iron was absent in control samples and present in the sequestra group (751 ± 336 vs. 0; p = 0.047; Figure 1B). Sodium, potassium, chloride, magnesium, phosphorus, calcium, sulfur, and zinc did not differ significantly between sequestra and control corneas (all p > 0.05; Figure 2). Median peak areas were numerically higher in the sequestra group for sodium, chlorine, calcium, sulfur, and zinc, whereas potassium, magnesium, and phosphorus showed similar or lower values compared with controls.
[图：Box‐and‐whiskers plots depicting peak areas for bromine (A) and iron (B) in corneal sequestra and healthy control cornea]
[图：Box‐and‐whiskers plots depicting peak areas for other trace elements (sodium, magnesium, phosphorus, sulfur, zinc, potas]
TABLE 1. Peak area values for elemental composition in corneal sequestra and healthy control corneas, as determined by particle‐induced X‐ray emission (PIXE).
| Element | Sequestra | Control | p (Mann–Whitney) |
|---|---|---|---|
| Na | 65 609 ± 3113 (14163–134 362) | 46 183 ± 3346 (29762–58 482) | 0.192 |
| Mg | 5849 ± 360 (2649–14 772) | 6014 ± 107 (5214–6149) | 0.896 |
| P | 139 427 ± 8544 (21117–303 829) | 143 325 ± 13 548 (60053–177 123) | 1.000 |
| S | 443 782 ± 6764 (264616–477 509) | 334 365 ± 10 924 (291231–391 789) | 0.090 |
| Zn | 105 ± 8 (25–316) | 72 ± 6 (45–91) | 0.192 |
| K | 82 572 ± 4324 (10868–157 687) | 123 635 ± 15 407 (57980–181 217) | 0.240 |
| Ca | 26 712 ± 6814 (10380–268 554) | 21 023 ± 1786 (15147–32 193) | 0.192 |
| Fe | 751 ± 336 (0–3333) | 0 (0–0) | 0.047 |
| Cl | 79 056 ± 2473 (6344–101 465) | 62 584 ± 8589 (39552–108 189) | 0.794 |
| Br | 110 ± 50 (39–625) | 0 (0–0) | 0.004 |
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Discussion
In the present study, elemental analysis using PIXE revealed the presence of bromine and iron in FCS but not in normal feline corneas. These findings suggest that sequestral tissue exhibits a distinct biochemical environment, potentially reflecting local inflammatory, oxidative, or degenerative processes associated with the development of this disease.
Importantly, no statistically significant differences were observed in the levels of sodium, potassium, chloride, magnesium, phosphorus, calcium, sulfur, or zinc between sequestral and normal corneal tissues. This observation indicates that FCS is not characterized by classical mineralization processes, which typically involve increased deposition of calcium or phosphate salts. Similar conclusions were previously reported by Featherstone et al., who did not identify significant calcium deposition within sequestral tissue [9]. Together, these findings provide strong evidence that the dark pigmentation and stromal degeneration characteristic of FCS do not result from conventional dystrophic mineralization.
Iron accumulation within sequestra may contribute to the pathophysiology of this disease. Iron is well recognized for its capacity to catalyze the formation of reactive oxygen species through Fenton chemistry, generating hydroxyl radicals capable of inducing oxidative damage to lipids, proteins, and nucleic acids [12]. Within the cornea, such oxidative stress may contribute to collagen degradation, keratocyte injury, and extracellular matrix remodeling, processes that are consistent with the stromal necrosis observed histologically in feline corneal sequestra [10]. In addition to its oxidative properties, iron has also been associated with pigmentary alterations in ocular tissues. Iron can bind to melanin and influence melanin oxidation pathways, potentially intensifying tissue pigmentation. This interaction may partially explain the characteristic brown‐to‐black discoloration observed clinically in sequestra, although previous investigations have produced conflicting findings regarding the presence of melanin within these lesions [9, 10]. In contrast to our findings, Featherstone et al. detected low iron levels in both normal corneas and sequestra using scanning electron microscopy with energy‐dispersive X‐ray spectroscopy (SEM–EDX) [9]. This discrepancy is primarily attributable to fundamental differences in sampling methodology, analytical depth, and sensitivity: while SEM–EDX evaluates only the outermost surface layer (1–2 μm) with a detection limit around 0.1% (1000 ppm), superficial iron signals in normal corneas may represent residual tear film components or surface artifacts rather than true stromal accumulation. Conversely, high‐energy proton PIXE evaluates the full thickness of the excised keratectomy specimen with trace elemental sensitivity as low as 1–10 ppm, capturing total elemental composition throughout the entire volume of the sample and confirming that iron accumulation is restricted to sequestral stroma while healthy control corneas are free of detectable iron.
Iron‐mediated oxidative stress is also central to ferroptosis, a recently described form of regulated cell death characterized by iron‐dependent lipid peroxidation [19, 20]. Ferroptosis has been implicated in various degenerative and inflammatory diseases and may play a role in tissue injury where iron accumulation and oxidative stress coexist. Although absolute concentration thresholds required to trigger ferroptosis or melanin binding in corneal stroma are currently unknown, the selective presence of iron in feline corneal sequestral tissue compared to its total absence in normal control corneas raises the hypothesis that iron‐dependent oxidative pathways could contribute to keratocyte death and stromal degeneration. Future spatially resolved quantitative studies are needed to determine whether iron is focally concentrated within specific cellular or extracellular compartments at levels sufficient to drive these proposed processes.
The detection of bromine exclusively in sequestral tissue is another intriguing finding. Bromine has recently been identified as a measurable component of the human tear film, indicating that it is present in the ocular surface environment [21]. Bromine also serves as a physiological substrate for eosinophil peroxidase (EPO), an enzyme released during eosinophil activation in inflammatory tissues. In the presence of hydrogen peroxide, EPO catalyzes the oxidation of bromide ions to hypobromous acid (HOBr), a potent oxidizing agent capable of brominating tyrosine residues within extracellular matrix proteins [22, 23, 24]. This reaction leads to the formation of bromotyrosine derivatives, which are widely regarded as specific molecular markers of eosinophil‐mediated tissue injury [25].
Although eosinophils are not commonly reported in histologic studies of feline corneal sequestra, eosinophil‐associated inflammatory processes could potentially occur during earlier stages of disease, or transiently during corneal injury and repair. Furthermore, eosinophilic ocular surface diseases, including eosinophilic keratitis and allergic conjunctivitis, are well recognized in cats [26, 27, 28], indicating that eosinophil‐mediated inflammatory pathways occur in the feline ocular surface environment.
Alternatively, bromine detection may reflect oxidative modifications of extracellular matrix components within necrotic corneal tissue, rather than a direct consequence of eosinophilic inflammation. Bromination reactions may occur during inflammatory oxidative stress and could contribute to protein modification, matrix instability, and stromal degeneration.
Taken together, the presence of bromine and iron in sequestral tissue suggests that oxidative biochemical processes may play an important role in the pathogenesis of FCS. Both elements are closely associated with oxidative reactions capable of modifying proteins and extracellular matrix components, potentially contributing to stromal necrosis and the progressive development of sequestral lesions.
The exact origin of the accumulated trace elements in sequestral tissue remains to be fully elucidated, and may involve potential endogenous and exogenous pathways. For iron, endogenous sources may include localized intracorneal hemorrhage, vascular leakage from neovascularization, tear film iron‐binding proteins (such as lactoferrin), or cellular ferritin accumulation resulting from sustained oxidative stress. Exogenous contribution from prolonged topical medication use must also be considered. Similarly, the bromine signal may originate endogenously from bromide present in the tear film or from brominated biomolecules generated through EPO‐mediated bromide oxidation during allergic or chronic inflammatory responses, or exogenously from topical ophthalmic formulations containing brominated compounds.
Iron deposition within the cornea is a well‐recognized phenomenon in human ophthalmology, most commonly manifesting as localized epithelial iron lines such as Fleischer rings in keratoconus, Hudson–Stähli lines, and Ferry lines adjacent to filtering blebs [29]. These lesions are typically confined to the corneal epithelium and are thought to result from localized alterations in tear film dynamics, epithelial turnover, and iron accumulation rather than tissue necrosis. Whether similar mechanisms contribute to the early stages of FCS development before stromal necrosis occurs remains unknown and warrants further investigation.
Several limitations of the present study should be acknowledged. First, the sample size was relatively small, reflecting the difficulty in obtaining surgically excised sequestra suitable for elemental analysis. Second, although PIXE analysis delivers high‐sensitivity trace element quantification, it identifies only elements; it does not provide information regarding the chemical forms, oxidation state, or precise spatial micro‐localization within corneal layers and cellular compartments. Third, the cross‐sectional design of the study precludes determination of whether the observed elemental alterations represent primary pathogenic mechanisms or secondary consequences of tissue necrosis and inflammation.
Future studies integrating spatial elemental imaging techniques, such as micro‐PIXE or synchrotron‐based X‐ray fluorescence microscopy [30, 31], together with histologic and molecular analyses, may help further elucidate the role of trace elements in feline corneal disease. Additionally, investigation of oxidative stress markers, ferroptosis pathways, and inflammatory mediators in sequestral tissue may provide further insights into the mechanisms underlying stromal degeneration.
In summary, FCS exhibited a distinct elemental profile characterized by selective accumulation of bromine and iron. Together with existing biochemical and clinical evidence, these findings support a potential role for oxidative tissue remodeling in sequestra formation. Further studies integrating spatially resolved elemental analysis, immunohistochemistry, and eosinophil‐associated markers are warranted to clarify the contribution of inflammatory and allergic pathways to this unique corneal disease.
Author Contributions
O. Pe'er: conceptualization, investigation, writing – original draft, methodology, writing – review and editing, data curation, formal analysis. R. Ofri: funding acquisition, methodology, data curation, writing – original draft, writing – review and editing. L. Sebbag: writing – original draft, writing – review and editing, methodology, data curation, formal analysis.
Disclosure
AI statement: The authors declare that no generative AI tools were used in the preparation of this manuscript.
Ethics Statement
This study was approved by the Institutional Animal Care and Use Committee of Israel's Ministry of Health (PET‐2024‐14‐NE).
Conflicts of Interest
The authors declare no conflicts of interest.