兽研简要总结
猫慢性龈口炎（FCGS）是一种严重的口腔炎症性疾病，可引起疼痛、进食困难和生活质量下降。拔牙被认为是标准治疗方法，但术后口腔组织和局部免疫反应如何变化知之甚少。这项前瞻性初步研究对五只猫治疗后进行了随访，评估其临床和血液参数，其中三只猫还接受了组织病理学变化和局部免疫细胞标志物的评估。大多数猫在拔牙后表现出临床改善。口腔活检中观察到炎症减轻。拔牙后受影响组织中B细胞标志物的表达降低。然而，其他免疫标志物的变化因个体而异。这些发现为标准手术治疗后的局部组织反应提供了初步信息，并增进了我们对这种慢性炎症性疾病的理解。
关键词：龈口炎、炎症、拔牙、组织学、猫
1. 引言
猫慢性龈口炎（FCGS）是一种疼痛性、致衰弱性、多因素炎症性疾病，以影响口腔黏膜的严重炎症为特征，显著降低患病猫的生活质量[1,2,3,4,5]。病变可累及牙龈黏膜、牙槽黏膜和颊唇黏膜，最具特征性的是腭舌褶外侧的尾侧口腔黏膜[3,4,5]。口外表现包括食管炎和气管支气管炎症也已有描述，进一步支持FCGS与广泛黏膜炎症相关的概念[6,7]。组织学上，该病以密集的淋巴浆细胞浸润为特征，伴有数量不等的中性粒细胞和巨噬细胞。免疫表型研究已证实受影响组织内存在多种免疫细胞群，包括CD20+ B淋巴细胞、CD3+ T细胞（包括CD4+辅助性和CD8+细胞毒性亚群）以及FoxP3+调节性T细胞[8,9]。近期，转录组学和免疫学研究提示FCGS以持续性免疫失调为特征，涉及T细胞功能和炎症通路的改变，支持该病代表异常宿主反应的假说[2,8,10]。
通过部分拔牙（PME）或全口拔牙（FME）的手术治疗仍是当前的标准治疗方案，尽管约30%的猫表现出部分临床反应或对治疗无反应[1,11]。既往评估治疗疗效的研究主要关注临床结局[11,12,13,14]，而近期研究则探索了与疾病进展和治疗反应相关的口腔微生物组、全身炎症生物标志物和转录组谱的改变[15,16,17,18,19]。特别是，循环CD8+ T细胞的转录组分析显示了耗竭样表型和线粒体生物能量学受损，而接受FME和间充质干细胞（MSC）治疗后的猫评估显示这些分子特征部分恢复[19]。
尽管取得了这些进展，关于FME或PME后口腔黏膜内炎症浸润和局部免疫反应的演变知之甚少。大多数表征口腔免疫微环境的研究集中于疾病发病机制或对FME无反应接受MSC治疗的猫的演变[2,8,20,21,22]。尽管在接受细胞治疗的治疗反应者中已有淋巴浆细胞浸润减少的报道，但描述标准手术治疗后组织病理学和免疫学变化的纵向信息仍然有限。由于获取连续口腔活检的实际困难，拔牙后的局部组织反应尚未得到充分表征。
本前瞻性研究的目的是描述FCGS猫接受PME后发生的临床、血液学、生化、组织病理学和局部免疫学变化。
2. 材料与方法
2.1. 研究设计
本初步前瞻性纵向研究旨在描述诊断为FCGS的猫在PME后所发生的临床、血液学、生化、组织病理学及局部免疫学变化（图1）。纳入标准如下：出现与FCGS相符的炎性病变、且无肿瘤性或免疫介导性疾病的猫。入选患者表现为累及前磨牙和/或磨牙区域的尾侧口腔黏膜炎及牙槽黏膜炎，未累及犬齿或切齿区域。对品种、年龄、体重或猫免疫缺陷病毒（FIV）或猫白血病病毒（FeLV）状态无限制。入组前已获得所有主人的书面知情同意。若此前使用过皮质类固醇治疗，则于手术前至少20天停药，且研究期间不允许使用。无猫接受长效注射型糖皮质激素。研究期间允许使用NSAIDs、普瑞巴林、加巴喷丁和阿片类药物。符合纳入标准的猫接受PME，包括拔除所有前磨牙和磨牙。术前，猫接受抗生素（阿莫西林-克拉维酸（15 mg/kg BID）或螺旋霉素-甲硝唑（分别为75,000 IU/kg和12.5 mg/kg））及NSAIDs（美洛昔康（0.1–0.02 mg/kg SID））。术后维持抗生素7至10天。PME前获取口内X线片，但拔牙后牙科X线片并非常规获取；仅在发生牙根折断时拍摄以确认牙根完全拔除。术后7至15天，猫接受阿莫西林-克拉维酸和美洛昔康（剂量从0.1 mg/kg减至0.03 mg/kg SID），以及普瑞巴林（2 mg/kg SID或BID）。在PME当天（D0）；第7天（D7）、15天（D15）、30天（D30）和60天（D60）；以及此后每月评估，直至治疗后6个月（D180）。除D0和D30时三只猫因活检采样而麻醉外，所有时间点的评估均在不镇静的情况下进行。
[图：研究设计概述。]
2.2. 临床评估
临床疾病严重程度采用口腔炎疾病活动指数（SDAI）评分系统评估，综合考虑研究者与主人的评估，评分范围为0（健康）至30（重度）[12,23]。每次随访时均进行评分。研究者对病变进行评分（0–3，从无病变到重度），猫主人对猫的食欲、活动水平、理毛行为及感知的口腔舒适度进行评分。SDAI改善百分比采用以下公式计算：[（初始SDAI D0 − 治疗后随访SDAI）/初始SDAI D0]×100；根据既往研究，改善阈值为>12%[23]。与初始SDAI评分相比降低>50%视为良好改善，>30–50%视为中度改善，12–30%视为轻度改善，−12至<12%视为无改善，低于−12%视为恶化[23]。
2.3. 血液参数
在整个随访期间的预定时间点进行全血细胞计数（ProCyte One分析仪，Idexx，美国缅因州）和血清生化（CM320，Weiner lab，阿根廷布宜诺斯艾利斯）分析。当以下红细胞参数中至少两项低于该物种既定参考范围时，患者被认为贫血：红细胞总数、血细胞比容和/或血红蛋白浓度。根据绝对网织红细胞计数将贫血分类为再生性或非再生性[24]。逆转录病毒感染状态采用市售即时检验检测FeLV抗原和FIV抗体（FASTest FIV/FeLV，Megacor Diagnostik GmbH，德国Hörbranz）确定。
2.4. 局部炎症评估
在全身麻醉下，于D0和D30从三只猫腭舌褶外侧的尾侧口腔黏膜采集0.5 cm全层活检。D0从口腔一侧采集活检样本，D30从对侧采集。对福尔马林固定、石蜡包埋的标本进行组织病理学和免疫组化（IHC）分析。组织样本用4%缓冲甲醛固定、处理并切成4 μm厚的切片。为评估炎症，切片用苏木精和伊红（H&E）染色。炎症程度采用显微炎症变化严重程度评分系统评估（0–3级，从正常到重度）[9]。组织学图像使用配备高分辨率数码相机和采集软件（AmScope MU853B USB 3.0高速数码相机，美国加利福尼亚州）的透射光显微镜（Olympus BX41，Olympus Corporation，日本东京）观察并获取。切片还使用全切片扫描仪（Motic EasyScan One扫描仪，Motic China Group Co., Ltd.，中国厦门）在默认设置和400×放大倍数（图像分辨率：0.25 µm/像素）的焦平面进行扫描，并使用查看器（Motic ‘DSAssistant’，Motic VM V1 Viewer 2.0，版本2019-08-02，中国）和扫描软件（Motic ‘EasyScanner’，中国香港）进行可视化。组织病理学评估由四名对患者和采样时间点不知情的观察者进行，最终评分通过共识确定。
为评估细胞浸润类型，对CD20、FoxP3和IL-17抗原的表达进行了检测。为此，组织切片经二甲苯脱蜡，并通过梯度乙醇系列复水。应用3%过氧化氢封闭液，随后在蒸锅中进行热诱导抗原修复，将切片浸入装有0.01 M柠檬酸盐缓冲液（pH 6）的聚丙烯Coplin缸（Deltalab，巴塞罗那，西班牙）中30分钟。使用猫淋巴结和尾侧口腔黏膜作为阳性对照对IHC条件进行了优化。阴性对照通过以磷酸盐缓冲盐水（PBS）替代一抗进行。未进行同型对照。遵循制造商建议并稍作修改。本研究使用的一抗列于表1。所选CD20抗体[25]和FoxP3克隆（221D）[26]已在猫组织中使用。IL-17A的IHC检测此前已有报道在其他猫组织中使用不同抗体[27]；然而，这并不构成本研究所用抗体在猫组织中验证的依据。将每种一抗应用于组织，然后在4 °C湿盒中孵育过夜。应用检测系统（Mach 4 Universal HRP-Polymer Detection System，Biocare Medical，加利福尼亚州，美国）和链霉亲和素-过氧化物酶。使用DAB溶液显色3–5分钟，然后切片用Mayer苏木精复染、脱水并封片。组织学图像按先前描述的方法采集。
Table 1. Details of antibodies for immunohistochemical labeling.
| Primary Antibody | Manufacturer | Dilution |
|---|---|---|
| Rabbit anti-CD20 (polyclonal) | Invitrogen/Catalog # PA5-16701 | 1:400 |
| Mouse anti-FoxP3 (clone 221D) | Donated by Giovanna Roncador, Spanish National Cancer Research Center (CNIO) | undiluted |
| Rabbit anti-IL-17A (polyclonal) | ABCAM/Catalog # ab79056 | 1:200 |
免疫标记区域使用数字图像分析软件进行定量，从而能够客观评估标记物表达并最大限度减少观察者相关偏倚。全切片图像分析由四名对患者和采样时间点不知情的观察者使用Motic SlideViewer软件（Motic VM V1 Viewer 1.0）协作完成，以确保判读的一致性。在低倍镜下检查整个切片后，从炎症浸润的代表性区域选取五个不重叠的高倍视野（400×）[28]对免疫标记细胞进行评估。坏死区域因可能产生非特异性染色和假阳性结果而被排除在评估之外，血管化明显、海绵水肿严重或与组织处理、切片或染色相关的技术伪影区域亦被排除。存在技术性染色伪影以致无法可靠区分特异性DAB信号与背景的区域被排除。定量使用FIJI（ImageJ 2.14.0 1.54p，NIH，马里兰州，美国）进行。应用颜色阈值分离特异性显色信号并将其与背景区分。然后将阈值化图像二值化，将所选信号转换为二进制格式。随后对阳性染色所占面积进行定量，并以占总图像面积的百分比表示。所有图像使用相同的亮度、对比度和阈值参数处理，以确保样本间的一致性。
2.5. 描述性分析
数据以每只猫和每个时间点的个体值进行描述性呈现。未进行推断性统计分析。
3. 结果
3.1. 患者特征
纳入五只客户拥有的混种猫，平均年龄7岁。四只为FCGS溃疡型，一只为增殖型。猫饲养于室内或室内外混合生活方式，每户平均与另外两只猫共同生活。个体患者特征和FIV/FeLV状态分别见表A1和表A2。
3.2. 临床评估
PME后的临床反应因个体而异；然而，与基线相比，所有猫在第30天时SDAI评分均有所降低（图2）。改善程度因猫而异。在D0时，猫的平均SDAI评分为12.6。在D30时，两只显示良好改善，两只显示中度改善，一只显示轻度改善。三只猫随访至D180，其中两只在该时间点显示良好改善，一只显示轻度改善。猫E的SDAI评分显示良好改善，但在研究期后死于FeLV相关并发症（淋巴母细胞白血病）。猫C在第90天后因肾病退出，尽管其在PME后SDAI评分有所改善。猫D在第30天后失访。猫A从D60至D120接受阿米替林控制猫口面部疼痛综合征（FOPS），之后使用CBD油，至D180达到完全缓解。猫C接受了奥美拉唑。
[图：（A）每只猫和每个时间点的口腔炎疾病活动指数（SDAI）。每个彩色圆点代表一只猫的]
3.3. 血液参数
五只猫中有三只在基线时表现为贫血（表A2，图S1）。猫D和猫E均观察到持续性非再生性贫血，这两只猫的FeLV和/或FIV检测均为阳性。相比之下，猫C在随访期间记录到一过性再生性贫血。猫D和猫E的单核细胞增多尤为显著。猫B在第15天观察到嗜酸性粒细胞增多和ALP升高，至第60天时下降，同时检测到体内寄生虫（复孔绦虫属），随后该猫接受了驱虫治疗。
高球蛋白血症是最一致的生化异常，在所有猫的一个或多个时间点均存在（图S2）。五只猫中有两只在第0天观察到低白蛋白血症，随后除一只猫外其余均恢复正常。此外，一只猫出现血清尿素和肌酐水平升高，因此接受了肾脏疾病治疗。
3.4. 组织病理学发现
在第0天和第30天从三只FeLV阳性猫（A、D和E）采集口腔活检（图3A）。猫B未在适合全身麻醉的条件下参加预定的第30天访视，猫C因检测到血清尿素和肌酐浓度升高而未进行活检。考虑到潜在的麻醉风险，为保障该猫的福利，未对其进行全身麻醉下活检采集。组织切片显示上皮增生，在最严重的病例中伴有大量细胞碎片。一致注意到中度至显著的淋巴浆细胞浸润，主要局限于黏膜下层。浸润灶内偶见莫特细胞。黏膜下层和血管周围区域可见中性粒细胞，尤其是在显示水肿和坏死的区域。在分级为最严重的样本中，可见明显的细胞间水肿、间质水肿以及包裹肌纤维的致密炎性浸润。猫E第0天和第30天口腔活检的组织病理学检查显示慢性炎性改变，无肿瘤浸润证据。在研究期结束后的第200天，诊断为淋巴母细胞性白血病。
[图：PME前后口腔活检中的炎性改变。(A) 口腔活检显微图像（H&E，100×，比例尺]
两只猫表现出显微炎性改变严重程度降低，并伴有临床改善（SDAI降低）（图3B），而一只猫的炎性评分不变，SDAI仅有轻微改善，且该猫为FIV和FeLV阳性。
3.5. 免疫组织化学发现
IHC分析显示三只猫连续活检中CD20、FoxP3和IL-17的标记模式各具特征（图4a）。CD20标记强烈，主要集中于固有层，并浸润至黏膜下层。在固有层少数细胞中观察到FoxP3的核和细胞质标记，主要位于显示炎症或水肿区域的上皮附近。在上皮、固有层和黏膜下层的细胞质和间质中检测到IL-17标记。
[图：免疫组织化学图像。(a) CD20、FoxP免疫组织化学染色的代表性显微图像（100×）]
免疫标记区域的定量分析表明，PME后所有猫的CD20表达均降低。这些变化的幅度存在个体差异，猫A、D和E分别降低46.3%、39.4%和18.6%。猫A和猫E还显示组织学炎性评分降低和临床改善。相比之下，FoxP3和IL-17免疫标记模式显示出明显的个体间差异。两只猫的FoxP3阳性区域减少，同时SDAI和组织学炎性评分也降低，但一只猫的FoxP3标记区域增加，而其组织学炎症评分保持不变，临床改善甚微。IL-17标记在两只猫中降低，在一只猫中升高。个体SDAI评分、组织病理学炎性评分和定量免疫组织化学测量结果汇总于图4b。
4. 讨论
本初步前瞻性研究提供了关于FCGS猫在PME后发生的临床、组织病理学和局部免疫学变化的纵向信息。传统上，患者在拔牙后需要超过14天的内科管理[11]。PME后1个月，猫表现出临床改善，尽管改善程度随时间和个体而异（以SDAI衡量）。拔牙后的临床反应可能在数月内持续演变，早期评估可能无法反映最终结果。有报道称术后六个月出现改善或临床痊愈[11,29]。因此，尽管对猫进行了长达180天的随访，本研究主要旨在描述PME后的早期变化，尤其是前30天内的变化。先前使用不同评分系统的报告观察到52%的猫在手术治疗后38天表现出临床改善[29]，而更近期的研究报告在60天时出现显著改善[18]。
在这个小型队列中，逆转录病毒感染阳性的猫表现出最明显的血液学异常。主要的血液学发现是贫血，主要见于FIV和/或FeLV阳性的猫。这些发现与近期报道一致，即18%的FeLV阳性猫和12%的FIV阳性猫出现贫血，且这两个群体中分别有16%和3%出现白血病[30]。同一研究报告称，11%的FeLV阳性猫和24%的FIV阳性猫患有FCGS。在三只FeLV阳性猫中，猫D和猫E在随访期间出现了具有临床相关性的血液学并发症。尽管FeLV阳性，猫A和猫E显示出临床改善（SDAI评分改善）。FeLV感染与拔牙后临床反应不良的可能性增加7.5倍相关[13]。在本研究中，一名患者在PME后第15天观察到嗜酸性粒细胞增多和ALP升高，可能与其并发的Dipylidium spp.感染有关。虽然嗜酸性粒细胞增多在FCGS猫中不常被报道，但一项回顾性研究记录了约25%的病例存在外周嗜酸性粒细胞增多[31]，凸显了排查并发感染的重要性。
第0天的主要生化发现是高球蛋白血症，这与既往报道描述的FCGS猫慢性抗原刺激和全身性炎症激活一致[5,32]。然而，PME后未观察到变化。近期研究表明，拔牙并未导致第60天α1-酸性糖蛋白水平显著下降，表明尽管临床改善，全身性炎症仍然持续[18]。相反，我们的结果显示，除一只猫外，所有猫在PME后15天血清白蛋白水平均恢复正常。术后血清白蛋白水平升高可能反映PME后全身性炎症减轻，因为白蛋白是猫体内重要的负性急性期蛋白，在慢性炎症性疾病中可观察到其浓度降低[33]。这些相互矛盾的结果强调需要进一步研究PME对猫免疫系统的影响。
在本研究中，一只猫显示尿素和肌酐水平升高，符合慢性肾病（CKD）。既往报道发现，约10%的FCGS猫患有CKD[34]。后续综述强调了筛查并发全身性疾病的重要性，因为CKD等合并症可能影响临床严重程度和治疗反应[4,5]。近期一项比较尾侧口炎猫与健康对照的横断面分析揭示了生化指标的轻微但显著差异，如肌酐较低和磷水平较高，但CKD患病率并未升高[35]。然而，既往研究发现26.5%的猫血清尿素浓度高于参考上限[31]。这些相互矛盾的结果表明，我们应考虑监测FCGS猫的肾功能生物标志物。
组织学检查结果显示了既往报道中提到的典型淋巴浆细胞性和中性粒细胞性浸润[5,9,32,36]。采用基于面积的定量方法对炎症浸润内免疫标记的总体范围进行标准化评估，特别是在染色密集或部分融合、无法始终可靠识别单个细胞边界的区域。所有标志物均一致采用相同方法。结果显示，两只猫的组织学炎症浸润评分降低，三只猫的CD20免疫标记减少。然而，三只猫的CD20免疫标记呈不同程度下降，与其SDAI反应无关，这引出了一个问题：这是否反映的是拔牙相关抗原刺激的消除，而非疾病的控制。有趣的是，FoxP3和IL-17免疫标记的变化呈异质性：两只组织学改善的猫显示FoxP3阳性面积减少，而组织学炎症持续严重的那只猫尽管临床改善甚微，却表现出FoxP3免疫标记增加。虽然这一观察的生物学意义仍不确定，任何解释都必然是推测性的，但FoxP3免疫标记降低可能与PME后抗原刺激减少相一致，而FoxP3免疫标记升高则可能反映对持续炎症的代偿性反应。此外，由于IHC检测提供的是表达和形态学信息，无法评估细胞功能。IL17-A显示出明显的个体间变异性。既往关于FCGS的转录组学研究提示，与IL-17家族相关的信号通路可能参与局部免疫反应[2,8,10]。然而，这些发现与本研究的免疫组化观察结果不能直接比较。在FCGS中已记录到促炎细胞因子升高，如IL-6、TNF-α和IFN-γ，它们构成了促进Th17细胞分化和功能的炎症环境的一部分[10,32]。尚未在尾侧口腔黏膜中使用免疫标记技术检测这些细胞因子。尽管既往已有使用不同抗体在其他猫组织中进行IL-17A免疫组化检测的报道[27]，但这并不构成本研究所用抗体在猫组织中的验证。因此，IL-17A免疫组化结果应谨慎解读。
本试点研究的主要局限性包括样本量小，以及仅有三只猫可获得配对的组织病理学和IHC样本。由于配对切片并非总能在同一IHC批次内处理，不能排除显色剂显色和染色强度方面的批间变异性。为避免愈合组织的影响，在D0和D30从对侧获取序贯活检；因此，组织组成的部位相关差异可能影响了基于面积的IHC测量。尽管最终组织病理学评分由四名盲法观察者协商一致确定，但未正式评估观察者间一致性。此外，应注意的是，碰巧进行活检的三只猫均为FeLV阳性，这可能对淋巴细胞群和拔牙后的临床反应产生负面影响[13]。本试点研究的另一局限性是未检测猫杯状病毒（FCV），因为本机构没有经过验证的FCV检测方法。美洛昔康在术后早期给药，并在D30活检前停药；然而，不能完全排除其对D30时炎症或免疫相关标志物的潜在影响。由于本研究涉及客户拥有的猫，当临床有指征时，尤其是为了疼痛管理和动物福利，无法停止伴随的药物治疗。此外，一只猫在随访期间发生FOPS，随后接受了额外药物治疗，这可能以研究设计未控制的方式潜在影响了免疫相关结果。然而，这些额外治疗是在D30活检之后开始的，因此不应影响D0和D30之间的早期组织病理学和IHC比较。尽管如此，在解读后期临床和免疫学发现时，应考虑其潜在影响。
有限数量的研究报告了患有FCGS的猫在PME后的临床随访情况。值得注意的是，关于细胞疗法的研究为接受MSC治疗难治性FCGS的猫的免疫学演变提供了宝贵见解[20,21,22]。然而，这些发现不应直接外推至接受PME的猫，因为MSC疗法代表一种主动的免疫调节干预，而PME主要减少局部抗原刺激。这种可比数据的缺乏也凸显了研究推荐标准护理手术治疗后免疫学变化的相关性。据我们所知，本初步研究是少数前瞻性评估PME后发生的纵向临床、组织病理学和局部免疫学变化的研究之一。更好地理解PME诱导的免疫变化可为该疾病的病理生理学提供关键见解，并有助于解释临床结局的异质性。需要更大规模的纵向研究来确定特定免疫模式是否与治疗结局相关。
5. 结论
五只猫在PME后临床SDAI评分下降，尽管改善的幅度和持续时间因个体而异。在三只具有配对活检的FeLV阳性猫中，无论临床和组织学反应如何，CD20免疫标记均下降，而FoxP3和IL-17水平显示可变变化。这些发现代表PME后早期组织变化的初步描述性观察，应在考虑小活检样本子集和本研究方法学局限性的情况下进行解读。
致谢
作者感谢参与本研究的每只猫的主人。作者使用ChatGPT（OpenAI，GPT-5.5）协助英语语言编辑和科学写作改进。所有建议的修订均由作者严格审查并批准。
补充材料
以下支持信息可从以下网址下载：https://www.mdpi.com/article/10.3390/vetsci13101038/s1，图S1。该图显示了关键血液学参数的个体数据点：红细胞、血红蛋白、血细胞比容、白细胞、淋巴细胞、中性粒细胞、单核细胞和嗜酸性粒细胞。测量在第0天至第60天的指定时间点进行。每个彩色圆点代表一只猫患者，编码如下：A（红色）、B（绿色）、C（蓝色）、D（粉色）和E（黑色）。误差棒表示均值的标准差（SD），虚线水平线表示各相应参数的既定参考范围。图S2。该图显示了关键生化参数的个体数据点：血清球蛋白、白蛋白、总蛋白、碱性磷酸酶（ALP）、尿素和肌酐。测量在第0天至第60天的指定时间点进行。每个彩色圆点代表一只猫患者，编码如下：A（红色）、B（绿色）、C（蓝色）、D（粉色）和E（黑色）。误差棒表示均值的标准差（SD），虚线水平线表示各相应参数的既定参考范围。
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附录A
Table A1. Characteristics of cats with feline chronic gingivostomatitis.
| Id | Age | Sex | Cohabiting Cats | FCGS Type | Life Style |
|---|---|---|---|---|---|
| A | 8 | Male | 1 | Ulcerative | Mixed |
| B | 4 | Female | 1 | Proliferative | Indoor |
| C | 9 | Male | 6 | Ulcerative | Indoor |
| D | 7 | Male | 4 | Ulcerative | Mixed |
| E | 7 | Male | 0 | Ulcerative | Mixed |
Table A2. Anemia status over time for each feline patient.
| ID | D0 | D7 | D15 | D30 | D60 | D90 | D180 | Anemia Type | FIV | FeLV |
|---|---|---|---|---|---|---|---|---|---|---|
| A | NO | NO | NO | NO | - | - | - | - | NEG | POS |
| B | NO | NO | NO | NO | + | - | NO | - | NEG | NEG |
| C | ++ | + | NO | NO | - | NO | - | R | POS | NEG |
| D | +++ | + | + | ++ | - | - | - | NR | POS | POS |
| E | + | - | + | ++ | NO | + | +++ | NR | NEG | POS |
作者贡献
概念化，A.A.和K.Y.；方法学，A.A.、G.T.和K.Y.；验证，B.V.、G.T.、A.W.、J.M.V.和K.Y.；形式分析，A.A.、L.T.、B.V.和K.Y.；调查，A.A.、L.T.、B.V.、G.T.、M.M.、J.E.、M.N.G.和K.Y.；资源，B.V.、G.T.、J.E.、J.M.V.和K.Y.；数据管理，A.A.、L.T.和M.M.；写作——初稿准备，A.A.、L.T.、B.V.、G.T.、M.M.、J.E.和M.N.G.；写作——审查和编辑，A.W.、J.M.V.和K.Y.；可视化，A.A.、J.M.V.和K.Y.；监督，G.T.、A.W.、J.M.V.和K.Y.；项目管理，A.A.和K.Y.；资金获取，A.A.、B.V.、J.M.V.和K.Y.。所有作者均已阅读并同意稿件的发表版本。
机构审查委员会声明
本研究经兽医学院机构动物护理和使用委员会批准（CEUAFVET批准方案代码1460），于2022年3月30日批准。
数据可用性声明
本研究中呈现的原始贡献包含在文章/补充材料中。进一步咨询可联系通讯作者。
利益冲突
作者声明无利益冲突。
资金声明
本研究由FMV-ANII FMV_3_2022_1_172705（Fondo María Viñas–Agencia Nacional de Investigación e Innovación）和CAP-Udelar研究生实习（Comisión Académica de Posgrado–Universidad de la República，乌拉圭）资助。
脚注
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参考文献
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关联数据
补充材料
[内容请查看原文]
数据可用性声明
本研究呈现的原创性贡献已包含在文章/补充材料中。进一步的咨询可联系通讯作者。
查看英文原文▾
Simple Summary
Feline chronic gingivostomatitis (FCGS) is a severe inflammatory disease of the oral cavity that causes pain, difficulty eating, and reduced quality of life. Dental extractions are considered the standard treatment, but little is known about how the oral tissues and local immune response change after surgery. This pilot prospective study followed five cats after treatment, evaluating their clinical and blood parameters, with three cats also being evaluated for histopathologic changes and local immune cell markers. Most cats showed clinical improvement after dental extractions. Reduced inflammation was observed in oral biopsies. Expression of B-cell markers in the affected tissues was reduced after dental extraction. However, changes in other immune markers varied among individuals. These findings provide preliminary information on the local tissue response following standard surgical treatment and improve our understanding of this chronic inflammatory disease.
Keywords: gingivostomatitis, inflammation, dental extraction, histology, cat
1. Introduction
Feline chronic gingivostomatitis (FCGS) is a painful, debilitating, and multifactorial inflammatory disease characterized by severe inflammation affecting the oral mucosa that markedly decreases the quality of life of affected cats [1,2,3,4,5]. Lesions may involve the gingival, alveolar, and buccolabial mucosae, and most characteristically, the caudal oral mucosa lateral to the palatoglossal folds [3,4,5]. Extraoral manifestations, including esophagitis and tracheobronchial inflammation, have also been described, further supporting the concept that FCGS is associated with widespread mucosal inflammation [6,7]. Histologically, the disease is characterized by a dense lymphoplasmacytic infiltrate accompanied by variable numbers of neutrophils and macrophages. Immunophenotypic studies have demonstrated the presence of multiple immune cell populations within affected tissues, including CD20+ B lymphocytes, CD3+ T cells (including CD4+ helper and CD8+ cytotoxic subsets), and FoxP3+ regulatory T cells [8,9]. More recently, transcriptomic and immunological studies have suggested that FCGS is characterized by persistent immune dysregulation involving alterations in T-cell function and inflammatory pathways, supporting the hypothesis that the disease represents an aberrant host response [2,8,10].
Surgical treatment through partial-mouth extraction (PME) or full-mouth extraction (FME) remains the current standard of care, although approximately 30% of cats exhibit partial clinical responses or remain refractory to treatment [1,11]. Previous investigations evaluating treatment efficacy have primarily focused on clinical outcomes [11,12,13,14], whereas more recent studies have explored alterations in the oral microbiome, systemic inflammatory biomarkers, and transcriptomic profiles associated with disease progression and treatment response [15,16,17,18,19]. In particular, transcriptomic analysis of circulating CD8+ T cells demonstrated an exhaustion-like phenotype and impaired mitochondrial bioenergetics, while cats evaluated after FME and mesenchymal stem cell (MSC) therapy showed partial recovery of these molecular signatures [19].
Despite these advances, relatively little is known regarding the evolution of the inflammatory infiltrate and local immune response within the oral mucosa following FME or PME. Most studies characterizing the oral immune microenvironment have focused either on disease pathogenesis or evolution in cats refractory to FME undergoing MSC therapy [2,8,20,21,22]. Although reductions in lymphoplasmacytic infiltration have been reported in treatment responders receiving cell-based therapies, longitudinal information describing the histopathologic and immunological changes after standard surgical treatment remains limited. Owing to the practical difficulties associated with obtaining sequential oral biopsies, the local tissue response to dental extractions has been insufficiently characterized.
The aim of the present prospective study was to describe the clinical, hematologic, biochemical, histopathologic, and local immunological changes that occur after PME in cats with FCGS.
2. Materials and Methods
2.1. Study Design
This pilot prospective longitudinal study was conducted to characterize the clinical, hematologic, biochemical, histopathologic, and local immunological changes that occur after PME in cats diagnosed with FCGS (Figure 1). The inclusion criteria were as follows: cats presenting inflammatory lesions compatible with FCGS, without oncologic or immune-mediated disease. Selected patients exhibited caudal oral mucositis and alveolar mucositis involving the premolar and/or molar regions, with no involvement of the canine or incisor regions. There were no restrictions regarding breed, age, body weight, or feline immunodeficiency virus (FIV) or feline leukemia virus (FeLV) status. Written informed consent was obtained from all owners before enrollment. Corticosteroid therapy, when present, was discontinued at least 20 days prior to surgery and was not permitted during the study period. No cat received a long-acting injectable glucocorticoid. NSAIDs, pregabalin, gabapentin, and opioids were allowed during the study period. Cats meeting the inclusion criteria underwent PME, comprising extraction of all the premolar and molar teeth. Before surgery, cats received antibiotics (amoxicillin–clavulanic acid (15 mg/kg BID) or spiramycin–metronidazole (75,000 IU/kg and 12.5 mg/kg, respectively)) and NSAIDs (meloxicam (0.1–0.02 mg/kg SID)). Antibiotics were maintained from 7 to 10 days after surgery. Intraoral radiographs were obtained before PME, but post-extraction dental radiographs were not routinely obtained; they were only performed when root fracture occurred to confirm complete root removal. Between 7 and 15 days after surgery, the cats received amoxicillin–clavulanic acid and meloxicam (dose reduced from 0.1 mg/kg to 0.03 mg/kg SID), as well as pregabalin (2 mg/kg SID or BID). The cats were evaluated on the day of PME (D0); at 7 (D7), 15 (D15), 30 (D30), and 60 (D60) days; and monthly thereafter until 6 months post-treatment (D180). Evaluations were performed without sedation at all time points, except at D0 and D30, when three cats were anesthetized for biopsy sampling.
[图：Overview of the study design.]
2.2. Clinical Evaluation
Clinical disease severity was evaluated using the Stomatitis Disease Activity Index (SDAI) scoring system, considering both investigator and owner evaluations, and ranged from 0 (healthy) to 30 (severe) [12,23]. Scoring was performed at every follow-up. Investigators scored the lesions (0–3, from No Lesion to Severe), and the cats’ owners scored the cat’s appetite, activity level, grooming behavior and perceived oral comfort. The percentage of SDAI improvement was calculated using the following formula: [(initial SDAI D0 − post-treatment follow-up SDAI)/initial SDAI D0] ×100; the threshold for improvement was >12% following previous studies [23]. A reduction of >50% compared to the initial SDAI score was considered good improvement, >30–50% was considered moderate improvement, 12–30% was considered mild improvement, −12 to <12% was considered no improvement, and less than −12% was considered worsening [23].
2.3. Blood Parameters
Complete blood counts (ProCyte One analyzer, Idexx, Maine, USA) and serum biochemical (CM320, Weiner lab, Buenos Aires, Argentina) analyses were performed at predetermined time points throughout follow-up. Patients were considered anemic when at least two of the following erythrocyte parameters were below the established reference ranges for the species: total red blood cell count, hematocrit, and/or hemoglobin concentration. Anemia was classified as regenerative or non-regenerative according to the absolute reticulocyte count [24]. Retroviral infection status was determined using commercially available point-of-care assays for FeLV antigen and FIV antibodies (FASTest FIV/FeLV, Megacor Diagnostik GmbH, Hörbranz, Germany).
2.4. Evaluation of Local Inflammation
A 0.5 cm full-thickness biopsy was collected under general anesthesia from the caudal oral mucosa lateral to the palatoglossal folds of three cats at D0 and D30. The biopsy sample was collected from one side of the oral cavity at D0 and from the contralateral side at D30. Histopathologic and immunohistochemical (IHC) analyses were performed on formalin-fixed, paraffin-embedded specimens. Tissue samples were fixed in 4% buffered formaldehyde, processed, and sectioned into 4 μm thick sections. To assess inflammation, the sections were stained with hematoxylin and eosin (H&E). The degree of inflammation was evaluated using a scoring system for the severity of the microscopic inflammatory changes (Grades 0–3, from normal to severe) [9]. Histological images were visualized and acquired using a transmitted light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japon) equipped with a high-resolution digital camera and capture software (AmScope MU853B USB 3.0 high-speed digital camera, CA, USA). The sections were also scanned using a whole-slide scanner (Motic EasyScan One scanner, Motic China Group Co., Ltd., Xiamen, China) at a focal plane with default settings and a magnification of 400× (image resolution: 0.25 µm/pixel) and visualized using a viewer (Motic ‘DSAssistant’, Motic VM V1 Viewer 2.0, version 2019-08-02, China) and scanning software (Motic ‘EasyScanner’, Hong Kong, China). Histopathologic evaluation was performed by four observers blinded to the patient and sampling time point, with final scores assigned by consensus.
To assess the type of cellular infiltrate, expression of CD20, FoxP3, and IL-17 antigens was evaluated. For this, tissue sections were deparaffinized with xylene and rehydrated through a graded ethanol series. A 3% hydrogen peroxide blocking solution was applied, followed by heat-induced antigen retrieval in a steamer, with slides submerged in 0.01 M citrate buffer (pH 6) in a polypropylene Coplin jar (Deltalab, Barcelona, Spain) for 30 min. IHC conditions were optimized using feline lymph nodes and caudal oral mucosa as positive controls. Negative controls were performed by replacing the primary antibody with phosphate-buffered saline (PBS). Isotype controls were not performed. Manufacturer recommendations were followed with minor modifications. The primary antibodies used in this study are listed in Table 1. The selected CD20 antibody [25] and FoxP3 clone (221D) [26] have been used in feline tissues. IHC detection of IL-17A has previously been reported in other feline tissues using a different antibody [27]; however, this does not constitute validation of the antibody used in the present study for feline tissue. Each primary antibody was applied to the tissue, which was then incubated overnight at 4 °C in a humid chamber. The detection system (Mach 4 Universal HRP-Polymer Detection System, Biocare Medical, CA, USA) and streptavidin–peroxidase were applied. Labeling was visualized using DAB solution for 3–5 min, and then the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Histological images were acquired as previously described.
Table 1. Details of antibodies for immunohistochemical labeling.
| Primary Antibody | Manufacturer | Dilution |
|---|---|---|
| Rabbit anti-CD20 (polyclonal) | Invitrogen/Catalog # PA5-16701 | 1:400 |
| Mouse anti-FoxP3 (clone 221D) | Donated by Giovanna Roncador, Spanish National Cancer Research Center (CNIO) | undiluted |
| Rabbit anti-IL-17A (polyclonal) | ABCAM/Catalog # ab79056 | 1:200 |
Immunolabeled areas were quantified using digital image analysis software, allowing for objective assessment of marker expression and minimizing observer-related bias. Analysis of whole-slide images was performed collaboratively by four observers blinded to the patient and sampling time point using Motic SlideViewer software (Motic VM V1 Viewer 1.0) to ensure consistency in interpretation. Immunolabeled cells were evaluated in five non-overlapping high-power fields (400×) [28] selected from representative areas of the inflammatory infiltrate after examination of the entire section at low magnification. Necrotic areas were excluded from the evaluation due to their potential to produce nonspecific staining and false-positive results, as were regions with prominent vascularization, severe spongiosis, or technical artifacts related to tissue processing, sectioning, or staining. Areas with technical staining artifacts that prevented reliable discrimination of specific DAB signals from the background were excluded. Quantification was conducted using FIJI (ImageJ 2.14.0 1.54p, NIH, MD, USA). A color threshold was applied to isolate the specific chromogenic signal and distin guish it from the background. The thresholded image was then binarized to convert the selected signal into a binary format. The area occupied by positive staining was subsequently quantified and expressed as a percentage of the total image area. All images were processed using identical parameters for brightness, contrast, and thresholding to ensure consistency across samples.
2.5. Descriptive Analysis
The data are presented descriptively as individual values for each cat and time point. No inferential statistical analyses were performed.
3. Results
3.1. Patient Characteristics
Five client-owned mixed-breed cats with a mean age of 7 years were included. Four had the ulcerative form of FCGS, and one had the proliferative form. Cats were housed indoors or had a mixed indoor–outdoor lifestyle and lived with a mean of two additional cats per household. Individual patient characteristics and FIV/FeLV status are shown in Table A1 and Table A2, respectively.
3.2. Clinical Evaluation
Clinical response after PME varied among individuals; however, all cats exhibited reductions in SDAI scores by day 30 compared with baseline (Figure 2). The degree of improvement differed among cats. At D0, cats showed a mean SDAI score of 12.6. At D30, two showed good improvement, two showed moderate improvement, and one showed mild improvement. Three cats were followed up to D180, with two of them showing good improvement and one showing mild improvement at this time point. Cat E showed good improvement in SDAI score but died due to complications associated with FeLV (lymphoblastic leukemia) after the study period. Cat C was withdrawn after D90 due to kidney disease, although it showed an improvement in SDAI score after PME. Cat D was lost to follow-up after D30. Cat A received amitriptyline for feline orofacial pain syndrome (FOPS) control from D60 to D120 and then CBD oil afterwards, achieving full remission by D180. Cat C received omeprazole.
[图：( A ) Stomatitis Disease Activity Index (SDAI) for each cat and timepoint. Each colored dot represents a single feline p]
3.3. Blood Parameters
Three of the five cats showed anemia at baseline (Table A2, Figure S1). Persistent non-regenerative anemia was observed in cats D and E, both of which were positive for FeLV and/or FIV. In contrast, transient regenerative anemia was documented in cat C during follow-up. Monocytosis was particularly pronounced in cats D and E. Eosinophilia and increased ALP were observed in cat B at D15, which declined by D60, coinciding with the detection of endoparasites (Dipylidium spp.), after which the cat received anthelmintic treatment.
Hyperglobulinemia represented the most consistent biochemical abnormality and was present in all cats at one or more time points (Figure S2). Hypoalbuminemia was observed in two of five cats at D0, which subsequently normalized in all but one cat. Additionally, one cat had elevated serum urea and creatinine levels and consequently received treatment for kidney disease.
3.4. Histopathological Findings
Oral biopsies were collected from three FeLV-positive cats (A, D, and E) at D0 and D30 (Figure 3A). Cat B did not attend the scheduled D30 visit under conditions suitable for general anesthesia, and cat C was not biopsied because elevated serum urea and creatinine concentrations were detected. Considering the potential anesthetic risk, general anesthesia for biopsy collection was not performed in the interest of this cat’s welfare. Tissue sections exhibited epithelial hyperplasia with abundant cellular debris in the most severe cases. Moderate-to-marked lymphoplasmacytic infiltrate was consistently noted, primarily localized within the submucosa. Mott cells were occasionally observed within the infiltrate. Neutrophils were identified in the submucosa and perivascular regions, particularly in areas displaying edema and necrosis. In samples graded as most severe, there was evident spongiosis, interstitial edema, and dense inflammatory infiltrate encasing muscle fibers. Histopathologic examination of the D0 and D30 oral biopsies from cat E showed chronic inflammatory changes without evidence of neoplastic infiltration. Lymphoblastic leukemia was diagnosed after completion of the study period, on day 200.
[图：Inflammatory changes in oral biopsies pre- and post-PME. ( A ) Microscopic images of oral biopsies (H&E, 100×, scale]
Two cats exhibited a reduction in severity of microscopic inflammatory changes accompanied by clinical improvement (decrease in SDAI) (Figure 3B), whereas one cat showed an unchanged inflammatory score and minimal improvement in SDAI, and was positive for FIV and FeLV.
3.5. Immunohistochemical Findings
IHC analysis revealed distinct labeling patterns for CD20, FoxP3, and IL-17 in sequential biopsies from three cats (Figure 4a). CD20 labeling was intense and predominantly concentrated in the lamina propria, with infiltration of the submucosa. Both nuclear and cytoplasmic FoxP3 labeling was observed in a few cells within the lamina propria, mainly located near the epithelium in areas showing inflammation or edema. IL-17 labeling was detected in the cytoplasm and interstitium in the epithelium, lamina propria, and submucosa.
[图：Immunohistochemistry images. ( a ) Illustrative microscopic images (100×) of immunohistochemical staining for CD20, FoxP]
Quantitative analysis of immunolabeled areas demonstrated reductions in CD20 expression in all cats following PME. The magnitude of these changes varied among individuals, with decreases of 46.3%, 39.4%, and 18.6% in cats A, D, and E, respectively. Cats A and E also showed a decrease in histologic inflammatory score and clinical improvement. In contrast, FoxP3 and IL-17 immunolabeling patterns showed marked interindividual variability. FoxP3-positive areas decreased in two cats that also exhibited a reduction in both SDAI and histologic inflammatory scores, but one cat showed an increase in FoxP3 labeling area while its histologic inflammation score remained unchanged, and it showed minimal clinical improvement. IL-17 labeling decreased in two cats and increased in one cat. Individual SDAI scores, histopathologic inflammatory scores, and quantitative immunohistochemical measurements are summarized in Figure 4b.
4. Discussion
The present pilot prospective study provides longitudinal information regarding the clinical, histopathologic, and local immunological changes that occur following PME in cats with FCGS. Traditionally, patients require medical management for more than 14 days following dental extraction [11]. The cats showed clinical improvement 1 month post-PME, though the degree varied over time and between cats as measured by SDAI. The clinical response after dental extractions may continue to evolve over several months, and early evaluations may not reflect the final outcome. Improvement or clinical resolution has been reported six months after surgery [11,29]. Therefore, although the cats were followed for up to 180 days, this study primarily aimed to characterize early post-PME changes, particularly during the first 30 days. Previous reports using different scoring systems observed that 52% of cats showed clinical improvement 38 days after surgical treatment [29], while more recent studies have reported significant improvement at 60 days [18].
In this small cohort, the cats positive for retroviral infections exhibited the most pronounced hematologic abnormalities. The main hematologic finding was anemia, observed predominantly in cats positive for FIV and/or FeLV. These findings are consistent with recent reports of anemia in 18% of FeLV and 12% of FIV-positive cats, as well as leukemia in 16% and 3% of these populations, respectively [30]. The same study reported FCGS in 11% of FeLV-positive cats and 24% of FIV-positive cats. Among the three FeLV-positive cats, cats D and E developed clinically relevant hematologic complications during follow-up. Despite FeLV positivity, cats A and E showed clinical improvement (improved SDAI score). FeLV infection has been associated with a 7.5-fold higher likelihood of poor clinical response after dental extraction [13]. In this study, eosinophilia and increased ALP were observed in one patient at D15 post-PME, possibly associated with its concurrent Dipylidium spp. infection. Although eosinophilia is not commonly reported in cats with FCGS, a retrospective study documented peripheral eosinophilia in approximately 25% of cases [31], highlighting the importance of investigating concurrent infections.
The main biochemical finding at D0 was hyperglobulinemia, consistent with previous reports describing chronic antigenic stimulation and systemic inflammatory activation in cats with FCGS [5,32]. However, no changes were observed post-PME. Recent studies suggested that dental extraction does not contribute to a significant decrease in alpha-1 acid glycoprotein levels at D60, indicating the maintenance of systemic inflammation despite clinical improvement [18]. In contrast, our results showed a correction of serum albumin levels 15 days after PME in all but one cat. The post-surgical increase in serum albumin levels may reflect a reduction in systemic inflammation following PME, as albumin is a prominent negative acute-phase protein in cats, and decreased concentrations are observed in chronic inflammatory diseases [33]. These contrasting results emphasize the need for further investigation into the effects of PME on the cat immune system.
In this study, one cat showed increased urea and creatinine levels compatible with chronic kidney disease (CKD). Previous reports found that approximately 10% of cats with FCGS presented with CKD [34]. Later reviews highlighted the importance of screening for concurrent systemic disorders, as comorbidities such as CKD may influence both clinical severity and treatment response [4,5]. A recent cross-sectional analysis comparing cats with caudal stomatitis and healthy controls revealed mild but significant differences in biochemical markers, such as lower creatinine and higher phosphorus levels, without a higher prevalence of CKD [35]. However, previous studies found that 26.5% of cats showed serum urea concentrations above the upper reference limit [31]. These conflicting results indicate that we should consider monitoring renal function biomarkers in cats with FCGS.
The histologic findings showed the classical lymphoplasmacytic and neutrophilic infiltrate mentioned in previous reports [5,9,32,36]. Area-based quantification was used to provide a standardized assessment of the overall extent of immunolabeling within the inflammatory infiltrate, particularly in regions with dense or partially confluent staining where reliable identification of individual cell boundaries was not always possible. The same approach was applied consistently across all markers. The results showed a reduction in the histologic inflammatory infiltrate score in two cats and a reduction in CD20 immunolabeling in three cats. However, CD20 immunolabeling decreased to different degrees in the three cats, irrespective of their SDAI response, which raises the question of whether it reflects the removal of the extraction-related antigenic stimulus rather than control of the disease. Interestingly, the changes in FoxP3 and IL-17 immunolabeling were heterogeneous: two cats exhibiting histologic improvement showed reductions in FoxP3-positive areas, while the cat with persistent severe histologic inflammation demonstrated increased FoxP3 immunolabeling despite minimal clinical improvement. Although the biological significance of this observation remains uncertain and any interpretation is necessarily speculative, decreased FoxP3 immunolabeling could be consistent with reduced antigenic stimulation following PME, whereas increased FoxP3 immunolabeling could reflect a compensatory response to persistent inflammation. Furthermore, since IHC detection provides expression and morphologic information, it does not allow for the assessment of cellular functionality. IL17-A showed marked interindividual variability. Previous transcriptomic studies on FCGS suggest that signaling pathways related to the IL-17 family may be involved in the local immune response [2,8,10]. However, these findings are not directly comparable with the immunohistochemical observations of the present study. Elevated pro-inflammatory cytokines, such as IL-6, TNF-α, and IFN-γ, have been documented in FCGS and constitute part of an inflammatory milieu that promotes the differentiation and function of Th17 cells [10,32]. The detection of these cytokines using immunolabeling techniques has not been performed in caudal oral mucosa. Although IHC detection of IL-17A has previously been reported in other feline tissues using a different antibody [27], this does not constitute validation of the antibody used in the present study for feline tissue. Therefore, the IL-17A IHC findings should be interpreted with caution.
The main limitations of this pilot study include the small sample size and the availability of paired histopathologic and IHC samples from only three cats. Because paired sections were not always processed within the same IHC run, between-run variability in chromogen development and staining intensity cannot be excluded. Sequential biopsies were obtained from contralateral sides at D0 and D30 to avoid healing tissue; therefore, site-related differences in tissue composition may have influenced the area-based IHC measurements. Interobserver agreement was not formally assessed, although final histopathologic scores were assigned by consensus among the four blinded observers. Furthermore, it should be noted that, by chance, the three cats biopsied were FeLV-positive, which may negatively affect the lymphocyte populations and the clinical response following dental extraction [13]. Another limitation of this pilot study is that feline calicivirus (FCV) was not tested because a validated FCV assay was not available at our institution. Meloxicam was administered during the early postoperative period and discontinued before the D30 biopsy; nevertheless, a potential influence on inflammatory or immune-related markers at D30 cannot be completely excluded. Because this study involved client-owned cats, concomitant medical treatment could not be withheld when clinically indicated, particularly for pain management and animal welfare. In addition, one cat developed FOPS during follow-up and subsequently received additional medication that could have potentially influenced the immune-related outcomes in a manner that was not controlled for by the study design. However, these additional treatments were initiated after the D30 biopsy and therefore should not affect the early histopathologic and IHC comparisons between D0 and D30. Nevertheless, their potential influence should be considered when interpreting the later clinical and immunologic findings.
A limited number of studies have reported clinical follow-up after PME in cats with FCGS. Notably, studies on cell therapy have provided valuable insight into the immunological evolution of cats with refractory FCGS treated with MSC [20,21,22]. However, these findings should not be directly extrapolated to cats undergoing PME because MSC therapy represents an active immunomodulatory intervention, whereas PME primarily reduces local antigenic stimulation. This lack of comparable data also highlights the relevance of investigating immunologic changes following the recommended standard-of-care surgical treatment. To the best of our knowledge, the present pilot study is among the few that prospectively evaluated the longitudinal clinical, histopathological, and local immunological changes that occur after PME. A better understanding of the immune changes induced by PME could provide critical insights into the pathophysiology of the disease and help explain the heterogeneity in clinical outcomes. Larger longitudinal studies are needed to determine whether specific immunological patterns are associated with treatment outcomes.
5. Conclusions
Clinical SDAI scores decreased after PME in the five cats, although the magnitude and duration of improvement varied among individuals. In the three FeLV-positive cats with paired biopsies, CD20 immunolabeling decreased irrespective of the clinical and histologic responses, whereas FoxP3 and IL-17 levels showed variable changes. These findings represent preliminary descriptive observations of early tissue changes following PME and should be interpreted considering the small biopsy subset and the methodological limitations of this study.
Acknowledgments
The authors thank every owner of cats who participated in this study. The authors used ChatGPT (OpenAI, GPT-5.5) to assist with English-language editing and improvement of scientific writing. All suggested revisions were critically reviewed and approved by the authors.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vetsci13101038/s1, Figure S1. This figure displays individual data points for key hematological parameters: red blood cells, hemoglobin, hematocrit, leukocytes, lymphocytes, neutrophils, monocytes, and eosinophils. Measurements were conducted at specified time points from day 0 to day 60. Each individual colored dot represents a single feline patient, coded as follows: A (Red), B (Green), C (Blue), D (Pink), and E (Black). Error bars indicate the standard deviation (SD) of the mean, and the dashed horizontal lines denote the established reference range for each respective parameter. Figure S2. This figure displays individual data points for key biochemistry parameters: serum globulin, albumin, total proteins, alkaline phosphatase (ALP), urea and creatinine. Measurements were conducted at specified time points from Day 0 to Day 60. Each individual colored dot represents a single feline patient, coded as follows: A (Red), B (Green), C (Blue), D (Pink), and E (Black). Error bars indicate the standard deviation (SD) of the mean, and the dashed horizontal lines denote the established reference range for each respective parameter.
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Appendix A
Table A1. Characteristics of cats with feline chronic gingivostomatitis.
| Id | Age | Sex | Cohabiting Cats | FCGS Type | Life Style |
|---|---|---|---|---|---|
| A | 8 | Male | 1 | Ulcerative | Mixed |
| B | 4 | Female | 1 | Proliferative | Indoor |
| C | 9 | Male | 6 | Ulcerative | Indoor |
| D | 7 | Male | 4 | Ulcerative | Mixed |
| E | 7 | Male | 0 | Ulcerative | Mixed |
Table A2. Anemia status over time for each feline patient.
| ID | D0 | D7 | D15 | D30 | D60 | D90 | D180 | Anemia Type | FIV | FeLV |
|---|---|---|---|---|---|---|---|---|---|---|
| A | NO | NO | NO | NO | - | - | - | - | NEG | POS |
| B | NO | NO | NO | NO | + | - | NO | - | NEG | NEG |
| C | ++ | + | NO | NO | - | NO | - | R | POS | NEG |
| D | +++ | + | + | ++ | - | - | - | NR | POS | POS |
| E | + | - | + | ++ | NO | + | +++ | NR | NEG | POS |
Author Contributions
Conceptualization, A.A. and K.Y.; methodology, A.A., G.T. and K.Y.; validation, B.V., G.T., A.W., J.M.V. and K.Y.; formal analysis, A.A., L.T., B.V. and K.Y.; investigation, A.A., L.T., B.V., G.T., M.M., J.E., M.N.G. and K.Y.; resources, B.V., G.T., J.E., J.M.V. and K.Y.; data curation, A.A., L.T. and M.M.; writing—original draft preparation, A.A., L.T., B.V., G.T., M.M., J.E. and M.N.G.; writing—review and editing, A.W., J.M.V. and K.Y.; visualization, A.A., J.M.V. and K.Y.; supervision, G.T., A.W., J.M.V. and K.Y.; project administration, A.A. and K.Y.; funding acquisition, A.A., B.V., J.M.V. and K.Y. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
This study was approved by the Institutional Animal Care and Use Committee of Faculty of Veterinary (CEUAFVET approved protocol code 1460) approved on 30 March 2022.
Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by FMV-ANII FMV_3_2022_1_172705 (Fondo María Viñas–Agencia Nacional de Investigación e Innovación) and CAP-Udelar posgraduate internships (Comisión Académica de Posgrado–Universidad de la República, Uruguay).
Footnotes
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References
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Associated Data
Supplementary Materials
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Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.