Pro-inflammatory cytokines in aqueous humor from dogs with anterior uveitis and post-operative ocular hypertension following phacoemulsification, primary glaucoma, and normal healthy eyes

Abstract

Background The aim of this study was to evaluate the levels of pro-inflammatory cytokines in aqueous humor (AH) from dogs with anterior uveitis and post-operative ocular hypertension (POH) following phacoemulsification, in AH from dogs with primary glaucoma, and in normal healthy eyes with no signs of anterior uveitis or other ocular diseases. Methods An exploratory study including 21 samples of AH collected from 15 dogs; post-phacoemulsification with anterior uveitis and POH (‘POH group’, n = 10 samples), primary glaucoma (‘glaucoma group’, n = 6 samples), and normal (‘normal group’, n = 5 samples). Target mass spectrometry via multiple reaction monitoring (MRM-MS) with the Canine Cytokine SpikeMix™ as internal standard was used to measure the pro-inflammatory cytokine levels. Results The MRM-MS method measured 15 pro-inflammatory cytokines. Tumor-necrosis-factor-alpha (TNFα) and interleukin-18 (IL-18) levels in AH were different between all three groups (glaucoma>POH>normal) (p = .05, p = .02, respectively). Additionally, IL-6 was higher in the ‘POH group’ compared to the ‘glaucoma group’ (p = .04) and IL-4 was higher in the ‘POH group’ compared to the ‘normal group’ (p = .04). Intraocular pressure (IOP) was positively associated with increased AH levels of IL-18 (Spearman correlation = .64, p = .03). Conclusions MRM-MS using the Canine Cytokine SpikeMix™ as an internal standard was established as a method to detect pro-inflammatory cytokine levels in canine AH. The study demonstrated increased levels of IL-4, IL-6, IL-18, and TNFα in AH from canines with POH following phacoemulsification. Primary glaucomatous eyes had the highest levels of IL-18 and TNFα which may indicate that inflammation plays a role in the pathogenesis of primary glaucoma in dogs.

Detection of pro‐inflammatory cytokines in healthy canine tears using Canine Cytokine SpikeMix™ mass spectrometry via multiple reaction monitoring

“…Samples were processed with Canine Cytokine SpikeMix™ and MRM-MS as previously described in Terhaar et al ( 2022), but with minor modifications. 6 The Canine Cytokine SpikeMix™ can detect 144 cytokines but this study only evaluated 16 pro-inflammatory cytokines due to cost constraints and a desire for consistency between studies. Terhaar et al (2022) used the SpikeMix™ to evaluate for all 144 although only 15 cytokines were detected in canine aqueous humor.…”

“…Terhaar et al. (2022) used the SpikeMix™ to evaluate for all 144 although only 15 cytokines were detected in canine aqueous humor 6 . IL‐2 was not found in Terhaar et al.…”

“…Other changes to the protocol were as follows: 30 μg of each sample were digested rather than 50 μg (due to the small volume of tears), and the trypsin digestion was incubated for 6 h instead of 3 h. The difference in digestion time had minimal effect on the samples, as the process is approved for a range of 3–18 h when using ProteaseMax (Promega Corporation, Madison, WI, USA). Without ProteaseMax, the process would take 12–18 h. Analysis of the samples by MRM‐MS was performed with the optimized method described by Terhaar et al 6 . Descriptive data from this study were reported as the normalized total peak area (nTPA) and median (range) using data imported from the online MRM‐MS Skyline program (https://skyline.ms/project/home/begin.view).…”

“…3 However, multiplexed bead assay is associated with quality control issues, variations between laboratories, unexpected molecular interactions causing unpredictable results, and it requires a larger volume of sample for evaluation relative to other methods. 6,7 In contrast, targeted mass spectrometry with multiple reaction method (MRM-MS) is highly specific, highly sensitive, requires low volume samples to screen for multiple cytokines even in low abundance. 6 The purpose of this study was to evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles.…”

“…6,7 In contrast, targeted mass spectrometry with multiple reaction method (MRM-MS) is highly specific, highly sensitive, requires low volume samples to screen for multiple cytokines even in low abundance. 6 The purpose of this study was to evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles. Canine aqueous humor was evaluated for pro-inflammatory cytokines using Canine Cytokine SpikeMix™ and MRM-MS in a recent study, 6 but canine tears have not yet been evaluated with MRM-MS. By evaluating canine tears with mass spectrometry, more detailed information regarding the presence of pro-inflammatory cytokines in the canine tear film may be attained.…”