Table 8.

Evaluation methods and parameters in experimental DED.

Types of Tests	Items	Animal	Methods	References
Clinical sings		Rabbit	Conjunctivitis (0–3), ocular discharge (0–3), corneal opacity (0–3)	Baudouin et al. [l] 2013, Silva et al. [74] 2017
	Modified severity score from Eaton J.S. et al. [137]; Silva D.A. et al. [74]		The scored varies from 0 to 16 and was defined as the sum of the individual scores graded from conjunctival hyperemia (0–3), conjunctival swelling (chemosis) (0–4), conjunctival discharge (0–3), corneal opacity (area) (0–4), and corneal vascularization (0–2).	Silva et al. [74] 2017, Eaton et al. [137]
Inflammatory index/scoring and markers	Macroscopic (visually) inflammatory scoring/index	Mice	Total score of 0–9; ciliary hyperemia (0–3); central corneal edema (0–3); peripheral corneal edema (0–3)	Lin et al. [83] 2011, Xiao et al. [84] 2013, De Paiva et al. [135] 2009, Laria et al. [136] 1997
	Microscopically evaluated by infiltration of inflammatory cells	Rats	Evaluated by counting PMN cells in the cornea and limbus by HE stains	Han et al. [138] 2017
	Inflammatory cytokines	Mice, Rat	TNF-α, IL-6, TNF- α, IL-1 a, IL-1β, and MMP-9 in corneas, CD11b+ (IHC)	Xiao et al. [84] 2012, Han et al. [138] 2017, Kwon et al. [139] 2016, De Paiva et al. [140] 2006
		Rabbit	TNF-α, IL-1 β, IL-6, IL-8 in corneas	Tseng et al. [81] 2016
		Rabbit	IL-1β, TNF-a, and MMP-9 in conjunctiva epithelium	Bhattacharya et al. [43] 2015
Test for detection of tear abnormality	Tear film osmolarity	Rats	Using Tearlab Osmolarity System® osmometer. Osmolarity increase in DED.	Marques et al. [87] 2014
	Schirmer’s I Test	Rabbit	Tear volume (mm)	Luo et al. [79] 2017, Tseng et al. [81] 2016
		Rats		Liu et al. [141] 2019
	Phenol red thread tests	Rabbit		Lin et al. [142] 2018
		Rat		Han et al. [138] 2017
		Mice		Dietrich et al. [143] 2019
	Tear breakup time (BUT) (Specific for tear film instability)	Mice, rats, rabbit	Usually, TBUT is ≤10 s for in DED	Liu et al. [141] 2019, Shinzawa et al. [144] 2019, Wei et al. [145] 2013
Surface structural damage	Corneal Fluorescein Staining Test	Rabbit		Luo et al. [79] 2017, Tseng et al. [81] 2016
		Rat		Liu et al. [141] 2019
		Mice	Oxford grading	Dietrich et al. [143] 2019
	Rose Bengal Staining Test	Rabbit		Luo et al. [79] 2017, Na et al. [88] 2017
		Rat		Beyazyildiz et al. [86] 2014
		Mice		Lin et al. [83] 2011
	Lisamine green staining	Rabbit		Chen et al. [133] 2021
		Rat		Song et al. [112] 2020
		Mice		Chen et al. [146] 2017
Changes of epithelial layers of cornea or conjunctiva	Corneal epithelial thickness	Rabbit		Luo et al. [79] 2017, Tseng et al. [81] 2016, Luo et al. [79] 2017, Tseng et al. [81] 2016
		Mice		Xiao et al. [84] 2013, Na et al. [88], Diego et al. [147] 2016
		Rat		Marques et al. [87] 2014
	Goblet cells count	Mice, Rabbit, Rats	Counting the goblet cells number by staining MUC5AC in the conjunctiva	Luo et al. [79] 2017, Lin et al. [83] 2011, Han et al. [138] 2017
		Rabbit	Conjunctival impression cytology (CIC) by hematoxylin and periodic acid-Schiff (PAS) reagent	Luo et al. [79] 2017, Jiang et al. [148] 2017
		Rabbit, mice	Counting goblet cells in conjunctiva by periodic acid-Schiff (PAS) staining	Chen et al. [44] 2011, Xiao et al. [84] 2013
Molecular analysis	MAP kinase pathways	Rabbit	p-ERK1/2 protein expression	Jiang et al. [148] 2017
		Mice	phospho-JNK/total JNK, phospho-ERK/total ERK, phospho-p38 (p-p38)/total p38 (p-38) in the corneal epithelia	De Paiva et al. [140] 2006
	NFkB	Mice, HCE cells	Western blot analysis of NFkB	Tan et al. [113] 2018, Zhao et al. [149] 2019
	Apoptosis	Rabbit, mice, rat	TUNEL assay (apoptotic cells count)	Tseng et al. [81] 2016, Luo et al. [79] 2017, Xiao et al. [84] 2013, Han et al. [138] 2017
		Mice	Bax, BCL2, Bax/BCL2	Na et al. [88] 2017
	Vascular dysfunction	Mice	VEGF-A in corneas; cell vascular endothelial cells on corneal flat mounts	Kwon et al. [139] 2016
		Rabbit	endothelial cell damage with dislocation of ZO-1, and disruption of PAMR	Liang et al. [63] 2008
	Fibrosis	Rabbit	TGF-β1	Yao et al. [150] 2010