Line system (Tet-On/Off system) [14], which equilibrates transgene expression to endogenous levels [15]. Excessive oxidative stress induces mitochondrial respiratory chain dysfunction and results in excessive apoptosis leading to low birth weight and growth retardation in Tet-mev-1 mice [14]. Using thismouse model, we found that the lacrimal gland of Tet-mev-1 mice produced more O22 and oxidative protein than the lacrimal gland of wild type mice. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease.Methods Animals and MaterialsC57BL/6L and Tet-mev-1 mice were bred and maintained under specially pathogen free (SPF) conditions in the Center of Genetic Engineering for Human Disease (CGHED) (Tokai University School of Medicine, Kanagawa, Japan). Doxycycline was administered in a drinking water mix (dose: 2 mg/ml). All mice used in analyses were 3 month old males.HistopathologyUnder the operating microscope, the lacrimal gland and submandibular salivary gland were surgically excised after death. A portion of each dissected specimen was immediately embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Miles Inc., Elkhart, IN, USA) and snap frozen in pre-cooled isopentane at 280uC. The remainder of the tissues was analyzed after being fixed in 4 paraformaldehyde or 10 neutral buffered formalin and embedded in paraffin wax.Oxidative Stress Induced Dry Eye DiseaseHE staining and Azan staining. Five micrometer-thick paraffin embedded sections fixed in 4 paraformaldehyde were cut and stained with HE. Additionally, 5 mm-thick paraffin embedded sections fixed in 10 neutral buffered formalin underwent Azan staining to evaluate the severity of fibrosis in the 18055761 lacrimal gland. Immunohistochemical analysis of DNA damage due to oxidative stress (8-OHdG). The 5 mm-thick paraffin embed-ded sections fixed in 4 paraformaldehyde were cut and stained with a mouse anti-8-OHdG monoclonal antibody (Japan Institute for the Control of Aging [JaICA], Shizuoka, Japan) to analyze DNA damage due to oxidative stress [16,17]. After removal of paraffin, the sections were placed in 10 mM citrate buffer solution and autoclaved at 121uC for 10 min. After blocking with 10 normal goat serum (Vector Laboratories, Burlingame, CA), sections were first blocked with Avidin/Biotin blocking reagent (Vector Labs) and then with a mouse on mouse blocking reagent (M.O.M.TM). Blocking with the anti-mouse IgG blocking reagent (Vector Laboratories) was completed overnight at 4uC. Sections were exposed to diluted mouse anti-8-OHdG monoclonal antibody (1:10). Antibody binding was detected with a horse anti-mouse IgG ABC kit (Vector Laboratories) according to the manufacturer’s protocol. The bound antibodies were visualized by the 101043-37-2 site addition of diaminobenzidine tetrahydroxychloride.Analysis of the mononuclear cell fraction using histochemical staining (CD4, CD8, CD19 and F4/80). Im-20 min at 4uC was again performed and the resulting RNA 
pellet was washed in 3 ml of 70 MedChemExpress 374913-63-0 ethanol and centrifuged at 7000 rpm for 20 min at 4uC. After centrifugation, the RNA pellet was airdried (1 h) at room temperature. After drying, 88 ml 0.1 diethyl pyrocarbonate (DEPC) in distilled water was added to the pellet. The solution was transferred to a 2 ml Eppendorf tube with 2 ml DNase (20 U), 10 ml DNase buffer and 0.5 ml RNase inhibitor (Pharmacia) and was heated for 30 min at 37uC. After cooling on ice.Line system (Tet-On/Off system) [14], which equilibrates transgene expression to endogenous levels [15]. Excessive oxidative stress induces mitochondrial respiratory chain dysfunction and results in excessive apoptosis leading to low birth weight and growth retardation in Tet-mev-1 mice [14]. Using thismouse model, we found that the lacrimal gland of Tet-mev-1 mice produced more O22 and oxidative protein than the lacrimal gland of wild type mice. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease.Methods Animals and MaterialsC57BL/6L and Tet-mev-1 mice were bred and maintained under specially pathogen free (SPF) conditions in the Center of Genetic Engineering for Human Disease (CGHED) (Tokai University School of Medicine, Kanagawa, Japan). Doxycycline was administered in a drinking water mix (dose: 2 mg/ml). All mice used in analyses were 3 month old males.HistopathologyUnder the operating microscope, the lacrimal gland and submandibular salivary gland were surgically excised after death. A portion of each dissected specimen was immediately embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Miles Inc., Elkhart, IN, USA) and snap frozen in pre-cooled isopentane at 280uC. The remainder of the tissues was analyzed after being fixed in 4 paraformaldehyde or 10 neutral buffered formalin and embedded in paraffin wax.Oxidative Stress Induced Dry Eye DiseaseHE staining and Azan staining. Five micrometer-thick paraffin embedded sections fixed in 4 paraformaldehyde were cut and stained with HE. Additionally, 5 mm-thick paraffin embedded sections fixed in 10 neutral buffered formalin underwent Azan staining to evaluate the severity of fibrosis in the 18055761 lacrimal gland. Immunohistochemical analysis of DNA damage due to oxidative stress (8-OHdG). The 5 mm-thick paraffin embed-ded sections fixed in 4 paraformaldehyde were cut and stained with a mouse anti-8-OHdG monoclonal antibody (Japan Institute for the 
Control of Aging [JaICA], Shizuoka, Japan) to analyze DNA damage due to oxidative stress [16,17]. After removal of paraffin, the sections were placed in 10 mM citrate buffer solution and autoclaved at 121uC for 10 min. After blocking with 10 normal goat serum (Vector Laboratories, Burlingame, CA), sections were first blocked with Avidin/Biotin blocking reagent (Vector Labs) and then with a mouse on mouse blocking reagent (M.O.M.TM). Blocking with the anti-mouse IgG blocking reagent (Vector Laboratories) was completed overnight at 4uC. Sections were exposed to diluted mouse anti-8-OHdG monoclonal antibody (1:10). Antibody binding was detected with a horse anti-mouse IgG ABC kit (Vector Laboratories) according to the manufacturer’s protocol. The bound antibodies were visualized by the addition of diaminobenzidine tetrahydroxychloride.Analysis of the mononuclear cell fraction using histochemical staining (CD4, CD8, CD19 and F4/80). Im-20 min at 4uC was again performed and the resulting RNA pellet was washed in 3 ml of 70 ethanol and centrifuged at 7000 rpm for 20 min at 4uC. After centrifugation, the RNA pellet was airdried (1 h) at room temperature. After drying, 88 ml 0.1 diethyl pyrocarbonate (DEPC) in distilled water was added to the pellet. The solution was transferred to a 2 ml Eppendorf tube with 2 ml DNase (20 U), 10 ml DNase buffer and 0.5 ml RNase inhibitor (Pharmacia) and was heated for 30 min at 37uC. After cooling on ice.
