1. Volume 129, Issue 6, Pages 1991-2008 (December 2005)
Targeted Deletion of Metalloproteinase 9 Attenuates Experimental Colitis in Mice: Central Role of Epithelial-Derived MMP 
Florencia E. Castaneda, Baljit Walia, Matam Vijay–Kumar, Neal R. Patel, Susanne Roser, Vasantha L. Kolachala, Mauricio Rojas, Lixin Wang, Gabriela Oprea, Pallavi Garg, Andrew T. Gewirtz, Jesse Roman, Didier Merlin, Shanthi V. Sitaraman 
Gastroenterology 
Volume 129, Issue 6, Pages (December 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
MMP-9 is induced after DSS-induced colitis. Mice were given water or 3% DSS (wt/vol) for 6 days, at which time mice were killed. Colon was harvested and processed as described in the Materials and Methods section. Colonic tissue homogenates from WT and MMP-9−/− mice were analyzed by gelatin zymography. Each lane shows protein (30 μg per lane) from an individual mouse with or without DSS treatment. (A and B) Representative zymogram of homogenates from WT and MMP-9−/− mice, respectively. Gelatinolytic bands of MMP-9 are indicated by arrows. (C and D) Representative Western blot of protein from the colon of WT and MMP-9−/− mice probed with anti–MMP-9. Each lane shows protein (30 μg per lane) from an individual mouse with or without DSS treatment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
MMP-9 knockout mice are resistant to the development of colitis. Mice were weighed and randomized into 4 groups, WT water or DSS (3%) and MMP−/− water or DSS (3%) for 6 days, at which time they were killed. Disease severity was assessed as described in the Materials and Methods section and expressed in terms of percentage body change (A), stool consistency (B), fecal blood (C), and clinical activity score (D). Values are representative of 3 individual experiments (mean ± SE); n = 5 for the DSS-treated groups and n = 4 for the control group in each of the experiments. Significant differences from mice given water are shown: *P < .05, **P < .0001, and #P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Targeted deletion of MMP-9 inhibits inflammation associated with DSS. Mice were weighed and randomized into 4 groups, WT water or DSS (3%) and MMP-9−/− water or DSS (3%), for 6 days, at which time they were killed. (A) Colons were removed from individual mice on day 6, fixed in formalin, paraffin-embedded, sectioned, and stained with H&E. Representative sections of colon from each group are shown. WT and MMP-9−/− mice given water show normal colonic mucosa and intact crypts; WT mice given DSS show severe colonic inflammation characterized by ulceration (arrows), a dense cell inflammatory infiltrate in the mucosa and submucosa, and disruption of the crypt architecture; and MMP-9−/− mice given DSS show minimal inflammatory cell infiltration and crypt architectural distortion. (B) One group of WT and MMP-9−/− mice was switched to plain drinking water after 7 days of DSS. Mice were killed after 7 days of drinking water. Representative histological sections from WT and MMP-9−/− littermates treated with DSS are shown. Note the extensive ulceration, inflammatory infiltrate, and granulation tissue in WT mice. MMP-9−/− mice showed healed mucosa and residual inflammatory infiltrate in the lamina propria.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Granulocyte accumulation in the colon in response to DSS is dependent on MMP-9. Mice were weighed and randomized into 4 groups, WT water or DSS (3%) and MMP-9−/− water or DSS (3%), for 6 days, at which time they were killed. (A) Colons were removed from individual mice on day 6, fixed in formalin, paraffin-embedded, sectioned, and stained with H&E. The numbers of granulocytes and lymphocytes in colonic sections from WT and MMP-9−/− mice with or without DSS administration were counted (mean ± SE; 4–6 fields per section with 3 individual sections from each animal; n = 5 for the DSS-treated group and n = 4 for the control group). Significant differences compared with the control group are shown: *P < .05; #P < .01. (B) Colons were snap-frozen in liquid nitrogen, and myeloperoxidase was measured as an index of neutrophil infiltration into the injured tissue, as described in the Materials and Methods section. Each bar represents mean ± SE; n = 6 animals for each group. **Significantly different from mice given water (P < .001).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Mice were weighed and randomized into 4 groups: WT water or DSS (3% wt/vol) and MMP-9−/− water or DSS (3% wt/vol) for 6 days. Mice were killed, and colon was processed for immunohistochemistry as described in the Materials and Methods section. Immunoreactive MMP-9 was shown by peroxidase reaction. Sections were counterstained with hematoxylin. Shown here are representative sections from 3 individual experiments (n = 5 for the DSS group and n = 4 for the control group). (A–C) WT mice fed DSS (original magnification, 40×) showing dense staining localized to epithelial cells bordering an ulcer (B) and away from an ulcer (A) and granulocytes (C). Inset: Section incubated with anti-rabbit immunoglobulin G primary antibody. (D) WT mice fed water. (E) MMP-9−/− mice fed with water showing no MMP-9 staining (original magnification, 40×). (F) MMP-9−/− DSS-exposed mouse colonic section showing a focal area of granulocyte accumulation and preserved crypt architecture (original magnification, 40×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Targeted deletion of MMP-9 inhibits Salmonella typhimurium–induced enterocolitis. Mice were randomized into 4 groups: WT vehicle or S typhimurium and MMP-9−/− vehicle or S typhimurium. Mice were pretreated with streptomycin before the administration of S typhimurium, as described in the Materials and Methods section. Mice were killed 24 hours after the administration of S typhimurium. Cecum and colon were harvested, photographed (A), and processed by MPO assay. (B) The photograph and histological results are representative of 2 individual experiments (n = 5 per group). (C) MPO values are represented as mean ± SE (n = 5); P < .001 compared with MMP-9−/− mice treated with S typhimurium. (D) MMP-9 immunohistochemistry performed on WT and MMP-9−/− mice infected with S typhimurium showing intense MMP-9 staining in the epithelial cells and inflammatory cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Lack of MMP-9 does not affect systemic Salmonella typhimurium infection or neutrophil transmigration. (A) WT and MMP-9−/− mice were administered S typhimurium SL3201 (104 colony-forming units per mouse). Mice were weighed daily and observed for clinical signs of sepsis, as well as mortality. Data represent the mean ± SE of 2 independent experiments (n = 10). (B) Neutrophils from WT and MMP-9−/− mice were isolated, and transmigration was performed as described in the Materials and Methods section. Neutrophil transmigration was initiated by 1 × 10−6 mol/L or 1 × 10−5 mol/L fMLP in HBSS+. After 120 minutes at 37°C, migrated neutrophils across monolayers into chemoattractant-containing lower chambers were quantitated by myeloperoxidase assay. Data represent the mean ± SE of 3 individual experiments, each consisting of duplicate determinations. Significant differences from the control group are shown: *P < .05; #P < .01; **P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
Neutrophil MMP-9 is not required for neutrophil migration, and mucosal MMP-9 mediates tissue damage. (A) Bone marrow transplantation was performed as described in the Materials and Methods section (D, donor; R, recipient; WT, wild type; KO, MMP-9−/− mice). Each bar represents the mean ± SE (n = 6). a or c vs b: P < .01; c vs a: P < .05. (B) Colons were snap-frozen in liquid nitrogen, and myeloperoxidase was measured as an index of neutrophil infiltration into the injured tissue as described in the Materials and Methods section. Each bar represents the mean ± SE (n = 6 animals for each group). a vs b or c and b vs c: significantly different from each other (P < .001 and P < .05, respectively). (C) Histological assessment of colitis after administration of 3% DSS for 5 days. Scores are presented as mean ± SD: 4–6 fields per section with 3 individual sections from each animal (n = 6). aP < .01, significantly different compared with MMP-9−/− donor and MMP-9−/− recipient.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 9
MMP-9 impairs cell attachment to matrix. (A) Caco2-BBE cells were seeded into ECIS plates. MMP-9 was added at the time of seeding, and capacitance was measured every few seconds for 16 hours (green, 100 ng/mL; red, 10 ng/mL; shown in duplicate). Representative values from 2 individual experiments performed in duplicate are shown (n = 4). (B) Photomicrograph of the wells taken at the end of the experiment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

11. Figure 10
MMP-9 impairs wound healing. (A) Caco2-BBE monolayers plated in ECIS plates were subjected to increased voltage pulses of 40-kHz frequency, 4.5-V amplitude, and 30-second duration to create a wound. Vehicle (black, phosphate-buffered saline) or MMP-9 (red, 100 ng/mL; green, 1 μg/mL) was added. The wound was then allowed to heal, and resistance was measured every few seconds for 16 hours. Representative normalized resistance values to a baseline resistance of 2 independent experiments are shown (n = 4). (B) Photomicrograph of the wells taken at the end of the experiment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions