Study of the roles of caspase-3 and nuclear factor kappa B in myenteric neurons in a P2X7 receptor knockout mouse model of ulcerative colitis

Table 1 Changes in weight, stool consistency, and the presence of blood in the stool were scored for the ulcerative colitis model mice and mice that received intrarectal injection of 35% ethanol according to the following scale

Disease activity index		Score
A	Weight change (< 1%)	0
	Weight change (1%-2%)	1
	Weight change (2%-4%)	2
	Weight change (4%-6%)	3
	Weight change (> 6%)	4
B	Stool consistency (normal)	0
	Stool consistency (normal and well-formed)	1
	Stool consistency (pasty that did not stick to the anus)	2
	Stool consistency (loose stool)	3
	Stool consistency (diarrhea)	4
C	Rectal bleeding (absent)	0
	Rectal bleeding (+)	1
	Rectal bleeding (++)	2
	Rectal bleeding (+++)	3
	Rectal bleeding (gross bleeding)	4

A: Weight; B: Stool consistency; C: Presence of blood in the stool.

Table 2 Scale used to score the severity of macroscopic and microscopic lesions in the distal colon of ulcerative colitis model mice and mice that received intrarectal injection of 35% ethanol

Macroscopic injury	Score
Normal	0
Hyperemia without ulcers	1
Ulcerations without hyperemia	2
Ulcerations at one site	3
Ulcerations at two or more sites	4
Sites of damage extending > 1 cm	5
Sites of damage extending > 2 cm; the score increases by 1 for each additional cm	6-10
A
No ulcer	0
Single ulceration not exceeding the lamina muscularis mucosa	1
Ulcerations not exceeding the mucosa	2
Ulcerations exceeding the submucosa	3
B
No submucosal edema	0
Mild edema	1
Moderate edema	2
Severe edema	3
C
No inflammatory cell infiltration	0
Mild infiltration	1
Moderate infiltration	2
Dense infiltration	3

The presence of ulcers (A), the severity of edema in the submucosal layer (B), and the degree of inflammatory cell infiltration (C) were assessed by histological analysis.

Table 3 Characteristics of primary and secondary antibodies

Antigen	Host	Dilution	Source
Calretinin	Goat	1:300	Swant
P2X7 receptor	Rabbit	1:200	Merck Millipore
Cleaved caspase-3	Rabbit	1:100	Cell Signaling
Total caspase-3	Rabbit	1:100	Cell Signaling
Phospho- NF-κB	Rabbit	1:100	Cell Signaling
Total NF-κB (p65)	Rabbit	1:100	Cell Signaling
Secondary antibodies
Alexa Fluor 488-conjugated donkey anti-rabbit IgG	Donkey	1:500	Molecular Probes
Alexa Fluor 594-conjugated donkey anti-sheep IgG	Donkey	1:100	Molecular Probes

NF-κB: Nuclear factor kappa B.

Table 4 Macroscopic and microscopic lesions in the distal colon of ulcerative colitis model mice and mice that received intrarectal injection of 35% ethanol

Groups	Macroscopic injury	Microscopic injury
		Ulcer	Submucosal edema	Inflammatory cell infiltration
WT sham 24 h	0.0 ± 0.0NS	0.0 ± 0.0NS	0.8 ± 0.2NS	1.2 ± 0.2NS
WT colitis 24 h	1.0 ± 0.0NS	0.0 ± 0.0NS	1.6 ± 0.2NS	2.0 ± 0.2NS
KO sham 24 h	0.0 ± 0.0NS	0.0 ± 0.0NS	0.8 ± 0.2NS	1.0 ± 0.0NS
KO colitis 24 h	0.4 ± 0.1NS	0.0 ± 0.0NS	0.8 ± 0.4NS	1.2 ± 0.2NS
WT sham 4 d	0.0 ± 0.0NS	0.0 ± 0.0NS	1.0 ± 0.2NS	1.0 ± 0.0NS
WT colitis 4 d	0.5 ± 0.2NS	0.0 ± 0.0NS	1.0 ± 0.0NS	1.2 ± 0.2NS
KO sham 4 d	0.0 ± 0.0NS	0.0 ± 0.0NS	0.6 ± 0.2NS	0.4 ± 0.2NS
KO colitis 4 d	0.0 ± 0.0NS	0.0 ± 0.0NS	0.8 ± 0.2NS	0.4 ± 0.2NS

The presence of ulcers, severity of edema in the submucosal layer, and degree of inflammatory cell infiltration were assessed by histological analysis. NS: Not significant; WT: Wild-type; KO: Knockout.