Ischemia/reperfusion injury in porcine intestine - Viability assessment

Table 1 Comparison of modified Swerdlow et al[21,27,28] and Park/Chiu et al[22,26] systems for grading of histological damage on the intestine

Grade	Modified Swerdlow	Park/Chiu
0	No pathological change	Normal mucosa
1	Focal loss of surface epithelium	Subepithelial space at villus tips
2	Mucosal infarction (extensive loss of surface epithelium, loss of variable amounts of lamina propria, sparing of basal glands, intact muscularis mucosae)	Extension of subepithelial space with moderate lifting
3	Submucosal infarction (variable necrosis of submucosa, complete mucosal necrosis, intact muscularis mucosae)	Massive lifting down the sides of the villi, some denuded tips
4	Mural infarction (loss of muscularis mucosae, complete necrosis of mucosa and submucosa)	Denuded villi, dilated capillaries
5	Mural infarction (involvement of inner layer of muscularis propria, complete necrosis of mucosa and submucosa)	Disintegration of lamina propria
6	Transmural infarction (complete necrosis of the bowel wall)	Crypt layer injury
7		Transmucosal infarction
8		Transmural infarction

Table 2 Clinical parameters during ischemia/reperfusion in porcine jejunum

Ischemia (h)	Observations on the ischemic jejunum	Minutes after reperfusion before color has returned (mean ± SD)	Observable peristalsis in No. of pigs	Reperfusion (h)	Observations on the reperfused jejunum	No. of pigs
0	Normal color					15
1	Purple	0.9 ± 0.1	15 of 15	8	Edema	15
2	Darker purple	2 ± 0.1	2 of 2	8	Edema, slight fibrinous coating	2
3	Darker purple	4 ± 0.3	13 of 13	8	Edema, fluid droplets, slight fibrinous coating	13
4	Darker purple	6 ± 0.7	4 of 4	8	Edema, fluid droplets, fibrinous coating, darker internal hue	4
5	Darker purple	15 ± 1.6	11 of 11	8	Edema, fluid droplets, fibrinous coating, darker internal hue	11
6	Darker purple	26 ± 3.3	3 of 4	8	Edema, fluid droplets, fibrinous coating, deeper red color, darker internal hue	4
8	Black	49 ± 91	0 of 4	8	Edema, fluid droplets, fibrinous coating, deeper red color, darker internal hue	4
12	Patches of paler color					4
16	Necrotic					4

¹There was a lot of internal bleeding in the jejunum, so determination of the time before return of color was difficult. Images in Figure 1.

Table 3 Summary of main findings from light microscopy of 128 biopsies from 5 pigs at selected intervals of ischemia/reperfusion time

Ischemia	1 h isc, 8 h rep	2 h isc, 8 h rep	3 h isc, 8 h rep	4 h isc, 8 h rep	6 h isc, 8 h rep	8 h isc, 8 h rep	Control
I-1: Early loss of SE1	I-1: Early loss of SE1	I-2: Total loss of SE2	I-3: Early loss of CE, congestion and bleeding LP2	I-4: Total loss of SE, focal damage to outer layer of MP2	I-6: Total loss of CE, damage to LP, MM. Bacteria in LP2	I-8: Damage to all components3	N-0: Normal1
I-2: Total loss of SE2	I-1/R-1: Total loss of SE, apoptosis in CE, light N2	I-2/R-1: Apoptosis in CE, light N, congestion and focal bleeding in LP2	I-3/R-1: Apoptosis in CE, N, wavy myocytes in MP2	I-4/R-1: Focal damage to both layers of MP (most to outer layer)2	I-6/R-1: Damage to all components3	I-8/R-1: Damage to all components3	N-6: Few instances of apoptosis in CE, light N and light edema in MP1
I-3: Early loss of CE2	I-1/R-3: Focal damage to outer layer of MP2	I-2/R-3: Early regeneration of SE, congestion, bleeding and necrosis in LP, apoptosis in CE, interstitial inflammation in MP2	I-3/R-3: Edema, inflammation, and focal necrosis in outer layer of MP2	I-4/R-3: Total loss of CE, NGR, cell disintegration in MM and MP3	I-6/R-3: Damage to all components3	I-8/R-3: Damage to all components3	N-12: Few instances of apoptosis in CE, light N and light edema in MP1
I-4: Focal damage to outer layer of MP2	I-1/R-6: SE regenerated. Focal damage to outer layer of MP1	I-2/R-6: Regeneration of SE, wavy myocytes and focal necrosis in MP2	I-3/R-6: Most of CE is lost, wavy myocytes and focal necrosis in MP2	I-4/R-6: Total loss of CE, NGR, loss of myocytes, disintegration3	I-6/R-6: Damage to all components3	I-8/R-6: Damage to all components3
I-5: Damage to inner layer of MP2	I-1/R-8: SE regenerated. Focal damage to outer layer of MP1	I-2/R-8: Regeneration of SE with focal loss and erosion, focal damage to the MP with wavy myocytes and necrosis2	I-3/R-8: Most of CE is lost, wavy myocytes and focal necrosis in both layers of MP2	I-4/R-8: Damaged SE, CE, MM, submucosa, MP, PM3	I-6/R-8: Damage to all components3	I-8/R-8: Damage to all components3
I-6: Total loss of CE, damage to LP, MM and bacteria in LP3
I-7: Hemorrhage in subserosa, peritonitis, and damage to all components3
I ≥ 8: Damage to all components3

Each column shows the results from a series of tissue samples, with time progression from the top to the bottom. The table is indexed using “I” for ischemia, “R” for reperfusion and “N” for normal perfusion, followed by a number showing the hour duration. The results are indexed by a superscript number by the end of each sentence.

¹Normal/light changes,

²Visible cell damage, but still probably viable,

³Probably irreversible cell damage. CE: Crypt epithelium; LP: Lamina propria; MM: Muscularis mucosae; MP: Muscularis propria; N: Neutrophils; SE: Surface epithelium.

Table 4 Summary of main findings from transmission electron microscopy of porcine jejunum at selected intervals of mesenteric occlusive ischemia and reperfusion

Ischemia (h)	Observations	Ischemia/reperfusion (h/h)	Observations
0	Intact musculature. Some variation in the electron density in the muscle cells, focal swollen mitochondria’s with vacuolized matrixes1
1	Intact musculature. Discrete intercellular edema. Lymphocytes in the interstitial space. Increased variation in the electron density in the muscle cells. Some cells have increased electron density (darker). Some of the mitochondria are more prominent. Some minimal fat vacuoles are visible2	1-3	Inflammation, cell death, sparse fine-vacuolization of the sarcoplasm, slightly swollen mitochondria2
2	More prominent variation in electron density between muscle cells. Increased number of vacuoles, some of them are fat vacuoles. Focal edema, thickening of the mitochondrial cristae. Some lysosomes with membrane fragments2	2-3	Inflammation, cell death, more comprehensive fine-vacuolization of the sarcoplasm, slightly swollen mitochondria2
3	Same results as at 2 h, but a few more interstitial immune response cells are visible. Monocytes, macrophages, and a few granulocytes. Vacuoles in the sarcoplasm. Slightly swollen mitochondria2	3-3	Inflammation, cell death, more comprehensive fine-vacuolization of the sarcoplasm, slightly swollen mitochondria, focal single cell necrosis, swollen cell nuclei2
4	Same changes as at 3 h, but the changes are more prominent as the cells with higher electron density are more condensed, and there are more vacuoles around the mitochondria2	4-3	Pronounced cell shrinking/cell death, swollen cell nuclei, loss of cohesion, interstitial edema3
5	Focal edema, variations in electron density, thickening of the mitochondrial cristae, vacuoles in the sarcoplasm, swollen mitochondria, interstitial lymphocytes/monocytes/granulocytes, loss of plasma-membrane and coherence, focal single cell necrosis3	5-3	Increased cell shrinking/cell death, swollen cell nuclei, loss of cohesion, interstitial edema3
6	Necrosis, focal large vacuoles in some mitochondria3	6-3	Increased cell shrinking/cell death, swollen cell nuclei, loss of cohesion, interstitial edema3
7	Necrosis with macrophages. Non-necrotic cells appear like the cells at time intervals 3-6 h3
8	Like the results at 7 h3

Changes in the muscularis propria and serosa are described (3 pigs, a total of 58 samples). The results are indexed by a superscript number by the end of each sentence.

¹Normal/light changes,

²Visible cell damage, but still probably viable,

³Probably irreversible cell damage.