behavioralmouseC57BL/10 (B10), CBA/ca, C57BL/6 (B6) H-2k, and (B10 X CBA)F1
Objective: To assess the systemic effects of GVHD and anti-TNF treatment by measuring body weight changes in lethally irradiated mice on day 18 post-transplantation
Materials & Equipment Checklist
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View Abstract
Lethally irradiated mice were injected with semiallogeneic, T-depleted bone marrow cells and an amount of peripheral T lymphocytes sufficient to induce graft-vs.-host disease (GVHD) becoming apparent on the second week after the graft and leading to an increasing mortality rate within the following weeks (greater than 90% mortality within 80 d). Mice receiving bone marrow cells alone had no GVHD and were used as controls. Beginning on day 8, mice with GVHD were injected weekly with 2 mg of either rabbit anti-mouse recombinant tumor necrosis factor/cachectin (TNF-alpha) IgG, or normal rabbit IgG. On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD. The anti-TNF treatment resulted in an almost complete prevention of the severe lesions seen in the mice treated with normal rabbit IgG, i.e., the skin epidermal cell necrosis, foci of lichenoid hyperplastic reactions, and loss of the hypodermic fat; in the gut dilatation with marked flattening of the villi and elevation of the crypts, with increased numbers of mitoses and isolated crypt cell necrosis. In addition to preventing these acute lesions, anti-TNF treatment resulted in a significantly decreased mortality (approximately 70% survival at 80 d). These results suggest that during acute GVHD, the activation of grafted lymphocytes leads to a local release of TNF in the cutaneous and intestinal mucosae, which induces epithelial cell alterations and increases the inflammatory reaction.
Protocol Steps
1
Lethal irradiation of recipient mice
Recipient mice greater than 3 months old were irradiated using a Cesium source
2-3 minutes
Note: Dose: 800 rad
View evidence from paper
“Recipient mice >3 mo old, were irradiated by a Cesium source (800 rad, delivered during 2-3 min)”
2
Intravenous injection of bone marrow cells and T lymphocytes
Lethally irradiated mice were injected intravenously with T-depleted bone marrow cells, either supplemented with T lymphocytes from lymph nodes or alone as control
Note: T lymphocytes: 2 X 10^6 B10 LN cells; Controls received BMC alone
View evidence from paper
“Recipient mice >3 mo old, were irradiated by a Cesium source (800 rad, delivered during 2-3 min) and injected intravenously with T depleted bone marrow cells (BMC), either supplemented with T lymphocytes prepared from lymph nodes (LN), or alone, as a control”
3
Begin anti-TNF or control IgG treatment
Starting on day 8 post-transplantation, mice with GVHD were injected with either rabbit anti-TNF IgG or normal rabbit IgG as control
Weekly injections until day 35
Note: Dose: 2 mg per injection; Treatment began before clinical symptoms appeared
View evidence from paper
“Beginning on day 8, mice with GVHD were injected weekly with 2 mg of either rabbit anti-mouse recombinant tumor necrosis factor/cachectin (TNF-alpha) IgG, or normal rabbit IgG”
4
Body weight measurement on day 18
Body weight was measured on day 18 post-transplantation to assess systemic effects of GVHD and anti-TNF treatment
Note: Weight loss was compared between GVHD mice treated with normal IgG, GVHD mice treated with anti-TNF, and control mice without GVHD
View evidence from paper
“On day 18, normal rabbit IgG-injected GVHD mice showed a weight loss of 11% (4%, mean SD) compared with control mice without GVHD (i.e., not injected with parental T cells), while there was no significant difference in weight between anti-TNF-injected GVHD mice and control mice”
5
Tissue collection and histological examination
On days 16-18, mice were killed to examine skin and intestinal lesions of acute phase GVHD
Note: Specimens were fixed in 2% formaldehyde/80% ethanol, embedded in paraffin or methyl methacrylate, and sectioned for staining
View evidence from paper
“On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD”
Subjects / Specimens
Species
mouse
Strain
C57BL/10 (B10), CBA/ca, C57BL/6 (B6) H-2k, and (B10 X CBA)F1
Age
greater than 3 months old
Sex
unknown
Weight
not specified at baseline
Recipient mice were lethally irradiated and injected with T-depleted bone marrow cells with or without T lymphocytes