Inhibition of the Continuum of Radiation-Induced Normal Tissue Injury by a Redox-Active Mn Porphyrin

Abstract

Normal tissue damage after head and neck radiotherapy involves a continuum of pathologic events to the mucosa, tongue and salivary glands. We examined the radioprotective effects of MnBuOE, a redox-active manganese porphyrin, at three stages of normal tissue damage: immediate (leukocyte endothelial cell [L/E] interactions), early (mucositis) and late (xerostomia and fibrosis) after treatment. In this study, mice received 0 or 9 Gy irradiation to the oral cavity and salivary glands ± MnBuOE treatment. Changes in leukocyte-endothelial cell interactions were measured 24 h postirradiation. At 11 days postirradiation, mucositis was assessed with a cathepsin-sensitive near-infrared optical probe. Stimulated saliva production was quantified at 11 weeks postirradiation. Finally, histological analyses were conducted to assess the extent of long-term effects in salivary glands at 12 weeks postirradiation. MnBuOE reduced oral mucositis, xerostomia and salivary gland fibrosis after irradiation. Additionally, although we have previously shown that MnBuOE does not interfere with tumor control at high doses when administered with radiation alone, most head and neck cancer patients will be treated with the combinations of radiotherapy and cisplatin. Therefore, we also evaluated whether MnBuOE would protect tumors against radiation and cisplatin using tumor growth delay as an endpoint. Using a range of radiation doses, we saw no evidence that MnBuOE protected tumors from radiation and cisplatin. We conclude that MnBuOE radioprotects normal tissue at both early and late time points, without compromising anti-tumor effects of radiation and cisplatin.

FIG. 1

Schematic of study methods. Panel A: Comprehensive timeline of treatment course for radioprotection studies. Mice received a loading dose of MnBuOE or saline on day 1, followed by TIW maintenance doses up through 12 weeks. Radiation was given as a single 9 Gy fraction on day 0. Panel B: Radiation field was targeted to include the oral cavity and salivary glands, found in the neck. Care was taken to exclude the central nervous tissue from the treatment field.

FIG. 2

Mucositis signal on day 11 postirradiation. Inflammatory tissue (pink) was identified using ProSense 750EX, which is cleaved to become activated in the presence of cathepsins to yield a near infrared signal. A 9 Gy dose significantly increased in NIR signal in mice treated with both saline and 2/1 mg/kg MnBuOE. NIR signal was decreased in mice treated with either 0.6/0.3 mg/kg or 0.2/0.1 mg/kg MnBuOE. Representative FMT images are shown for all groups. N = 4–6 mice/group. Data is reported as mean with SEM. *Significant difference (P < 0.05) vs. saline/0 Gy control **Significant difference (P, 0.05) vs. 2/1 mg/kg MnBuOE/0 Gy control.

FIG. 3

Intravital quantification of L/E interactions and physical properties of blood vessels 24 h postirradiation. Panel A: Example image obtained from intravital microscopy of the inferior surface of a mouse tongue after i.v. administration of 0.1% acridine orange solution 24 h postirradiation. The three white arrows indicate rolling leukocytes visible along the venule. Panel B: Sub-panel i: Mean number of rolling leukocytes decreases in response to a combination of 0.2/0.1 MnBuOE and radiation. Sub-panel ii: Radiation increased the mean velocity of free-flowing leukocytes in the blood (V_L) in mice treated with saline. This effect was not observed in mice treated with MnBuOE. Sub-panel iii: Radiation or MnBuOE treatment alone (2/1) led to an increase in the average diameter (D) of blood vessels. Combined radiation + MnBuOE treatment (0.2/0.1) did not lead to an increase in diameter vs. the saline control. Sub-panel iv: Shear rates (g_s) were calculated with the formula g_s = 8V_L/D. Only mice treated with MnBuOE alone (2/1) experienced a decrease in shear rate, primarily driven by increased vessel diameter. Data points are presented for each vessel, along with mean and SEM. N = 8–13 mice/group *Significant difference (P < 0.05) vs. saline/0 Gy control.

FIG. 4

Radiation-induced xerostomia at 11 weeks postirradiation (post-RT). To reduce variability, all mice were normalized to salivation levels for their appropriate control. Mice receiving saline or 2/1 mg/kg MnBuOE treatment experienced significant (P < 0.05) decreases in salivation in response to radiation, but mice treated with either 0.6/0.3 or 0.2/0.1 mg/kg MnBuOE experienced smaller, nonsignificant decreases in saliva production compared with unirradiated controls. N = 5–6 mice/group *Significant difference (P < 0.05) vs. saline/0 Gy control **Significant difference (P < 0.05) vs. 2/1 mg/kg MnBuOE/0 Gy control.

FIG. 5

Analysis of fibrosis in salivary glands and tongues at 12 weeks postirradiation. Panel A: Masson’s trichrome staining in the salivary glands and tongue results in bright turquoise staining of fibrotic tissue and red staining of healthy tissue. Panel B: Plot of fibrosis in the salivary glands normalized to the control value of either saline or 2/1 mg/kg MnBuOE control. A 9 Gy dose increases fibrosis in mice that received saline only. MnBuOE attenuates fibrosis in irradiated mice, with all doses of MnBuOE equally effective at reducing fibrosis. N = 4–6 mice/group. *Significant difference (P < 0.05) vs. saline/0 Gy control. Panel C: Plot of fibrosis in the tongue normalized to the control value of either saline of 2/1 mg/kg MnBuOE control. As in the salivary glands, the 9 Gy dose led to an increase in fibrosis. Both 0.6/0.3 and 0.2/0.1 mg/kg MnBuOE treatment reduce fibrosis to a greater degree than 2/1 mg/kg. N = 4–6 mice/group. *Significant difference (P < 0.05) vs. saline/0 Gy control.

FIG. 6

MnBuOE did not interfere with radiation/cisplatin-mediated tumor growth delay in a murine xenograft model. The human pharyngeal carcinoma tumor cell line (FaDu) was transplanted to flanks of nude mice randomized to receive fractionated radiation + cisplatin along with MnBuOE treatment or saline control injections. MnBuOE was started one week after transplanted and continued TIW for the duration of the experiment. Radiation + cisplatin treatments were initiated as tumors reached 200 mm³. Differences in tumor growth rate were compared by assessing time to tripling of tumor volume, using the initial treatment volume of reach mouse as baseline.

FIG. 7

MnBuOE protects mice against normal tissue injury after radiation + cisplatin treatments. Panel A: Mice were treated with 1× 9 Gy + 6 mg/kg cisplatin. Saline or MnBuOE treatment (0.6 mg/kg loading dose) began 24 h prior to radiation + cisplatin treatments and maintenance dosing of MnBuOE (0.3 mg/kg) continued TIW for five weeks (post-RT). Panel B: Radiation + cisplatin treatments increased NIR signaling in saline-treated mice compared to unirradiated controls (P < 0.01) and to MnBuOE-treated mice that received radiation + cisplatin (P < 0.01). Mice treated with MnBuOE and/or radiation + cisplatin did not show significantly greater NIR signal than unirradiated controls [P=0.002, across all groups (ANOVA), N = 2–4/group]. Panel C: All radiation + cisplatin-treated mice showed initial weight loss 8–14 days postirradiation. At 10 weeks postirradiation + cisplatin, the body weight of the MnBuOE-treated mice were not significantly different from unirradiated control mice. Radiation + cisplatin-treated saline mice had significantly lower body weight compared to sham-irradiated mice (P < 0.0001), and radiation + cisplatin-MnBuOE-treated (P < 0.01) mice. Two-way ANOVA showed a significant interaction between time and treatment group (P = 0.018). Panel D: MnBuOE lessened long-term ulcerations and moist desquamation in the irradiated area (right side panels) compared to mice that received radiation + cisplatin with saline (left side panels).