Figure 6. Treatment with an agonist for β2 adrenergic receptor attenuates the anti-tumor effect of Oxt^PVN neuron activation.

(A) Control and AAV-hSyn-DIO-hM3Dq-mCherry (hM3Dq) viruses were injected into the paraventricular nucleus (PVN) of male adult Oxt^Cre mice. Colitis-associated cancer (CAC) was then induced using azoxymethane (AOM) and dextran sodium sulfate (DSS). These mice were i.p. administered with clozapine-N-oxide (CNO) every other day and i.p. injected with saline or isoprenaline, a β2 adrenergic receptor agonist, on a daily basis. After 3 weeks of treatment, mice were perfused with 4% paraformaldehyde (PFA). For control, double immunofluorescence staining for c-Fos (gray) and oxytocin (Oxt) (red) was performed. For hM3Dq, immunofluorescent staining for c-Fos (green) was performed and Oxt neurons were identified using hM3Dq-mCherry (red). Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (B) The percentage of Oxt neurons expressing c-Fos in the PVN. ISO, isoprenaline. n = 7 (control, saline) or 6 (all other groups) mice per group. (C) Adult Oxt^Cre mice were injected with adeno-associated viruses (AAVs) into the PVN. CAC was then induced using AOM and DSS. Subsequently, these mice were i.p. administered with CNO every other day and i.p. injected with saline or isoprenaline on a daily basis. These treatments were continued for 3 weeks (see also Figure 6—figure supplement 1C). Representative images of colorectal tissue after the treatments are shown. White eclipse outlines individual tumor. (D and E) Tumor number (D) and diameter (E). n = 7 (control, saline) or 6 (all other groups) mice per group. (F) Colorectal length. n = 7 (control, saline) or 6 (all other groups) mice per group. (G and H) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) of tumor tissue. Representative images (G) and the density of PCNA-positive cells (H) are shown. Scale bars, 50 μm. n = 4 (saline) or 5 (ISO) mice per group. (I and J) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of tumor tissue. Representative images (I) and the density of TUNEL-positive cells (J) are shown. TUNEL labeling is in red. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 μm. n = 4 mice per group. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s post hoc test (B, D, E, H, J).

Figure 6—figure supplement 1. Body weight and food intake in mice.

(A) Male C57 BL/6 mice (2 months of age) were i.p. administered with saline or isoprenaline (10 mg kg^–1). Two hours later, celiac-superior mesenteric ganglion (CG-SMG) were dissected and fixed in 4% paraformaldehyde (PFA). Double immunofluorescence staining of c-Fos (red) and tyrosine hydroxylase (TH, in green) of the CG-SMG was performed. Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (B) The percentage of TH-positive cells expressing c-Fos in the CG-SMG. n = 5 (saline) or 4 (isoprenaline) mice per group. (C) A schematic diagram illustrating the experimental design. The adult Oxt^Cre mice were injected with control or hM3Dq adeno-associated viruses (AAVs) into the paraventricular nucleus (PVN). The colitis-associated cancer was then induced using azoxymethane (AOM) and dextran sodium sulfate (DSS). Subsequently, these mice were i.p. administered with clozapine-N-oxide (CNO) (3 mg kg^–1) every other day, as well as saline or isoprenaline (ISO), a β2 adrenergic receptor agonist, on a daily basis. These treatments were continued for 3 weeks. (D and E) Body weight (D) and food intake (E) in mice throughout the experiment. n = 7 (control+ CNO + saline), 6 (control+ CNO + ISO), or 5 (hM3Dq) mice per group. Data are presented as means ± SEM.