J Vet Sci.  2017 Sep;18(3):267-272. 10.4142/jvs.2017.18.3.267.

Inoculation of Lewis lung carcinoma cells enhances formalin-induced pain behavior and spinal Fos expression in mice

• Choi JG
• Kim JM
• Kang DW
• Choi JW
• Park JB
• Ahn SH
• Ryu Y
• Kim HW

• KMID: 2412443
• DOI: http://doi.org/10.4142/jvs.2017.18.3.267

Abstract

The incidence of lung cancer has rapidly increased and cancer patients at a later cancer stage frequently suffer from unbearable cancer-associated pain. However, the pathophysiology of lung cancer pain has not been fully described due to a lack of appropriate animal models. This study was designed to determine the effect of Lewis lung carcinoma (LLC) cell inoculation on formalin-induced pain behavior and spinal Fos expression in C57BL/6 mice. LLC cells (1.5 × 10⁵, 2.5 × 10⁵, 3.0 × 10⁵ or 5.0 × 10⁵) were inoculated into back or peri-sciatic nerve areas. Back area inoculation was adopted to determine the effect of cancer cell circulating factors and the peri-sciatic nerve area was used to evaluate the possible effects of cancer cell contacting and circulating factors on formalin-induced pain. At postinoculation day 7, LLC cell (5.0 × 10⁵) inoculations in both back and peri-sciatic nerve area significantly increased formalin-induced paw-licking time and spinal Fos expression over those in cell-media-inoculated (control) mice. Enhanced pain behavior and spinal Fos expression were significantly suppressed by ibuprofen pretreatment (250 mg/kg). The results of this study suggest that LLC cell circulating factors and inflammatory responses may be critical in enhancing pain sensation in the early stage of lung cancer cell inoculation.

Keyword

Fos; Lewis lung carcinoma; formalin; lung neoplasms; pain

MeSH Terms

Analgesics, Non-Narcotic/therapeutic use
Animals
Cancer Pain/drug therapy/*etiology
Carcinoma, Lewis Lung/*complications
Formaldehyde/pharmacology
Ibuprofen/therapeutic use
Male
Mice
Mice, Inbred C57BL
Neoplasm Transplantation
Oncogene Proteins v-fos/*metabolism
Pain/chemically induced/etiology/psychology
Spinal Cord/drug effects/*metabolism
Spinal Cord Dorsal Horn/drug effects/metabolism
Analgesics, Non-Narcotic
Oncogene Proteins v-fos
Formaldehyde
Ibuprofen

Figure

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  Fig. 1 Effect of Lewis lung carcinoma (LLC) cell inoculation on formalin-induced pain behavior. LLC cells were administered into mouse back (Back) or peri-sciatic nerve (Sciatic) areas at four different inoculated cell numbers: 1.5 × 105 (A), 2.5 × 105 (B), 3.0 × 105 (C), and 5.0 × 105 (D). At 7 days postinoculation, formalin-induced first phase (0–10 min) pain was not affected in all groups of mice. Only the highest number of injected LLC cells (5.0 × 105) significantly increased formalin-induced late phase (10–40 min) pain in Back and Sciatic areas (D). Data are presented as mean ± SEM values. *p < 0.05 and **p < 0.001 in comparison to media + formalin group.

• 

  Fig. 2 Effect of pretreatment with ibuprofen (Ibu, 250 mg/kg, intraperitoneally) on Lewis lung carcinoma (LLC; 5.0 × 105) cell-enhanced formalin pain behavior. Formalin test was performed 7 days after the inoculation of LLC into back (Back) or peri-sciatic nerve (Sciatic) areas. Ibu treatment 30 min prior to the formalin injection significantly decreased late phase (10–40 min) LLC-enhanced formalin pain behavior during the second phase (10–40 min after formalin injection). Data are presented as mean ± SEM values. *p < 0.05 in comparison to vehicle (V)-treated control group.

• 

  Fig. 3 Representative photomicrographs illustrating Lewis lung carcinoma (LLC) cell-enhanced and formalin-induced Fos expressions in the ipsilateral spinal cord dorsal horn of lumbar 4–6 spinal segments. (A) formalin-injected control; (B) LLC cells (5.0 × 105) injected into back area + formalin injection; (C) LLC cells (5.0 × 105) injected into peri-sciatic nerve area + formalin injection; (D) media only injected into peri-sciatic nerve area + formalin injection + ibuprofen pretreatment (250 mg/kg, intraperitoneally); (E) LLC cells injected into back + formalin injection + ibuprofen pretreatment; (F) LLC cells injected into peri-sciatic nerve area + formalin injection + ibuprofen pretreatment. SDH, superficial dorsal horn (laminae I–II); NP, nucleus of proprius (laminae III–IV); NECK, dorsal horn neck (laminae V–VI). Scale bar = 200 µm.

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  Fig. 4 Effect of pretreatment with ibuprofen (Ibu, 250 mg/kg, intraperitoneally) on Lewis lung carcinoma (LLC) cell-enhanced and formalin-induced expression of Fos-like immunoreactive (FLI) neurons in the spinal dorsal horn. LLC inoculation into back (Back) or peri-sciatic nerve (Sciatic) areas significantly increased the number of FLI neurons in each segment of the spinal dorsal horn; that is, superficial dorsal horn (SDH; laminae I–II), nucleus of proprius (NP; laminae III–IV) and dorsal horn neck (NECK; laminae V–VI). Ibu treatment occurred 30 min prior to formalin injection and suppressed the LLC- and formalin-induced increase in the number of FLI neurons. ***p < 0.001.

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