Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat

Abstract

This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray (PAG) and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund's adjuvant (CFA). Matrix-Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1 (HK-1), which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the PAG and cuneiform nucleus. SubP content in homogenates of the PAG and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.

Keywords: hemokinin-1; hyperalgesia; inflammation; neurokinin-1 receptor; rostral ventromedial medulla; substance P.

Fig. 1

MALDI-TOF mass spectra of Substance P (SubP) and hemokinin-1 (HK-1) in the rostral ventromedial medulla (RVM). periaqueductal gray (PAG) and spinal cord of the rat. (A) SubP and HK-1 (1 fmol each) separated with m/z values of 1347.68 and 1413.69, respectively. Representative spectra indicate the presence of SubP and absence of HK-1 in samples of (B) RVM, (C) PAG, and (D) spinal cord. Panels E and F illustrate spectra for a sample from the RVM that was split into two after homogenization, of which one half was spiked with 50 pmol HK-1 prior to further processing. Note that the plate was spotted with an aliquot containing ~ 1 pmol HK-1.

Fig. 2

Expression of Tac1 in the rostral ventromedial medulla (RVM) and periaqueductal gray (PAG) of rats does not differ between saline (SAL)- or complete-Freund's adjuvant (CFA)-treated rats either four days or two weeks after intraplantar injection. Experiments for Tac1 in the RVM were run using 96 well plates and normalized to Actb, while those for the PAG were run on 394 well plates and normalized to the geometric mean of Actb and Mapk6. As a result, the normalized values cannot be compared between regions. Values are the mean ± S.E.M. of determinations in 6-8 samples. * P = 0.04 compared to four day CFA-treatment group.

Fig. 3

Total SubP content as measured by enzyme immunoassay in the rostral ventromedial medulla (upper panels) of rats did not differ between saline (SAL)- or complete Freund's adjuvant (CFA)-treated rats four hours, four days or two weeks after intraplantar injection. In contrast, SubP content increased in the periaqueductal gray and cuneiform nuclei (lower panels) in a time-dependent manner in CFA-treated rats. Values are the mean ± S.E.M. of determinations in 8-15 samples for the RVM and 4-7 for the PAG. * P < 0.05, ** P < 0.01 compared to saline at the corresponding time point. † P < 0.05 compared to CFA-treated rats at 4 hrs or 2 weeks.

Fig. 4

Peripheral inflammatory injury increased substance P (SubP) immunoreactivity in the rostral ventromedial medulla (RVM) in a time-dependent manner. Representative images of SubP immunoreactivity in the RVM four hours, four days and two weeks after intraplantar injection of saline or complete Freund's adjuvant (CFA). Scale bar is 200 μm and applies to each image. SubP immunoreactivity in (A) ipsilateral, (B) contralateral and (C) combined RVM increased four hours (n = 5) and four days (n = 6), but not two weeks (n = 3) after intraplantar injection. Data were normalized to the respective ipsilateral, contralateral, or total values in saline-treated rats and expressed as a ratio, where 1 indicates no difference from saline values. Ratios are the mean ± S.E.M. of determinations in 3-6 samples per treatment group. * P < 0.05; ** P < 0.01 compared to 1 (one-sample t-test).