A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain

Abstract

The treatment of acute and chronic pain is still deficient. The modulation of glial cells may provide novel targets to treat pain. We hypothesize that astrocytes and microglia participate in the initiation and maintenance of both, acute surgical and chronic neuropathic pain. Rats underwent paw incision, L5 nerve exposure or L5 nerve transection surgery. Behavioral mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry was performed using anti-ionized calcium binding adaptor protein, Iba-1 (microglia), and anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) on day 1, 4 and 7 after surgery. Following paw incision and at spinal L5 segment GFAP expression was increased in laminae I-II and Iba1 in deep laminae on day 1, in the entire dorsal horn on day 4 and dissipated on day 7 after paw incision in parallel with the allodynia. L5 nerve transection induced mechanical allodynia from day 1 to 7 which correlated with Iba-1 increases on day 1, 4 (entire dorsal horn) and day 7 after nerve injury (deep laminae of the dorsal horn) at spinal L5 segment. Conversely, GFAP increased at later time points from day 4 (deep laminae) and on day 7 (entire dorsal horn). Our data demonstrates that astrocytes (GFAP expression) play a role in the initiation of acute pain and the maintenance of chronic pain while Iba-1 increases closely correlated with the early phase of neuropathic pain. Iba1 and GFAP increased rostrally, at L3 segment, after paw incision (day 4) and only Iba1 increased following L5 nerve transection (day 7).

Figure 1

50% withdrawal thresholds ipsilateral to paw incision (A), L5 exposure sham surgery (B) or L5 nerve transection (L5NT, C) before (base line = BL) and one, four and seven days (D1, D4 and D7 respectively) after surgery. *P<0.05 vs. base line value, t tests or Mann–Whitney U test when normality failed. N=4 for all groups.

Figure 2

Representative images and quantification of Iba1 and GFAP immunofluorescence staining at dorsal horn of L5 spinal cord. (A) L5 spinal cord sections of day 1 after surgery groups in normal naïve animals (column 1), contralateral (column 2) and ipsilateral (column 3) to paw incision, ipsilateral to sham surgery (column 4), and ipsilateral to L5 nerve transection (column 5). Quantification of normalized Iba1 and GFAP staining intensity on day 1 following paw incision surgery in normal, contralateral and ipsilateral to paw incision dorsal horn (B and C) and in L5 nerve transection (L5NT) and L5 nerve exposure sham surgery dorsal horn (D and E). The staining was quantified as pixels both at laminae I-II and at entire dorsal horn (whole). Deep dorsal horn (deep) data were calculated by subtracting the value of laminae I-II from the value of whole dorsal horn. Data were normalized using contralateral group (for paw incision model) or L5 nerve exposure sham group (for L5 nerve transection) values as 100%. *P<0.05 (t tests or Mann-Whitney U test when normality failed) vs. ipsilateral to paw incision group in B and D, and vs. L5 nerve transaction group in C.

Figure 3

Representative images and quantification of Iba1 and GFAP immunofluorescence staining at dorsal horn of L5 spinal cord. (A) L5 spinal cord sections of day 4 after surgery groups in contralateral (column 1) and ipsilateral (column 2) to paw incision, ipsilateral to sham surgery (column 3) and ipsilateral to L5 nerve transection (column 4). Quantification of normalized Iba1 and GFAP staining intensity on day 4 following paw incision surgery in contralateral and ipsilateral to paw incision dorsal horn (B and D) and in L5 nerve transection (L5NT) and L5 nerve exposure sham surgery dorsal horn (C and E). The staining was quantified and normalized as described in Figure 2. *P<0.05 (t tests or Mann-Whitney U test when normality failed) vs. ipsilateral to paw incision group in B and D, and vs. L5 nerve transaction group in C and E.

Figure 4

Representative images and quantification of Iba1 and GFAP immunofluorescence staining at dorsal horn of L5 spinal cord. (A) L5 spinal cord sections of day 7 after surgery groups in contralateral (column 1) and ipsilateral (column 2) to paw incision, ipsilateral to sham surgery (column 3) and ipsilateral to L5 nerve transection (column 4). Quantification of normalized Iba1 and GFAP staining intensity on day 7 following paw incision surgery in contralateral and ipsilateral to paw incision dorsal horn (B and D) and in L5 nerve transection (L5NT) and L5 nerve exposure sham surgery dorsal horn (C and E). The staining was quantified and normalized as described in Figure 2. *P<0.05 (t tests or Mann-Whitney U test when normality failed) vs. L5 nerve transaction group in C and E.

Figure 5

Representative images and quantification of Iba1 and GFAP immunofluorescence staining at dorsal horn of L3 spinal cord. (A) L3 spinal cord sections of day 4 in contralateral (column 1) and ipsilateral (column 2) to paw incision, and of day 7 in ipsilateral to sham surgery (column 3) and ipsilateral to L5 nerve transection (column 4). Quantification of normalized Iba1 and GFAP staining intensity on day 4 following paw incision surgery in contralateral and ipsilateral to paw incision dorsal horn (B and D) and on day 7 following L5 nerve transection (L5NT) and L5 nerve exposure sham surgery dorsal horn (C and E). The staining was quantified and normalized as described in Figure 2. *P<0.05 (t tests or Mann-Whitney U test when normality failed) vs. ipsilateral to paw incision group in B and D, and vs. L5 nerve transaction group in C.