3.4.1. Methods
3.4.1.1. Surgical Procedures for Inflammatory Insult
In order to examine whether BSCB breakdown is altered after an inflammatory
insult, a separate group of rats underwent a painful inflammatory insult to the nerve root
(chromic, n=11) using previously developed and characterized procedures (Chang and
Winkelstein 2011, Rothman and Winkelstein 2007). Identical surgical procedures were
followed as those described in Section 3.3.1.1 to expose the right C7 dorsal nerve root in the rat. Following root exposure, four 1mm pieces of 3-0 chromic gut suture (Surgical
Specialties; Reading, PA) were placed directly on top the nerve root. Chromic gut suture
pieces were kept in contact with the root and the wound was closed using 3-0 polyester
suture and surgical staples. On day 1 (n=7) or day 7 (n=4) after surgery, fixed C7 spinal
cord tissue was harvested and processed for immunolabeling as described in Sections
3.3.1.1 and 3.3.1.3.
3.4.1.2. Assessment of Mechanical Hyperalgesia
For this study, ipsilateral mechanical hyperalgesia was assessed only on days 0, 1
and 7 after the inflammatory nerve root insult, since those time points correspond to the
development and maintence of nerve root-induced pain in this model. Using behavioral
hypersensitivity methods described previously (Section 3.3.1.2), mechanical hyperalgesia
normalized to baseline (day 0) levels. The paw withdrawal threshold in rats treated with
chromic gut suture were compared to thresholds of rats that received a 15-minute
compression or sham operation (see Section 3.3.1). Ipsilateral paw withdrawal thresholds
at day 1 (chromic, n=11; 15min, n=11; sham, n=10) and day 7 (chromic, n=4; 15min,
n=5; sham, n=4) were normalized to baseline (day 0) thresholds. Differences in the
normalized paw withdrawal threshold between groups over time were determined using a
repeated measures two-way ANOVA (group x time) with Tukey’s test. This two-way
ANOVA also tests for differences between the 15-minute compression group and sham
over time, which is also analyzed in the characterization of BSCB breakdown after nerve
root compression (Section 3.3).
3.4.1.3. Spinal Immunohistochemistry for IgG
IgG expression was labeled using methods described in Section 3.3.1.3 in the
bilateral spinal cord from a subset of rats that underwent inflammatory nerve root injury.
In order to assess the influences of injury type on BSCB permeability, spinal IgG
expression in tissue from rats receiving an inflammatory insult via chromic gut suture
application to the root was compared to IgG expression in the spinal cord from 15-minute
compression and sham at day 1 (chromic, n=3; 15min, n=5; sham, n=5) and day 7
(chromic, n=4; 15min, n=5; sham, n=4). A two-way ANOVA (group x day) with Tukey’s
test was used to test differences in spinal IgG expression between painful compression
and inflammatory injuries. This two-way ANOVA compares IgG expression for the 15-
minute compression group and sham over time, which is also analyzed in the
3.4.1.4. Serum Collection & Cytokine Multiplex Assay
Procedures for blood draw and elution of serum from whole blood samples were
performed according to the methods described in Section 3.3.1.4. For analysis of cytokine
levels after an inflammatory nerve root insult, blood was collected from rats on day 1
after exposure to chromic gut suture (chromic, n=7) and assayed. Concentrations of 23
cytokines and chemokines on day 1 were normalized to the corresponding baseline levels
and were correlated to the normalized ipsilateral paw withdrawal threshold at day 1 for
each rat. Each set of bivariate data (normalized threshold versus normalized cytokine
concentration) was fit using a linear regression as described in Section 3.3.1.4.
3.4.2. Results
Overall, an inflammatory nerve root insult induces a significant decrease in paw
withdrawal threshold compared to sham (p=0.0148), as well as on day 1 compared to
sham (p=0.0435) and its own baseline (day 0) levels (p=0.0307) (Figure 3.3A). Similarly,
the 15-minute root compression significantly reduces withdrawal threshold in the
ipsilateral forepaw compared to sham on days 1 (p<0.0001) and 7 (p=0.0021) and overall
(p<0.0001), as well as compared to its own baseline levels (p<0.0001) (Figures 3.2A &
3.3A). The inflammatory and compressive insults do not induce different withdrawal
thresholds on any individual day probed (Figure 3.3A); yet, a 15-minute compression
does induce a significant drop in withdrawal threshold compared to an inflammatory
chromic insult overall (p=0.0159) (Figure 3.3A). The paw withdrawal thresholds for each
A painful inflammatory nerve root insult does not induce a substantial increase in
spinal IgG labeling and its expression is not different from sham levels (Figures 3.3B &
3.3C). A painful 15-minute compression significantly increases (p<0.0001) ipsilateral
spinal IgG expression compared to sham and IgG expression after an inflammatory insult
on day 1 (Figures 3.3B & 3.3C). Taken together, both inflammatory and compressive
nerve root insults induce behavioral sensitivity (Figure 3.3A), but only the compressive
injury also induces BSCB breakdown (Figures 3.3B & 3.3C). A summary of the
quantified spinal IgG expression for each rat in this study is detailed in Appendix B.
Figure 3.3. A painful inflammatory nerve root insult does not induce BSCB breakdown at day 1. (A) The normalized paw withdrawal threshold is significantly decreased at 1 day after an inflammatory root insult induced by chromic gut (chromic) compared to sham (**p=0.0435) and baseline (day 0, #p<0.0307). A 15-minute root compression (15min) significantly decreases the withdrawal threshold at days 1 and 7 compared to sham (*p<0.0021) and day 0 (#p<0.0307). (B) Spinal IgG labeling is relatively low at day 1 in the ipsilateral dorsal horn for both sham and a chromic root insult in contrast to the 15min group, which exhibits robust IgG labeling. (C) The percent of IgG labeling normalized to expression in naïve rats in the ipsilateral spinal cord is significantly elevated (*p<0.0001) at day 1 after a 15min compression compared to both sham procedures and a chromic insult. Data are mean±SD.
Serum levels of pro- and anti-inflammatory cytokines and chemokines were
assayed at day 1 after an inflammatory nerve root injury in order to determine if
inflammatory components also modulate systemic inflammation similarly to a
compressive insult. Unlike nerve root compression (Table 3.1), an inflammation-induced
nerve root insult does not significantly alter any of the serum factors probed in a manner
that correlates to mechanical hyperalgesia (Table 3.2). Serum analyte expression for all
rats in each group can be found in Appendix C.
Table 3.2. Serum levels of pro- and anti-inflammatory cytokines and chemokines (x) and equations of their correlation to ipsilateral forepaw withdrawal threshold (y) on day 1 after inflammatory root insult.
cytokine correlation R2 p-value
pro-inflammatory GM-CSF y=-0.06x+0.73 0.186 0.5690 MCP-1 y=0.30x+0.08 0.176 0.3487 IL-17 y=-0.24x+0.84 0.164 0.3673 MIP-3α y=0.15x+0.36 0.042 0.6569 IL-2 y=0.11x+0.41 0.040 0.6675 TNF-α y=0.04x+0.46 0.037 0.6782 GRO/KC y=-0.12x+0.67 0.034 0.6914 RANTES y=-0.13x+0.66 0.016 0.7890 IL-1β y=0.03x+0.49 0.014 0.7982 IL-7 y=-0.05x+0.59 0.011 0.8269 VEGF y=0.04x+0.48 0.009 0.8382 IL-12 y=0.03x+0.50 0.006 0.8645 G-CSF y=0.02x+0.50 0.006 0.8648 IL-18 y=-0.03x+0.57 0.004 0.8898 IL-1α y=-0.01x+0.55 0.001 0.9443 IFN-γ y=-0.01x+0.55 0.001 0.9375 anti-inflammatory EPO y=0.11x+0.40 0.048 0.6361 IL-13 y=0.02x+0.50 0.006 0.8655 IL-10 y=0.04x+0.49 0.005 0.8821 IL-5 y=-0.06x+0.59 0.002 0.9223 IL-4 y=-0.00x+0.54 0.000 0.9772
pro- and anti-inflammatory
M-CSF y=-0.07x+0.63 0.011 0.8197
IL-6 y=-0.00x+0.54 0.001 0.9618
Note: Shaded cells indicate R2 greater than 0.5. Bolded values represent coefficients of variation greater