Study Design & Surgical Groups

4.4.1.1. Quantifying Spinal Astrocytic PAR1 Expression

Since compression-induced spinal thrombin activity is hypothesized to activate

astrocytes in the spinal cord through PAR1, the spinal astrocytic expression of PAR1 was

measured over time after root compression injuries. Briefly, a 15-minute (15min, n=9), 3-

minute (3min, n=8) or 0-minute (sham, n=10) compression was separately applied to the

right C7 nerve root using previously described surgical procedures on day 0 (Section

Figure 4.8. Intrathecal inhibition of PAR1 activation with SCH79797 does not reduce spinal fibrin(ogen) expression. (A) Increased spinal immunolabeling of fibrin(ogen) and IgG is evident at day 1 after 15-minute compression either with SCH79797 (SCH+15min) or vehicle (DMSO+15min) treatment compared to expression in normal tissue. At day 7, spinal fibrin(ogen) decreases for both SCH+15min and DMSO+15min relative to corresponding levels at day 1 and is similar to DMSO- treated sham (DMSO+sham). (B) Quantification of fibrin(ogen) labeling relative to expression in normal rats shows significant decreases (#p<0.001) at day 7 relative to day 1 after SCH+15min and DMSO+15min. Data are mean±SD.

4.3.2.2) (Rothman et al. 2010). Spinal cord tissue at C7 was harvested on day 1 (15min,

n=5; 3min, n=4; sham, n=4) or day 7 (15min, n=4; 3min, n=4; sham, n=6) in separate

groups, in order to evaluate the temporal expression of the co-localization of GFAP and

PAR1 in the ipsilateral spinal dorsal horn. Spinal GFAP was co-immunolabeled with

PAR1 and the co-localization was quantified; co-localized GFAP and PAR1 labeling was

normalized to labeling in naïve tissue and differences between groups over time were

determined using a two-way ANOVA (group x day) with Tukey’s test.

4.4.1.2. Measuring the Effects of Compression-Induced Spinal Thrombin Activity on BSCB Breakdown & Astrocyte Activation

In order to assess the effect of compression-induced spinal thrombin activity on

BSCB breakdown and glial activation, thrombin was blocked intrathecally with hirudin

prior to imposing a painful 15-minute nerve root compression. The methods for the

intrathecal injection of hirudin and for the surgical methods of the root compression

injury are detailed in Section 4.3.2.4. Briefly, rats received an intrathecal injection of

hirudin (4.2U/rat) 1 day prior to a 15-minute C7 nerve root compression (hir+15min,

n=6). In order to measure BSCB breakdown, fixed C7 spinal cord tissue was harvested at

day 1 since BSCB breakdown is evident at day 1 after a 15-minute compression (Figure

3.2)(Smith et al. 2015). IgG immunolabeling was quantified in the ipsilateral spinal cord

and was compared between compression with hirudin treatment and matching tissue from

that same time point after an untreated 15-minute compression (15min, n=5). Spinal IgG

hir+15min and 15min groups were determined using a one-way ANOVA with Tukey’s

test.

A separate group of rats receiving hirudin treatment and a painful root

compression were perfused at day 7 (hir+15min, n=5) to measure astrocyte activation,

since spinal GFAP is robustly elevated at day 7 after painful nerve root compression

(Rothman et al. 2010, Rothman and Winkelstein 2007). GFAP expression in the

ipsilateral spinal cord was immunolabeled and quantified. Details about the specific

methods for the GFAP immunolabeling are the same as described in Section 4.4.2.3.

Spinal GFAP expression was normalized to expression in naïve tissue and compared to

normalized GFAP expression in tissue from matching rats at day 7 after undergoing a 15-

minute compression (15min, n=6) without hirudin treatment. Differences in spinal GFAP

labeling between groups were determined using a one-way ANOVA with Tukey’s test.

4.4.1.3. Measuring the Effects of Spinal PAR1 on BSCB Breakdown & Astrocyte Activation

In order to determine the effects of spinal thrombin and its activation of PAR1 on

non-injury induced, but painful, BSCB breakdown and astrocyte activation, rat thrombin

was intrathecally administered (4U/rat; RTh, n=5) to naïve rats or to rats receiving an

intrathecal injection of the PAR1 inhibitor, SCH79797 (50µg/kg) at 1 day earlier

(SCH+RTh, n=6). Additionally, rats receiving an intrathecal injection of a PBS vehicle

were included for immunohistochemical comparisons (PBS, n=2). Because rat thrombin

induces the most substantial increase in mechanical hyperalgesia at day 1 (Figures 4.1 &

Bilateral C7 spinal cord tissue was harvested and immunolabeled for IgG and GFAP. IgG

and GFAP expression were separately quantified and compared between the RTh,

SCH+RTh, and PBS groups using separate one-way ANOVAs with Tukey’s test for each

label of neuroinflammation.

4.4.1.4. Defining the Role of PAR1 in Compression-Induced Spinal Astrocyte Activation

In order to determine if spinal PAR1 activation contributes to BSCB breakdown

at day 1 or astrocyte activation at day 7 after a painful nerve root compression,

SCH79797 (50µg/kg) was administered 1 day before a 15-minute nerve root compression

(SCH+15min, n=13). Vehicle controls were included in which DMSO in PBS (1:15) was

administered intrathecally at 1 day before either a 15-minute root compression

(DMSO+15min, n=8) or a sham surgery (DMSO+sham, n=4). Methods for the surgeries

and the intrathecal injections are detailed in Section 4.3.2.4. Fixed C7 spinal cord tissue

was harvested at day 1 (SCH+15min, n=6; DMSO+15min, n=4) or day 7 (SCH+15min,

n=7; DMSO+15min, n=4; DMSO+sham, n=4) and immunolabeled for IgG and GFAP,

respectively. Details about the methods for the quantification of spinal IgG and GFAP

expression can be found in Sections 4.4.2.2 and 4.4.2.3, respectively. IgG expression in

the ipsilateral spinal cord at day 1 after injury was compared for a compression with

SCH79797 or vehicle treatment using a one-way ANOVA with Tukey’s test. Spinal

GFAP labeling was measured at day 7 after injury and compared between a compression

treated with SCH79797 or vehicle, and a sham operation with vehicle treatment, using a