Chapter 2. Rationale, Context, Hypotheses & Aims
3.3. Characterization of BSCB Breakdown after Nerve Root Compression
3.3.1. Methods
3.3.1.1. Surgical Procedures for Nerve Root Compression
For all animal studies, male Holtzman rats (Harlan Sprague-Dawley; Indianapolis,
Agriculture (USDA) and the Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC) with free access to water and food. All experimental
procedures were approved by the University of Pennsylvania Institutional Animal Care
and Use Committee (IACUC) and carried out under the guidelines of the Committee for
Research and Ethical Issues of the International Association for the Study of Pain (IASP)
(Zimmermann 1983).
Surgical procedures were performed under inhalation isoflurane anesthesia (4%
for induction, 3% for maintenance). Previously defined protocols were used for nerve
root compression injury (Rothman et al. 2010). Briefly, rats were placed in a prone
position; a midline incision was made along the back of the neck and the paraspinal
muscles were removed to expose the C6 and C7 vertebrae. A C6/C7 hemilaminectomy
and facetectomy was performed on the right side to expose the right C7 dorsal nerve root.
A small incision was made in the dura over the C7 nerve root and a 10gf microvascular
clip was applied to the exposed root (Figure 3.1).
Figure 3.1. Nerve root compression injury. Cross-section schematic (A) and dorsal image (B) of nerve root compression applied unilaterally to the right C7 dorsal nerve root in the rat. A microvascular clip (10g force) is applied transiently to the root, between the DRG and spinal cord, for 3 or 15 minutes. Shaded boxes indicate the dorsal horns where the compressed axons synapse.
Compression was applied to the C7 dorsal nerve root via the clip for 3 minutes
(3min, n=12) or 15 minutes (15min, n=10) after which the wound was closed using 3-0
polyester suture and surgical staples. Rats were allowed to recover in room air with
continual free access to food and water. Sham operated rats (sham, n=9) underwent
identical surgical procedures except they did not undergo nerve root compression. Spinal
cord tissue from rats was harvested on day 1 (15min, n=5; 3min, n=7; sham n=5) or day 7
(15min, n=5; 3min, n=5; sham n=4) in order to measure temporal responses of BSCB
permeability.
Spinal cord tissue was harvested from rats either on day 1 or day 7 after surgery.
Rats received an overdose of sodium pentobarbital (65mg/kg), administered
intraperitoneally. Once unconscious, rats were transcardially perfused with 1%
phosphate-buffered saline (PBS; Mediatech, Inc.; Manassas, VA) until blood ran clear
and were subsequently perfused by 300ml of 4% paraformaldehyde (Sigma; St. Louis,
MO). The C7 bilateral spinal cord was exposed via a bilateral C5-T1 laminectomy and
facetectomy and harvested en bloc. Spinal tissue was post-fixed overnight in 4%
paraformaldehyde, transferred to 30% sucrose for one week at 4°C and then embedded in
optical cutting temperature (OCT) compound (Sakura Finetek USA, Inc.; Torrance, CA)
for cryosectioning. Fixed spinal cord tissue was sectioned at 14µm along the long-axis of
the spinal cord to create cross-sections that were mounted directly onto slides for
immunolabeling. Spinal cord tissue at C7 also was harvested from naïve rats (n=2) and
included in tissue processing for comparison of expression of IgG in tissue from rats that
3.3.1.2. Assessment of Mechanical Hyperalgesia
In this study, mechanical hyperalgesia was measured as the response threshold, in
grams, of the forepaw to a mechanical stimulus. Mechanical thresholds were measured in
the bilateral forepaws for 7 days post-surgery. Prior to each testing round, rats were
acclimated for 15 minutes to the testing apparatus, which consisted of an elevated mesh-
floored cage with walls providing a separate testing chamber for each rat. A series of
calibrated von Frey filaments (1.4g-26g) (Stoelting; Wood Dale, IL) was applied in
ascending order to the plantar surface of the forepaw until a filament induced a positive
response (Chang and Winkelstein 2011, Chaplan et al. 1994, Lee and Winkelstein 2009).
A positive response was defined as a withdrawal of the stimulated forepaw and was often
accompanied with a shaking or licking of the paw. Each filament was applied for five
consecutive stimulations and the filament strength that elicited a positive response was
recorded as the withdrawal threshold if the next consecutive filament also elicited a
positive response. If no response was evoked by a filament, then the highest magnitude
filament (26g) was recorded as the threshold. Each series of stimulations was repeated
three times for each testing round with at least 10 minutes between series; the withdrawal
threshold of each forepaw was taken as the average of the three series.
Mechanical hyperalgesia in the bilateral forepaws was assessed prior to surgery
on day 0 (baseline) and every other day for 7 days (day 1, 3, 5 and 7) after surgery.
Behaviors from rats that were terminated on day 7 were compared over time between a
15-minute compression (15min, n=5), a 3-minute compression (3min, n=5) and sham
(n=4). The mechanical threshold of each paw was normalized by the corresponding
over time in forepaw mechanical hyperalgesia were determined separately for the
ipsilateral and contralateral forepaws using two-way repeated measures ANOVAs (group
x day) with Tukey’s Honestly Significant Difference (HSD) test.
3.3.1.3. Spinal Immunohistochemistry of IgG Expression
Spinal cord sections at C7 were fluorescently immunolabeled for rat IgG as a
proxy for BSCB breakdown since this serum-derived protein is not present in the CNS
under normal conditions (Poduslo et al. 1994). Briefly, slide-mounted tissue sections
were blocked in 5% normal goat serum (Vector Laboratories; Burlingame, CA) with
0.3% Triton-X100 (Bio-Rad Laboratories; Hercules, CA) for 1 hour at room temperature.
Slides were then incubated with goat anti-rat IgG Alexa Fluor 568 (1:200; Life
Technologies; Carlsbad, CA) for two hours at room temperature and then washed with
PBS and cover slipped with fluoro-gel with TRIS buffer (Electron Microscopy Sciences;
Hatfield, PA). The ipsilateral and contralateral spinal dorsal horns were digitally imaged
at 10x in 2-6 spinal sections for each rat.
Spinal IgG labeling was quantified in uniformly cropped images of the dorsal
horn using a custom densitometry MATLAB script (Nicholson et al. 2012, Rothman et al.
2010, Rothman and Winkelstein 2007). The densitometry script quantified the percent of
the total tissue pixels that were above a pre-defined threshold; that threshold was chosen
using normal naïve spinal tissue to include pixels that represented positive IgG labeling
and was kept constant to analyze all images(Nicholson et al. 2012, Rothman et al. 2010,
Rothman and Winkelstein 2007). Levels of spinal IgG were then normalized to labeling
represented as expression relative to normal levels. Differences in normalized percent
positive IgG between 15-minute compression, 3-minute compression and sham were
tested on day 1 (15min, n=5; 3min, n=7; sham n=5) and day 7 (15min, n=5; 3min, n=5;
sham n=4), using two-way repeated measures ANOVA (group x day) with Tukey’s test.
3.3.1.4. Serum Collection & Cytokine Multiplex Assay
Rats were anesthetized with 4% isoflurane anesthesia by inhalation for blood
collection procedures. Blood samples were taken from a subset of rats undergoing either
a 15-minute (n=6), 3-minute (n=4) or 0-minute (sham, n=3) nerve root compression.
Blood was collected (~0.5ml) via a 25G needle syringe from the tail vein on day 0
(baseline) before, and on day 1, after surgery. Whole blood was allowed to clot at room
temperature and serum was separated using consecutive spins at 4°C, the first at 1000rcf
for 15 minutes and the second at 10,000rcf for 10 minutes. Serum samples were assayed
in duplicate for a panel of 23 pro- and anti-inflammatory cytokines and chemokines using
a multiplex bead-based Luminex assay kit (#L80-01V11S5; Bio-Rad; Hercules, CA). The
analytes measured within this pre-made kit are: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7,
IL-10, IL-12, IL-13, IL-17, IL-18, MCP-1, TNF-α, erythropoietin (EPO), granulocyte
colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor
(GM-CSF), keratinocyte-derived chemokines/growth-related oncogene (GRO/KC),
interferon-gamma (IFN-γ), macrophage colony-stimulating factor (M-CSF), macrophage
inflammatory protein-3 alpha (MIP-3α), regulated on activation, normal T cell expressed
and secreted (RANTES) and vascular endothelial growth factor (VEGF). For each rat, the
correlated to the normalized paw withdrawal threshold at day 1. Each pair of bivariate
data – the normalized withdrawal threshold and the normalized cytokine concentration –
was fit with a linear regression and analyzed to identify those cytokines that strongly
(R2>0.5) and significantly (p<0.05) correlate to paw withdrawal threshold (Cohen 1988).
3.3.1.5. Spinal Immunohistochemistry for TNF-α
TNF-α was chosen from the four cytokines that were found to strongly correlate
to forepaw withdrawal thresholds (IL-7, IL-12, IL-1α, TNF-α) and was co-
immunolabeled with IgG in the ipsilateral spinal cord. The IgG protocol described in
Section 3.3.1.3 was adapted to include TNF-α in order to visually assess whether its
spinal expression co-localizes to spinal regions where there is also BSCB breakdown.
Tissue sections harvested from two rats in each of the compressive insult and
corresponding control groups (15-minute compression, 3-minute compression, sham) at
day 1 were blocked in 5% goat serum in 0.3% TX-100 and incubated over night with
rabbit anti-TNF-α (1:200; Cell Signaling; Danvers, MA). Those slides were then
fluorescently labeled with goat anti-rabbit Alexa Fluor 488 (1:1000; Invitrogen; Carlsbad,
CA) and goat anti-rat IgG Alexa Fluor 568 (1:200; Invitrogen; Carlsbad, CA). The
ipsilateral dorsal horn was digitally imaged at 10x and visually inspected for the presence
of TNF-α and its co-localization with IgG.