The immunohistochemical and behavioral studies in this chapter demonstrate that
the development of pain after a nerve root compression depends on the breakdown of the
BSCB that occurs transiently, and early, after injury (Figures 3.2 & 3.4). Behavioral
sensitivity and BSCB breakdown are induced in parallel within 1 day of a 15-minute
nerve root compression (Figure 3.2), and are both evident only after that longer
compressive injury and only in the ipsilateral spinal cord, supporting their association.
Since pain is still present on day 7 after that compression, when BSCB permeability has
returned to normal (Figure 3.2), BSCB breakdown may be associated with the onset,
rather than the maintenance, of nerve root compression-induced pain. Early breakdown in
the BSCB is unique to compression-mediated pain and not pain in general, since the
inflammatory nerve root insult does not increase BSCB permeability at any of the time
points probed, despite inducing pain (Figure 3.3). The 15-minute nerve root compression
utilized in these studies has been shown to deform the macroscopic nerve root structure
immediately after compression and to decrease the structural integrity of the compressed
axons within the root for up to 2 weeks (Chang and Winkelstein 2011, Hubbard et al.
2008b, Nicholson et al. 2011). Neither a 3-minute compression nor an inflammatory
insult to the root induces the axonal disruption that is typically observed at later times
after the painful compression (Chang and Winkelstein 2011, Nicholson et al. 2011,
Rothman et al. 2010). The lack of BSCB breakdown induced by both a non-painful
compression and an inflammatory insult suggests that BSCB breakdown might be
compression-induced BSCB breakdown with APC completely prevents pain from
developing (Figure 3.4C), suggesting that the rapid targeting of the APC pathway after
neural injury might eliminate the need for chronic use of analgesics later.
The treatment window for blocking BSCB is expected to be on the order of hours
to a day after the initial injury since increased BSCB permeability is observed by day 1
after painful nerve root compression (Figure 3.2). In agreement, a painful chronic sciatic
nerve ligation induces BSCB breakdown in the lumbar spinal cord between 6 and 24
hours (Beggs et al. 2010, Echeverry et al. 2011). However, that nerve ligation induces
breakdown for up to 30 days in some cases (Echeverry et al. 2011), whereas spinal IgG
levels return to normal by day 7 after a transient root compression (Figure 3.2). This
discrepancy in the duration of BSCB breakdown after a nerve root compression and a
sciatic nerve ligation is most likely due to duration of local mechanical injury; the nerve
root compression is only applied transiently (for 15 minutes) in contrast to a nerve
ligation, which imposes a sustained neural compression.
Deforming neural tissue compromises both neuronal and vascular integrity
transiently during the applied compression (Igarashi et al. 2005, Rothman et al. 2010,
Rydevik et al. 1981, Yoshizawa et al. 1989). Compression of the nerve root blocks blood
flow through the root inducing a transient state of ischemia (Igarashi et al. 2005,
Olmarker et al. 1989, Rydevik et al. 1981). Although blood flow is restored within hours
after the compression is removed (Igarashi et al. 2005, Yoshizawa et al. 1989), evidence
of decreased axonal integrity is still present for up to 2 weeks (Chang and Winkelstein
2011). Since neural compression-induced ischemia occurs transiently (Yoshizawa et al.
Hubbard et al. 2008b), it is possible that the transient disruption in the BSCB is due to the
ischemic, rather than neuronally-mediated effects. A nerve root compression applied for a
longer duration, which may be more clinically relevant due to a bulging disc or spinal
stenosis, might induce BSCB breakdown that lasts longer, and, therefore, may offer a
longer treatment window after compression. It is also possible that chronic compression
of the nerve root may induce BSCB breakdown that is so robust that a single treatment or
even repeat treatments with APC would not adequately fortify the vasculature.
Administering APC or some other vascular protecting drug repeatedly would provide
more information about the regulatory mechanism of root-induced BSCB disruption.
A limitation of the current study is that the mechanism by which a painful nerve
root compression, which is remote from the spinal cord, is capable of disrupting spinal
vasculature was not investigated. The nerve root, where the compression is applied, is
physically connected to the spinal cord through afferent axons and the vasculature,
suggesting that injury to, and therefore dysfunction of, axons or vasculature might
mediate the breakdown. The effect of the neural compression duration on spinal
pathology is important. By day 7, a painful 15-minute compression induces axonal
degeneration in the nerve root, whereas a 3-minute compression does not induce any
nerve root pathology (Nicholson et al. 2011, Rothman et al. 2010). In the current study,
BSCB breakdown occurs only after the longer 15-minute, and not the shorter 3-minute,
compression (Figure 3.2). Electrophysiological activity in the ipsilateral dorsal horn is
maximally reduced at 6.6±3.0 minutes after the start of root compression (Nicholson et
al. 2011). That threshold of altered spinal neuronal signaling falls between the two
Since spinal IgG expression increases exclusively after a compression that is held for
longer than that threshold required to immediately disrupt spinal neuronal firing (Figure
3.2), it is possible that BSCB breakdown may also be influenced by early changes in
neuronal signaling. Yet, neuronal hyperexcitability in the spinal cord and axonal
degeneration in the nerve root remain at day 7 after a 15-minute compression(Zhang et
al. 2013) when BSCB integrity has recovered (Figure 3.2), suggesting that the breakdown
is not entirely controlled by neuronal signaling.
Compression applied to the nerve root, and the vasculature within, disrupts blood
flow to the spinal cord and dorsal root ganglia (Igarashi et al. 2005, Kobayashi et al.
2008, Olmarker et al. 1989, Yoshizawa et al. 1989), which may also influence BSCB
breakdown experienced after a compressive injury. This study shows that only the 15-
minute compression induces changes in permeability of the spinal vasculature (Figure
3.2). Previous studies have reported that compression of the lumbar nerve roots or cauda
equina also induce duration-dependent changes in blood flow. Blood flow within the root
is reduced within 10 minutes of compression and continuously decreases as the duration
of compression increases (Igarashi et al. 2005, Olmarker et al. 1989), suggesting that only
the 15-minute compression might disrupt blood flow to the spinal cord. The compression-
induced blockage of blood flow in the nerve root is similar to ischemic stroke models that
transiently block cerebral arteries for different periods of time to induce different patterns
of cerebral ischemia and reperfusion (Dobbin et al. 1989, Ek et al. 2015, Sage et al.
1984). Occluding the carotid artery for 15 minutes induces transient BBB breakdown as
soon as 3 hours that lasts for up to 24 hours (Dobbin et al. 1989, Ek et al. 2015),
compression (Figure 3.2). A compressed root undergoes more structural deformation and
load-relaxation after 15 minutes of compression than after just 3 minutes (Rothman et al.
2010); it is possible that the shorter duration of compression may not deform the root
tissue to an extent that induces physical compression of the vasculature within, thereby
not blocking blood flow to the spinal cord and not inducing ischemia. An inflammatory
insult to the root similarly does not mechanically disrupt the nerve root and, therefore,
does not block blood flow to the spinal cord. Since an inflammatory injury also does not
induce BSCB breakdown (Figure 3.3), BSCB breakdown may be at least partially
controlled by ischemia due to physical impingement of the root vasculature.
Systemic inflammation may be one mechanism by which compression-induced
BSCB breakdown facilitates pain. Serum concentrations of IL-7, IL-12, IL-1α and TNF-α
correlate to the severity of pain at day 1 (Table 3.1), which is when BSCB breakdown is
most robust (Figure 3.2). Of these cytokines, spinal IL-1α and TNF-α have been shown to
mediate pain after the nerve root compression injury examined in this chapter (Rothman
et al. 2009, Rothman and Winkelstein 2010). Painful nerve root compression increases
IL-1α and TNF-α transcription in the ipsilateral spinal cord as early as 1 hour after
compression; both are likely produced by glial cells since IL-1α expression is exclusively
localized to astrocytes in the spinal cord at 1 hour of compression (Rothman and
Winkelstein 2010). By day 1 after compression, IL-1α and TNF-α transcription levels in
the spinal cord return to normal (Rothman et al. 2009), but at that time spinal TNF-α
protein expression is elevated (Figure 3.2D). Since cells within the spinal parenchyma are
not transcribing TNF-α by day 1 after nerve root compression (Rothman et al. 2009) and
2015), peripheral TNF-α is likely transported from the blood into the spinal parenchyma
rather than being produced by cells within spinal cord at this time.
The early breakdown of the BSCB after painful compression may also promote
the extravasation of peripheral immune cells into the spinal parenchyma. Spinal
microglial expression is elevated by day 7 exclusively after a painful compression, and
not a non-painful compression (Rothman et al. 2010). Increases in spinal microglia can
be attributed to the migration of microglia from other locations within the CNS, division
of local microglia, or through the transmigration of peripheral immune cells into the CNS
(Inoue 2006, McMahon et al. 2005, Milligan and Watkins 2009, Stollg and Jander 1999,
Sweitzer et al. 2002, Vallejo et al. 2010, Watkins et al. 2001). The early BSCB
breakdown induced after a 15-minute compression may facilitate the extravasation of
peripheral cells into the spinal parenchyma and contribute to the increase in spinal
microglia that is evident by day 7. In agreement, a chromic gut-induced inflammatory
nerve root insult, which does not induce BSCB breakdown (Figure 3.3), also does not
increase the spinal microglia population at day 7 after insult (Rothman and Winkelstein
2007). Although that chromic gut nerve root insult is inflammatory in nature, systemic
concentrations of inflammatory cytokines are not elevated at day 1 when pain is present
(Table 3.2; Figure 3.3). Since pro-inflammatory serum cytokines can induce BSCB
breakdown de novo (Echeverry et al. 2011, Huber et al. 2001, Pan and Kastin 2007), the
lack of systemic inflammation produced after an inflammatory root insult may be
responsible for the lack of BSCB breakdown after that painful inflammatory insult
Although the roles of TNF-α and IL-1β in the development of radicular pain are
well established, the contributions of IL-7 and IL-12 to pain are controversial(Chen et al.
2013, Heitzer et al. 2012, Matute Wilander et al. 2014, Zhang et al. 2015). Clinically, IL-
7 transcription and expression were shown to be elevated in intervertebral disc cells from
patients experiencing low back pain (Zhang et al. 2015). However, serum concentrations
of IL-7 in patients with cancer-related pain increase within 3 hours of an analgesic opioid
treatment (Heitzer et al. 2012). Additionally, serum levels of IL-12 are elevated in
females with work-related musculoskeletal pain (Matute Wilander et al. 2014); in
contrast, administration of IL-12 subcutaneously reduces mechanical allodynia and
hyperalgesia for up to 4 hours when administered 9 days after a painful chronic
constriction of the sciatic nerve (Chen et al. 2013). In the current study, serum
concentrations of both IL-7 and IL-12 positively correlate to the severity of pain at day 1
following root compression (Table 3.1). Together with previous literature, it is clear that
the roles of serum IL-7 and IL-12 in pain severity likely depend on the cause of pain.
Studies blocking IL-7 and IL-12 systemically following painful root injury would provide
more information on whether elevated serum concentrations of those cytokines contribute
to the development of behavioral sensitivity.
The studies presented in this chapter emphasize the significance of BSCB
breakdown in pain and support targeting this early injury-induced phenomenon as a novel
and promising therapeutic route. The vascular-stabilizing agent, APC, was administered
at 1 hour after a compressive-injury that induces BSCB breakdown (Figure 3.4). This
time point was chosen as a conservative estimate based on a previous study reporting that
(Beggs et al. 2010). In a complementary pilot study (n=3 rats), BSCB breakdown was
measured at 6 hours after a painful nerve root compression in order to investigate whether
BSCB breakdown occurs prior to day 1. Spinal cord tissue was harvested from rats at 6
hours a painful nerve root compression, immunolabeled for IgG and compared to tissue
harvested at day 1 after a painful compression or a sham surgery. IgG labeling was not
apparent in the ipsilateral spinal cord at 6 hours after a painful compression and does
resemble expression in sham tissue at day 1 (Figure 3.5). The marked increase in IgG
labeling by day 1 after a painful compression (Figures 3.1 & 3.5) suggests that the BSCB
is disrupted between 6 and 24 hours after a 15-minute root compression. Since blood
flow to the surrounding neural tissue is still not completely re-established by 3 hours after
a 2-second root compression (Igarashi et al. 2005), it is possible that the 15-minute
compression reduces blood flow to the spinal parenchyma on the order of hours after
injury. Blood reperfusion after cerebral ischemia induces a breakdown of the BSCB for
up to 1 day (Dobbin et al. 1989, Ek et al. 2015, Sage et al. 1984). It is possible that
reperfusion of the ipsilateral spinal cord does not become completely restored until after
6 hours following a painful root compression and that such reperfusion contributes to
BSCB breakdown, similar to the ischemia, and subsequent reperfusion, that is observed
in parallel with the BBB breakdown after ischemic stroke.
Although treating a painful compression with intravenous APC completely
inhibits pain development (Figure 3.4), this enzyme is a potent anticoagulant (Bernard et
al. 2001, Finfer et al. 2008, Marti-Carvajal et al. 2012) and, therefore, is clinically unsafe
for use after traumatic injuries such as a nerve root compression. Determining a treatment
modality that activates the APC pathway to rescue vascular permeability, while at the
same time promotes coagulation would be an ideal candidate to block BBB disruption
and prevent development of pain after traumatic neural injury. This hypothesis is further
explored in Chapter 5 through investigations of salmon thrombin’s effects on painful
nerve root compression-induced BSCB through its preferential activation of protein C.