Overview

Cervical radiculopathy is a neuropathic condition that induces long-term pain that

radiates from an injured or irritated cervical nerve root to the shoulders and arms (Abbed

and Coumans 2007, Wall and Melzack 1994). Cervical disc herniation is a common

cause of radicular pain in the cervical spine and often involves both compressive and

inflammatory insults to the cervical nerve roots; a bulging disc can physically impinge on

the nerve root and also release materials from its nucleus pulposus, which is

inflammatory to the root (Abbed and Coumans 2007, Caridi et al. 2011, Wall and

Melzack 1994). Rat models imposing compressive or inflammatory nerve root insults

separately have established that while both components of injury can individually induce

sustained mechanical behavioral sensitivity, these stimuli differentially modulate

Winkelstein 2011, Colburn et al. 1999, Hashizume et al. 2000, Hou et al. 2003,

Kawakami et al. 1994, Kawakami et al. 1994, Rothman and Winkelstein 2007). For

example, a transient C7 nerve root compression for 15 minutes in the rat upregulates the

transcription of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in the

spinal cord at 1 hour after compression and activates spinal glia for at least 7 days

(Hubbard and Winkelstein 2005, Rothman et al. 2009, Rothman et al. 2010, Winkelstein

and DeLeo 2002). An inflammatory insult imposed by chromic gut suture does not

induce the same early spinal transcription of IL-1β and TNF-α nor does it lead to the

persistent glial activation that is induced after a root compression (Rothman et al. 2009,

Rothman and Winkelstein 2007, Rothman and Winkelstein 2010). This same nerve root

compression disrupts axonal myelination and promotes phagocytotic macrophage

infiltration at the injured root by two weeks after injury, while an inflammatory insult

only induces macrophage infiltration and to a much less degree than compression (Chang

and Winkelstein 2011). Since both compressive and inflammatory insults to the nerve

root produce indistinguishable mechanical hypersensitivity in the ipsilateral forepaw for

up to 2 weeks (Chang and Winkelstein 2011, Rothman and Winkelstein 2007), but a

compressive insult induces more robust spinal and nerve root pathologies (Chang and

Winkelstein 2011, Rothman and Winkelstein 2007), it is hypothesized that different

cellular cascades are induced by compressive and inflammatory stimuli to produce and

maintain the associated pain responses.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are

comprised of endothelial cells and act as physical obstacles between the circulatory

inflammatory molecules and cells under healthy conditions (Abbott et al. 2010, Ballabh

et al. 2004). Despite being remote from the CNS, chronic nerve ligation injury, which

induces sustained pain, disrupts the BSCB for up to 30 days in spinal regions where the

injured afferents synapse in both rats and mice (Beggs et al. 2010, Echeverry et al. 2011,

Gordh et al. 2006). This increased BSCB permeability facilitates the transmission

radioiodine-labeled IL-1β and green fluorescent protein positive (GFP+_{) bone marrow-}

derived monocytes into the spinal cord (Echeverry et al. 2011); both the presence of pro-

inflammatory cytokines and peripheral immune cells centrally can exacerbate

neuroinflammation. Different types of neural injuries can increase spinal microglial

activation through either resident spinal microglial division or through facilitated

infiltration of peripheral monocytes that express markers of microglia activation once

they are in the spinal cord (DeLeo et al. 2004, Rothman et al. 2009, Rutkowski et al.

2002, Stollg and Jander 1999, Sweitzer et al. 2002). A compressive, but not an

inflammatory, insult to the nerve root increases microglia populations in the spinal cord

both early (day 1) and at later times (day 7) after injury (Rothman and Winkelstein 2007).

It is possible that the lack of spinal microglial activation induced by an inflammatory root

insult might be due to its lack of induction of BSCB breakdown preventing peripheral

immune cell transmigration into the spinal cord. Studies in this chapter evaluate whether

compressive or inflammatory insults to the nerve root induce different extents of BSCB

breakdown and whether such barrier breakdown directly mediates pain.

Systemic concentrations of pro- and anti-inflammatory cytokines and chemokines

are upregulated in a variety of neuropathic and autoimmune disorders andinfluence the

Kastin 2007, Pedersen et al. 2015, Sharief and Thompson 1992). Intravenous pro-

inflammatory IL-1β, TNF-α or monocyte chemotactic protein-1 (MCP-1), independently,

increase BSCB permeability in normal naïve rats, which also has been shown to permit

the extravasation of serum proteins into the spinal parenchyma where the BSCB is

compromised (Echeverry et al. 2011, Pan and Kastin 2007, Sharief and Thompson 1992).

In contrast, anti-inflammatory transforming growth factor-beta (TGF-β) and IL-10 rescue

the BSCB breakdown that is induced after painful sciatic nerve ligation (Echeverry et al.

2011); this increased BSCB integrity is also achieved by anti-MCP-1 given intrathecally

over 3 days after ligation (Echeverry et al. 2011). Increased serum levels of some of these

BBB-disrupting cytokines are also increased in patients with painful disc herniation

(Kraychete et al. 2010, Pedersen et al. 2015); in particular, serum concentrations of IL-6,

IL-8 and TNF-α are elevated in those patients experiencing more intense chronic pain

compared to those with “mild” or no pain (Kraychete et al. 2010, Pedersen et al. 2015).

Despite a strong implication of peripheral inflammation in both BSCB breakdown and

pain, the relationship between systemic inflammation at times of increased BSCB

permeability after compressive and/or inflammatory nerve root insults and pain has not

yet been studied.

This chapter summarizes a multi-part study that tests two separate hypotheses.

The first two studies (Section 3.3 & Section 3.4) summarize a subset of the experiments

under Aim 1 and test the hypothesis that painful nerve root injury induces BSCB

breakdown and systemic inflammation facilitating the accumulation of serum molecules

in the spinal parenchyma where the BSCB is compromised (Aim 1a). To test this

BSCB breakdown over time after painful and non-painful nerve root compressions

separately (Section 3.3). The comparative effects of compressive and inflammatory nerve

root insults on BSCB breakdown and pain were also investigated (Section 3.4). BSCB

breakdown was measured in the bilateral spinal cord by immunolabeling for the serum

protein, immunoglobulin G (IgG), which is not typically present in the CNS(Echeverry

et al. 2011, Poduslo et al. 1994), at times early and later after the neural insults (Aim 1a).

To define the relationship between expression of inflammatory serum molecules and pain

at times of BSCB breakdown, the concentration of 23 pro- and anti-inflammatory serum

cytokines and chemokines was correlated to pain severity after a compressive injury

(Section 3.3) or an inflammatory insult (Section 3.4) to the nerve root (Aim 1b). Of the

cytokines found to correlate to pain intensity after a nerve root compression, TNF-α

expression was immunolabeled in the spinal dorsal horn at the time point when BSCB

reaches a maximum after painful and non-painful compression injuries.

The last set of studies in this chapter (Section 3.5) investigates the role of BSCB

breakdown in pain development after a nerve root compression and was designed to test

the hypothesis that blocking BSCB breakdown can inhibit the development of nerve root

compression-induced pain. Activated protein C (APC), a serum protease that strengthens

endothelial tight junctions and effectively reduces vascular leakiness in studies of sepsis

and ischemic stroke (Bernard et al. 2001, Petraglia et al. 2010, Zlokovic and Griffin

2011), was administered intravenously at 1 hour after a painful nerve root injury that

induces BSCB breakdown. The inhibition of BSCB breakdown by APC was confirmed

time to determine if BSCB breakdown contributes to the development of nerve root-

induced pain (Aim 1c).

3.2. Relevant Background

BBB disruption is characteristic of many neurological disorders, including

ischemic stroke, Parkinson’s disease and amyotrophic lateral sclerosis (ALS), and

contributes to the associated neuroinflammation and neurodegeneration within the central

nervous system (Sandoval and Witt 2008, Winkler et al. 2014, Zlokovic 2011). The

neurovasculature of the BBB is comprised of endothelial cells that are trophically

coupled to nearby neurons via glial cells, which together, make up the ‘neurovascular

unit’ and interact closely to maintain inflammatory dysfunction in disease states (Abbott

et al. 2010, Abbott et al. 2006, Hawkins and Davis 2005). The healthy BBB endothelium

is bound together by tight junctions, which inhibit the transmission of serum components

and blood-borne cells into the CNS (Abbott et al. 2010, Ballabh et al. 2004). Disruption

of the BBB permits entrance of neurotoxic factors into the CNS that impair normal

neuronal function and exacerbate inflammation (Webb and Muir 2000, Zlokovic 2008).

Peripheral nerve injuries themselves have also been shown to increase the permeability of

the BSCB at the same spinal level as the injury where the injured afferents synapse

(Beggs et al. 2010, Echeverry et al. 2011, Radu et al. 2013). Nerve injuries also induce

chronic neuropathic pain which is, at least partially, maintained by spinal

neuroinflammation (Echeverry et al. 2011, Rothman and Winkelstein 2007, Watkins et

Further, if BSCB breakdown is related to development of chronic pain, therapeutics

targeting and preventing vascular permeability would be ideal candidates for pain

prevention after neuropathic injury.

Systemic inflammation contributes to spinal neuroinflammation both indirectly

and directly. Pro-inflammatory molecules indirectly influence spinal homeostasis by

stimulating peripheral neuronal receptors (Szelenyi 2001). These primary afferents

synapse centrally in the spinal dorsal horn and become hyperexcitable due to constant

peripheral activation (Szelenyi 2001). Circulating pro-inflammatory cytokines, such as

TNF-α and IL-1β, also activate endothelial cells leading to increased vascular

permeability, which promotes their own entrance into the CNS via a compromised BBB

(Echeverry et al. 2011, Hoffmann et al. 2004, Huber et al. 2001, Pan and Kastin 2007,

Sharief and Thompson 1992). Once in the CNS, pro-inflammatory cytokines and

chemokines can directly interact with and stimulate neurons and glia, which further

accentuate nociceptive pathways by increasing synaptic concentration of excitatory

neurotransmitters and cytokines (DeLeo and Yezierski 2001, Milligan and Watkins

2009). Of note, independently blocking the actions of either spinal TNF-α or IL-1β

attenuates pain after compressive nerve root injury (Rothman et al. 2009), highlighting

the strong influence of these pro-inflammatory cytokines in pain signaling. Systemic and

central inflammation play important roles in pain and are integrated through BBB

permeability(Ballabh et al. 2004, Hawkins and Davis 2005, Huber et al. 2001, Sharief

and Thompson 1992, Szelenyi 2001, Webb and Muir 2000). Yet, it is not known whether

there is a relationship between nerve root-induced pain and systemic levels of

Vascular permeability, including that of the BBB and BSCB, is controlled

through various endothelial pathways. The serine protease, thrombin, most notably

recognized for its role in coagulation, also regulates a variety of endothelial processes

including permeability via its enzymatic activation of cell-bound receptors (Coughlin

2000, Di Cera 2008). Thrombin initiates distinct cellular signaling cascades depending on

the cofactor it binds, and therefore the substrate it activates (Coughlin 2000, Di Cera

2008, Jacques et al. 2000, Komarova et al. 2007, Riewald and Ruf 2005, Xu et al. 2005).

In its unbound state, mammalian thrombin increases vascular permeability by directly

cleaving protease-activated receptor-1 (PAR1) on the endothelial surface (Gandhi et al.

2011, Jacques et al. 2000, Komarova et al. 2007). In contrast, when bound to

thrombomodulin, thrombin activates endothelial-bound protein C into APC, which

stabilizes vascular integrity (Esmon 1993, Fuentes-Prior et al. 2000, Komarova et al.

2007, Xu et al. 2005). Both clinical trials and animal studies have tested APC for its

enhancement of endothelial barriers in sepsis and ischemic stroke (Zlokovic and Griffin

2011); but, its strong anticoagulant effects hamper its clinical safety (Bernard et al. 2001,

Finfer et al. 2008, Marti-Carvajal et al. 2012). For this reason, a major effort in thrombin

mutagenesis and protein engineering has identified peptide domains in thrombin’s

structure that control protein C activation which they can manipulate in order to increase

thrombin’s innate affinity for protein C instead of PAR1 (Dang et al. 1997, Gibbs et al.

1995, Marino et al. 2010). Although APC is not a clinically relevant treatment option for

neural trauma, administering APC after painful nerve root injury to fortify the BSCB

This three-part study was used to test the hypotheses that a painful nerve root

compression induces BSCB breakdown, which facilitates the accumulation of serum

molecules in the spinal parenchyma in regions of breakdown and that the formation of

nerve root-induced pain is inhibited when BSCB breakdown is blocked. Section 3.3

details the methods and results of a characterization study measuring BSCB breakdown

after painful and non-painful nerve root compression by immunolabeling IgG, a serum

protein that is not expressed in the CNS under normal conditions (Poduslo et al. 1994). In

that study, behavioral responses were induced using two previously defined durations of

nerve root compression: 15-minutes to induce sustained pain and 3-minutes to serve as a

loading control in which the nerve root undergoes injury, but pain does not develop

(Nicholson et al. 2012, Rothman et al. 2010). Forepaw mechanical hyperalgesia and

spinal IgG were assessed on days 1 and 7 as time points corresponding to the

development and maintenance of pain, respectively. Expression of serum cytokines and

chemokines were assayed at the time point (day 1) corresponding to BSCB breakdown

using a multiplex assay in order to characterize systemic inflammation and its

relationship to pain.

The second part of this chapter investigates the influence of the type of pain-

inducing nerve root insult (i.e. compressive versus inflammatory) on BSCB breakdown

and behavioral hypersensitivity (Section 3.4). A painful inflammatory insult was imposed

using a rat model of chromic gut suture application to the nerve root (Chang and

Winkelstein 2011, Rothman et al. 2011). Following the same procedures as from the

characterization study (Section 3.3), ipsilateral forepaw mechanical hyperalgesia and

investigate the comparative effects of compressive and inflammatory nerve root insults

on BSCB breakdown (Section 3.4).

The third part of the study defined the role of BSCB breakdown in the

development of pain by blocking BSCB after painful root compression injury (Section

3.5). APC was administered after painful nerve root compression to block BSCB

breakdown, based on its clinical and pre-clinical effectiveness at reducing vascular

disruption (Bernard et al. 2001, Marti-Carvajal et al. 2012). Spinal IgG levels were

measured at day 1 after injury with treatment to confirm its effectiveness and then

behavioral responses were measured at this early time point. The goal of the studies in

this chapter was to identify if BSCB breakdown is induced, and if so, to what

spatiotemporal extent within the spinal cord, after both compressive and inflammatory

nerve root insults. Further, the influence of nerve-root induced BSCB breakdown on pain

development and/or maintenance was investigated. The relationship between serum

inflammatory markers and pain severity was also defined at times of increased BSCB

permeability.

3.3. Characterization of BSCB Breakdown after Nerve Root

In document Defining the Role of Blood-Spinal Cord Barrier Breakdown and Spinal Thrombin Signaling tn the Development of Pain Following Neural Trauma (Page 70-79)