C H A P T E R

10
Peripheral nervous system toxicity biomarkers
T.V. Damodaran

INTRODUCTION to known and unknown toxic mechanisms becomes

imperative for effective treatment and management pro-
tocols. Even though the clinical presentation of the dis-
The nervous system is the most complex organ system
ease condition may be useful for preliminary diagnosis
in humans and many of the vertebrates and can be
for symptomatic treatment approaches, permanent cure
defined as the master controlling and communicating
may be possible only after disease-specific treatments.
system of the body. Its activity is the basis for every
The developing nervous system may be more or less
thought, action, and emotion. Along with the endocrine
susceptible to neurotoxic insult depending on the stage
system, the nervous system plays a major role in the
of development. Biomarkers used in adults are often
body’s homeostasis in human and other vertebrates.
applicable for use during development, but develop-
Because of the need to be rapid and immediate, its cells
mental stage-specific and aging related differences may
communicate by electrical and chemical signals that are
be important (Slikker and Bowyer, 2005). While both
highly specific. The dynamic nature of responses of var-
inherited and acquired peripheral nervous system
ious organisms to their extrinsic (external environment)
(PNS) disorders may have common pathways that can
and other intrinsic (homeostatic) factors to ensure their
be identified by biomarker identification and validation,
survival depends on the structural and functional integ-
there may be unknown layers of complexity that need
rity of the nervous system. The unique status of this
to be understood for better treatment protocols.
organ system also makes it relatively more susceptible
Thus, biomarkers (measurable end points by cell,
to toxic mechanisms arising from various day to day
molecular, and clinical parameters) are very important
life activities. These neurotoxic mechanisms can be of
for various applications connected with the accurate
short-term or long-term duration as well as either acute
diagnosis, treatment, and management of neurological
or chronic. The drastic outcome from these neurotoxic
disorders. These biomarkers are also useful for asses-
mechanisms can be immediate or delayed. These
sing clinical responses, identification of risks, selection
mechanisms may have general effects or may affect
of doses, and other aspects in drug development and
very specific parts of this multi-unit hierarchical struc-
evaluation. Positive correlation between symptoms at
ture. Some of these neurotoxic mechanisms may get
all levels (molecular, cellular, and phenotypical) of
resolved quickly by the body’s repair mechanisms,
abnormal and/or normal presentations (both qualita-
while other types may need rigorous therapeutic proto-
tively and quantitatively) of chemicals, metabolites, and
cols. Because of the nervous system’s omnipresent role
other clinical and measurable morphological features
in many other organ systems, the toxic mechanisms that
can help define the biomarker of choice. As per the NIH
affect the nervous system may have direct or indirect,
biomarker working group, a biomarker is a characteris-
mild, or profound effects on other organ systems. Thus,
tic that can be objectively measured as an indicator of
accurate and prompt identification of toxic mechanisms
normal biological processes, pathogenic processes, or a
responsible for any neurological disease conditions due
pharmacological response to a therapeutic intervention.

R. Gupta (Ed): Biomarkers in Toxicology. © 2014 Elsevier Inc. All rights reserved.
ISBN: 978-0-12-404630-6 DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00010-5
169
170 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
Hence, biomarkers can be effectively used to identify
(a) whether the exposure has occurred, (b) the route of
exposure, (c) the pathway of exposure, and (d) resulting
short-term or long-term effects, as well as outcomes of
therapeutic interventions to bring the organism to its
original healthy status. Thus, biomarkers of PNS can
define structural and functional aspects of the periph-
eral nervous system in both health and disease. In this
chapter, emerging trends in the broad field of PNS
related biomarkers in health and disease status, with
major emphasis on humans and related animal models,
are presented.
ORGANIZATION OF THE NERVOUS
SYSTEM
The nervous system is divided into two distinct types:
(a) Central Nervous System (CNS) and (b) Peripheral
Nervous System (PNS). The central nervous system con-
sists of the brain (with all of its highly specialized
regions, such as medulla oblongata, brainstem, mid-
brain, and cerebral cortex) and the spinal cord, which is
connected to the brain structure via the medulla oblon-
gata. The peripheral nervous system, on the other hand,
consists of the rest of the nervous tissue outside the
CNS. It includes sensory receptors, nerves (bundles of
nerve fibers called axons and their sheath), ganglia (col-
lection of cell bodies located outside the CNS), and
plexuses (extensive network of axons and cell bodies
outside the CNS). Highly specialized cells to detect vari-
ous stimuli such as temperature changes; sensation of
pain, pressure, touch; odor perception; light and sound
sensing, constitute the broader definition of sensory
receptors. The endings of neurons along with the previ-
ously mentioned specialized cells also constitute sen-
sory receptors.
The peripheral nervous system has two subdivisions:
(a) the sensory division and (b) the motor division. The
sensory division which is also called “afferent” 5 “to-
wards,” transmits electrical signals called action poten-
tials (which are nothing but net charge changes at the
site of action on the membrane) to the CNS (to the cell
bodies of sensory neurons). The motor division (also
called “efferent” 5 “away”) on the other hand, transmits
action potentials from the CNS to effector organs (such
as muscle) (Figure 10.1).
The motor division consists of two subdivisions: (a)
somatic nervous system, which is the voluntary division;
and (b) autonomic nervous system, which is involuntary
(ANS). The autonomic nervous system is further subdi-
vided into: (a) sympathetic division, which is more active
during physical activity, and (b) parasympathetic division,
which regulates functions during resting stages such as
digesting food and emptying the urinary bladder, etc.
(Figure 10.2). The enteric nervous system (ENS) consists
of plexuses within the wall of the digestive tract, and it
controls the digestive system through local reflexes (inde-
pendent of CNS).
The PNS includes cranial nerves (12 pairs) and spinal
nerves (31 pairs) and their ganglia. While the neuron is
the basic structural unit of the nervous system, the
reflex arc is the basic functional unit capable of receiv-
ing a stimulus. A reflex is an automatic response (that
occurs without conscious thought) formed by the reflex
arc. There are three major reflexes: (1) the stretch
reflex, (2) the Golgi tendon reflex, and (3) the with-
drawal reflex that takes care of two types of responses
from the nervous system. The first one is called the
autonomic reflex, for maintaining many of the homeo-
static mechanisms such as constant blood pressure,
blood carbon dioxide levels and water intake, etc. The
second one is called the somatic reflex, which removes
the body from painful stimulation, body imbalances,
and other external forces.
Schwann cells are the major neuroglia in the PNS.
They simply wrap around axons to form the myelin
sheath. Another specialized cell type called satellite cells
protect the PNS from heavy metal poisoning by simply
absorbing them, thereby preventing their entry into
neurons. The myelin sheath along with the nodes of
Ranvier are highly efficient in rapid propagation of
action potentials. The myelin sheath is also involved in
several aspects of axonal transport, ion channel activity
of the axonal membrane, and phosphorylation of the
cytoskeletal proteins. Most axonal components such as
synaptic vesicles precursors, cytoskeletal elements, and
mitochondria-like organelles are very important for the
proper functioning of sensory and motor neurons.
Human beings, like many other life forms on the
earth, are constantly bombarded with multitudes of che-
micals (from household items, from workplace materi-
als, and from other habitats connected with their lives),
radiation (of all known and unknown types), and bio-
logical materials (a variety of microbes, viruses, etc.).
Yet, a good majority of these life forms seem to be living
in comparatively stable and evolving ecosystems.
However, there has been an increasing realization of the
existence of both negative and positive feedback
mechanisms that silently balance the molecular mechan-
isms required for maintaining homeostasis in these
ecosystems. Spontaneous or induced disruption of
homeostatic mechanisms has been identified to be the
initial triggering and contributing aspect of disease initi-
ation and progression. Early intervention has been
advocated as the primary step for successful treatment
of any disease conditions. Hence, exposure to chemical,
physical, and biological agents needs to be identified
 TYPES OF BIOMARKERS OF THE PNS 171

Receptor → Sensory NS → CNS → Motor NS → Effector

FIGURE 10.1 Organization of the nervous system: The sensory division of the peripheral nervous system (PNS) detects stimuli and
conducts action potentials to the central nervous system (CNS). The CNS interprets incoming action potentials and initiates action potentials
that are conducted through the motor division to produce a response. The motor division is divided into the somatic nervous system and
the autonomic nervous system.

promptly. Similarly, identifying the dose and duration
of exposure may help to select the appropriate interven-
tion. Since genetic susceptibility also plays a significant
role in minimizing or exacerbating the pathological out-
come, genomic and genetic data become an important
aspect of diagnostic and prognostic treatment and man-
agement aspects of the PNS disorders.
TYPES OF BIOMARKERS OF THE PNS
The complexity of PNS structure and function makes it
a daunting task to enlist the potential targets, mechan-
isms of neurotoxicity, valid biomarkers, and candidate
biomarkers from many biological and clinical end-
points. The complexity and extensive nature of the PNS
makes it comparatively more susceptible and at the
same time endowed with more resilience for recovery
from injuries (compared to the CNS). In general the
biomarkers can be identified by carefully studying the
following aspects of PNS structure and function:
1. Integrity of the myelin structure that includes the
regulation of the development either during embryo-
genesis or as a part of the repair process in an
injured PNS structure. Defective PNS structures in
inherited PNS disorders are good examples of devel-
opmental dysregulation, while injury response can
be a good example of maintenance of the PNS
2. Protein biosynthesis, sorting, and degradation are
the cyclic events in PNS structures that involve a
multitude of macromolecules, organelles, and other
molecular mechanisms
3. Endocytosis, membrane integrity, vesicle dynamics,
and recently identified exosome dynamics that
involve several molecules and organelles such as
mitochondria
4. Prominent role of the cytoskeleton in axonal trans-
port and in many other known and unknown func-
tions of the PNS
172 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS

Preganglionic neuron
Postganglionic neuron
Lacrimal gland Ciliary ganglion
III
Eye

Nasal Pterygopalatine
mucosa ganglion

Sublingual and
submandibular
Submandibular VII
glands
ganglion
Parotid gland IX

Otic ganglion Medulla

Sympathetic X
nerves
Spinal Trachea
cord

Lung

Celiac Heart
ganglion
Greater
splanchnic Liver
Superior
nerve
mesenteric
ganglion Stomach

Lesser Adrenal Spleen

splanchnic gland
nerve Pancreas
L2 Small
intestine
Lumbar
splanchnic Inferior Kidney
nerves mesenteric
ganglion Large
intestine
S2
Sacral Pelvic S3
splanchnic Hypogastric splanchnic S4
nerves ganglion nerve
Sympathetic
chain
Urinary
system Preganglionic neuron
and genitalia Postganglionic neuron

Sympathetic (thoracolumbar) Parasympathetic (craniosacral)

FIGURE 10.2 Innervation of organs by the ANS. Preganglionic fibers are indicated by solid lines and postganglionic fibers are indi-
cated by dotted lines.

Bearing in mind the concept of multilevel-
organizational and functional complexity of the PNS
from the subject matter discussed above, the following
distinct aspects have been shown to be useful for devel-
oping reliable biomarkers:
The biomarkers of the PNS can be obtained from:
(a) molecular, (b) cellular, (c) tissue level, and (d) phe-
notypic data sets. These biomarkers are identified by
their specific functions in normal and abnormal scenar-
ios of the PNS’s structure and function. Based on the
type and location of injuries of the following categories,
several potential biomarkers of various categories have
been identified by use of the following:
a. Studies on mechanisms of formation of injuries to
nerve fibers and axons as well as their repair
mechanisms
b. Studies on the mechanisms of induced or inherited
forms of altered axonal transport
c. Studies on demyelination injuries
d. Studies on the effect of neuromuscular junction
injuries
e. Surrogate tissue analysis for underlying or ongoing
acute or chronic peripheral neurotoxic mechanisms.
PNS BIOMARKERS BASED ON
MECHANISMS
The following time-tested mechanistic approaches have
been found to be very useful to identify and validate
biomarkers of various categories:
1. By identifying potential disease-associated biomar-
kers by comparing gene and protein expression pro-
files between normal and diseased samples
2. By characterizing post-translational modifications
such as phosphorylation and de-phosphorylation
cycles
3. By confirming the tissue-specific changes at all levels
and their relevance from a disease progression point
of view
 PNS BIOMARKER METHODOLOGY
4. By identifying the prevalent biomarkers in the popu-
lation under study
5. By developing efficacy biomarkers from drug-
associated expression profiles to investigate the
downstream mechanisms of activities
6. By identifying potential biomarkers for cross-species
specificities that can be used to identify the ecosys-
tems impacted by an ongoing onslaught of PNS tox-
icity that affects vulnerable species more readily and
frequently. Extrapolation to humans can be done
using data from model animals such as rats, canines,
and birds
7. By developing a variety of biomarkers that can be
used to test clinical specimens, additional leads in
the potential compounds of similar molecular struc-
tures, and for comparing clinical specimens
8. By identifying gene expression profiles in untreated
clinical samples that correlate with a positive
response to therapy, providing outcome-based bio-
marker discovery.
PNS BIOMARKER METHODOLOGY
As mentioned earlier, applying appropriate methodol-
ogy is the major factor in the rapid identification and
validation of biomarkers of PNS neurotoxicity. In order
to make provisional diagnosis of the nature of toxic
mechanisms in any PNS toxicity related clinical presen-
tation, universal methods to collect clinical data using
standard methods of neurology clinic settings are essen-
tial. Biomedical informatics with its ever-increasing
branches of special categories has become an important
tool for collection, storage, retrieval, and dissemination
of relevant information.
The following are the commonly employed methodol-
ogies of such categories, providing valuable clinical bio-
marker data sets.
Neurological and physical examination
Protocol: In humans, standard neurological physical
examinations (NPxs) (Starks et al., 2012) include the fol-
lowing: Assessments of vibration perception and pro-
prioception are generally performed on the great toes,
bilaterally. Achilles deep tendon reflexes are examined
bilaterally, and Romberg test performance, tandem gait,
and postural tremor are routinely assessed. Clinical
examination results are recorded as normal, equivocal,
or abnormal. For all tests performed bilaterally (ankle
reflex, toe proprioception, and toe vibration), examina-
tion results are classified as “abnormal” if ratings are
173
abnormal or equivocal bilaterally, abnormal unilaterally
and equivocal on the contralateral side, or abnormal
unilaterally and missing (because of injury/amputation)
on the contralateral side. For tests without laterality
(postural tremor, Romberg test, and tandem gait), com-
bined abnormal and equivocal results are used to create
a dichotomized variable (normal vs. not normal) for
each outcome (Starks et al., 2012).
Electrophysiological measures. Standard noninvasive
electrophysiological measures of the dominant peroneal
motor nerve are performed with a factory-calibrated
TECA Sapphire II electromyograph (TECA Corp.,
Pleasantville, NY) (Starks et al., 2012). Foot temperature
is maintained above 32 C during testing. Distal motor
amplitude (millivolts), distal and proximal motor
latency (milliseconds), and short F-wave latency (milli-
seconds) are obtained and nerve conduction velocity
(meters per second) is routinely calculated.
Hand strength. Gross grip strength and key and pal-
mar pinch strength are obtained bilaterally using digital
grip and pinch dynamometers (JTech Medical, Salt Lake
City, UT) (Starks et al., 2012). A mean z-score for all six
hand strength tests (three tests performed bilaterally)
combined is calculated to create one summary measure
for hand strength.
Sway speed. Standing sway speed is measured with
a CATSYS 2000 Force Plate (Starks et al., 2012). Four 80-
sec trials are administered, two with eyes open and two
with eyes closed. Average sway speed (millimeters per
second) is reported separately for eyes open and eyes
closed.
Vibrotactile threshold. The Vibratron II (Sensortek,
Inc., Clifton, NJ) is used to measure bilateral great toe
120-Hz vibrotactile threshold with a standard protocol
(Starks et al., 2012). A single bilateral mean vibrotactile
threshold is reported in log micrometers. Any abnormal
results from the above parameters can be a useful clini-
cal biomarker that can be further tested using other
modalities.
Sample Requirements for Biomarker Testing
Adequate quantity of tissue (by biopsy) of investigation
can be obtained for various biomarker testing proce-
dures. The tissues include: blood, cerebrospinal fluid
(CSF), nerve tissue and target (end organ) tissue. CT
guided needle biopsy and other standard blood collec-
tion procedures are used for tissue sampling. A nerve
biopsy may help distinguish between demyelination
(damage to the myelin sheath covering the nerve) and
axon degeneration (destruction of the axonal portion of
the nerve cell). It may also help to identify an inflamma-
tory neuropathy or confirm specific diagnoses. A skin
biopsy is helpful to differentiate certain disorders that
174 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
might affect the small nerve fibers, as may be the case
with painful sensory axonal neuropathies. A muscle or
other tissue biopsy is used to diagnose and identify
damage caused to muscles and organs as a result of var-
ious disorders.
Biomedical imaging and informatics
Various images spanning the scale from microscopic
and molecular to whole body visualization, encompass-
ing many areas of clinical medicine, are important for
selecting, validating, and biomonitoring PNS disorders.
Recent years have witnessed impressive advances in the
use of magnetic resonance imaging (MRI) for the assess-
ment of patients with PNS and CNS disorders. MRI is
an imaging technique used to produce high quality
images of the inside of the human body. A scanner
emits a strong magnetic field inside the brain (or else-
where in the body) and produces signals that are ana-
lyzed by a computer to produce detailed images.
Complementary to the clinical evaluation, conventional
MRI provides crucial pieces of information for the diag-
nosis of acquired and hereditary PNS and CNS disor-
ders (Filippi and Agosta, 2010). Newer quantitative
MR-based techniques, such as magnetization transfer
MRI, diffusion tensor MRI, proton MR spectroscopy,
and functional MRI, are contributing to elucidate the
mechanisms that underlie injury, repair, and functional
adaptation in patients with nervous system disorders
(Filippi and Agosta, 2010).
Electrodiagnostic tests
Electrodiagnostic tests measure the electrical activity
of muscles and nerves. By measuring the electrical
activity, one can determine if there is nerve damage,
the extent of the damage, and potentially the cause of
the damage. Frequently, noninvasive neurological eva-
luations such as electromyography (EMG) and nerve
conduction velocity (NCV) testing are used.
Quantitative sensory testing (QST) and autonomic
testing are also commonly used.
Blood tests
Blood tests are commonly employed to check for vari-
ous factors that affect the nervous system directly or
indirectly (vitamin deficiencies, toxic elements, evidence
of an abnormal immune response). Depending on the
individual situation, certain laboratory tests to identify
potentially treatable causes for neuropathy by the fol-
lowing tests are employed: (1) Antibodies to nerve
components (e.g. anti-MAG antibody), (2) antibodies
to cytoskeletal components (NF; GFAP; and tubulin,
etc.); (3) vitamin B12 and folate levels; (4) thyroid, liver,
and kidney functions; (5) vasculitis evaluation; (6) oral
glucose tolerance test; (7); antibodies related to celiac
disease; (8) Lyme disease; (9) HIV/AIDS; and (10)
hepatitis C and B.
Testing for toxins
Toxins, poisons, and chemicals can cause peripheral
neuropathy. This can happen through drug or chemical
abuse or through exposure to industrial chemicals in
the workplace or in the environment (after either lim-
ited or long-term exposure). Common causes include:
exposure to lead, mercury, arsenic, and thallium. Some
organic insecticides and solvents can result in neuropa-
thies. Sniffing glue or other toxic compounds can also
cause peripheral neuropathy. Certain herbal medicines,
especially those that are particularly rich in mercury
and arsenic, can lead to peripheral neuropathy. Hence
blood profiling for the presence of parent or metabolic
products or metabolites of the drug pathways is com-
monly used to identify the basis of toxicity.
Molecular diagnostics
Molecular profiling techniques such as (a) Sanger
sequencing, (b) quantitative real-time PCR,
(c) amplification of refractory mutation system (allele
specific PCR), (d) fragment length analysis (to detect
any deletions within the coding portion of the gene),
(e) high performance liquid chromatography (to distin-
guish between wild type and mutated DNA strands
using heteroduplex formation), (f) immunohistochemis-
try (IHC) and fluorescence/chromogenic in situ hybrid-
ization (FISH/CISH), are used to detect any defects at
gene sequence level.
Molecular, cellular, and histological techniques
Analysis of patient samples from various medical clinics
as well as from animal model systems using various
combinations of molecular, cellular, and histological
techniques has become part of the routine laboratory
protocols. Details of nucleic acid extractions (DNA and
RNA), northern blotting, semi quantitative RT-PCR, and
real-time PCR protocols (Figure 10.3) have been
described in detail in several fairly recent publications
(Damodaran, 2009; Damodaran et al., 2011). Similarly,
methods of protein extraction and analysis using western
blotting (Figure 10.4) have also been described in several
 PNS BIOMARKER METHODOLOGY
FIGURE 10.3 Semiquantitative RT-PCR of candidate marker
genes of sarin exposure in the nervous system of hen (Damodaran
et al., 2006b). Representative photographic gel pictures of PCR pro-
ducts (one control and one treated) are provided here for the vali-
dated genes. An agarose gel (1%) was prepared using 1X TAE
buffer and PCR products (from each of the three animals) from
both control and treated groups were run in the same buffer for
2 h at 70 volts. Photographs of these bands were scanned into Tiff
files and quantified in IP lab gel quantification software
(Damodaran et al., 2006b).
publications (Damodaran et al., 2009, 2011). Classical his-
topathological analysis and other nervous system related
structure specific staining techniques (Figures 10.5 and
10.6) were also described in detail in recent articles
(Damodaran, 2009; Damodaran et al., 2011).
Toxicogenomics and proteomics approaches
RNA purity verification by Agilent analysis, micro array
chip hybridization, data capturing, data analysis, Tree
view analysis and clustering (Figure 10.7), data mining
for classifications, principal component analysis (PCA)
of gene expression (Figure 10.8), and gene validation
approaches are described in detail in many publications
(Damodaran et al., 2006 a,b). Functional analysis of the
proteins expressed in a specific tissue and/or stage
of toxic exposure is an essential step towards under-
standing biological processes. Once produced, the pro-
tein can then potentially undergo post-translational
175
FIGURE 10.4 Autoradiograms of sciatic nerve cytoskeletal
proteins (Gupta and Abou-Donia, 1995). Data from SDS-PAGE gel
and western blotting, followed by quantitative immunoreactivity
analysis using antibodies for NF-H, NF-HP, vimentin, GFAP, tubu-
lin, GAP-43, dynein and tau after treatment of hens with DFP
(diisopropylphosphorofluoridate).
modifications that can be caused by phosphorylation,
glycosylation, protein processing, and other proteolytic
cleavage (Cahill et al., 2001). Both 2DG (two dimensional
gels) as well as automated mass spectrometry based
approaches for proteome and phosphorylation site anal-
ysis as well as proteome data analysis have been
described in detail from several publications (Dunn and
Gorg, 2001). Binding assays to unravel the mechanics of
protein-protein and protein-enzyme interactions (Gupta
and Abou-Donia, 1995) as well as methodology for
studying axonal transport (Gupta et al., 1997) have been
described in detail in numerous articles.
Metabonomics
Neurotoxicity occurs after the neurotoxicants enter into
the blood circulation, travel to sites of vulnerability, and
gain access to tissues. Metabolic transformation is a key
mechanism by which the toxicants become either more
176 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS

FIGURE 10.5 (A) Photomicrograph of a longitudinal section in the proximal part of the sciatic nerve of a control hen showing normal
appearance of nerve fibers (Marchi’s stain 3132). (B) Photomicrograph of a longitudinal section in the proximal part of the sciatic nerve
showing degenerative myelin 17 days post-treatment with DFP (Marchi’s stain). (C) Photomicrograph of a longitudinal section in the proxi-
mal part of the sciatic nerve showing degenerating myelin 21 days post-treatment (Marchi’s stain, 3132) (Damodaran et al., 2011).

toxic or less toxic. These processes predominantly pro-
duce more polar metabolites that facilitate the increased
excretion (Abou-Donia and Nomeir, 1986). Thus, the
process called toxicokinetics encompassing absorption,
tissue distribution, storage, biotransformation, and elim-
ination plays a major role in PNS neurotoxicity.
Metabolic status during these stages can be assessed by
measuring the quantity and type of metabolites.
Detailed information about the toxicological outcome of
toxicant exposure is required to make decisions about
the long-term therapy protocols. Global profiling techni-
ques are rapid in gathering data on the impact made on
transcripts, proteins, and metabolites within the cell, tis-
sue, organ, and organ systems of the living organism.
Metabonomics/metabolomics is one such approach that
can provide a global profile of metabolites (Griffin and
Waters, 2006). High resolution Nuclear Magnetic
Resonance (NMR) along with statistical pattern recogni-
tion analysis can provide valuable data, and detailed
step-wise protocols are provided in several
publications.
Biomarkers of nerve tissue injury in the PNS
The pathologies of PNS tissue injury are extremely
diverse, with different etiologies such as genetic, toxic-
ity from various exposures including chemicals of ther-
apy, infectious agents such as HIV and leprosy, etc.
PNS injuries have very diverse clinical pictures and
disease courses. Peripheral neuropathies can thus be
acute or chronic, symmetrical or not, and be axonal or
involving demyelination and involve only parts of the
neuron or its whole. Some of the complex and chronic
disease conditions such as diabetes and alcoholism are
commonly seen in clinical scenarios.
A great number of peripheral neuropathies involve
either sensory neurons or motor neurons, or both, and
other cell types. The axon degenerates after a nerve
injury, and the myelin part of the Schwann cells around
it also degenerates. Those Schwann cells that enlarge
undergo mitosis to form a column of cells along the
regions once occupied by the disintegrated axons. The
process of repair at the site of injury is complex, involv-
ing the formation of axonal sprouts followed by selective
growth of one axon among the many that eventually dis-
integrate. Because of the higher abundance of Schwann
cells in the PNS, regeneration after an injury is much fas-
ter and with a higher rate of nerve repair. The proximity
of the cut ends of the nerve after an injury decides the
higher likelihood of repairing of the injured nerve in the
PNS. Clearing up irregular segments and other pieces of
debris at the site of injury by macrophages facilitate the
new sprout of synthesis of essential proteins and other
cytoskeletal structures at the site of injury.
Besides Schwann cells, SGCs (satellite glial cells) also
undergo mitosis and other changes in the PNS. These
cell types have been implicated in the genesis and main-
tenance of pain in injured nerves of the PNS (Jasmin
et al., 2010). Thus the coordinated functions of several
 PNS BIOMARKER METHODOLOGY 177

A B C

D E F

FIGURE 10.6 (A) Photomicrograph of a cross section in the distal part of the sciatic nerve of a control hen showing the appearance of
nerve fiber (Sevier-Munger, 3132). (B) Photomicrograph of a cross section in the proximal part of the sciatic nerve showing a region exhibit-
ing complete loss of axons (arrows) at 15 days post-treatment with DFP. This section is stained with H & E. (C) Photomicrograph of a cross
section in the distal part of the sciatic nerve showing almost complete loss of some of the affected fibers (empty myelin layers) 21 days post-
treatment. Seiver-Munger staining; 3132. (D) Photomicrograph of a longitudinal section in the proximal part of the sciatic nerve showing
Schwann cell hyperplasia (arrows), 15 days post-treatment with DFP; H & E staining, 3132. (E) Photomicrograph of a longitudinal section
in the proximal part of the sciatic nerve showing focal gliosis 10 days post-treatment (H & E, 3132). (F) Photomicrograph of the distal part
of the sciatic nerve showing focal gliosis 21 days post-treatment period. (Damodaran et al., 2011)

important molecules in various cell types are required
for this repair pathway. Table 10.1 provides a list of the
biomarker genes/proteins identified by several studies.
female mice. These changes were in correlation with
ultrastructural changes. These results from electrophys-
iological methods provide valuable data about the sta-
tus of the PNS nerves after acute or chronic
neurotoxicity.

Biomarkers of changes in electrophysiology

Dai et al. (2012) showed that the compound action
potential durations (CAPDs), compound muscle action
potential amplitudes (CAPAs), and conduction veloci-
ties of sciatic-tibial nerve (NCVs) exhibited progres-
sively abnormal changes over time in colistin treated
Ion channels as biomarkers
Peripheral sensory neurons are adapted to recognize
danger to the organism by virtue of their sensitivity
to intense mechanical, thermal, and irritant chemical
178 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
FIGURE 10.7 Tree view analysis of global gene expression at
the 2 h time point is presented here. Hierarchical clustering was
applied through the Cluster software program, and the results
were visualized in Treeview (check http://www.microarrays.org/
software for computational details). The expression level of each
gene relative to the median expression level across all samples was
represented by color: one representing log expression ratios of
overexpressed genes, and the other representing log expression
ratios of underexpressed genes. The remarkable homogeneity of
the coloring pattern validates the reproducibility of the
replicate data from the control and treated groups (Damodaran
et al., 2006b).
stimuli. Although CNS-expressed sodium channels also
occur in peripheral nerves, several additional channels
occur mainly in dorsal root ganglion cells.
Complementary roles for Nav1.7, Nav1.8, and Nav1.9
(encoded by SCN9A, SCN10A, and SCN11A, respec-
tively) in sensory transduction and nociception are
beginning to emerge (Kullmann, 2010). Transient
receptor potential (TRP) ion channels are the most
widely studied molecular mediators of nociception,
conducting nonselective entry of cations upon activa-
tion by various noxious stimuli. TRPV1 is activated by
high temperatures, low pH, and capsaicin, the valli-
noid irritant component of chili peppers. TRPA1 med-
iates the detection of reactive chemicals including
environmental irritants such as tear gas and industrial
isothiocyanates, but more importantly, it is also
activated during tissue injury by endogenous molecu-
lar signals including 4-hydroxynonenal and prosta-
glandins (Chiu et al., 2013). An extensive list of
nervous system related ion channel genes used for
expression profiling (SABiosciences, 2013) is provided
in Table 10.2. It is noteworthy to mention here that the
highest number of altered gene expression and highest
level of up- or down regulation were recorded for ion
channel genes, in global gene expression studies on
sarin exposure at early time points (Damodaran et al.,
2006a,b).
Biomarkers of axonal regeneration
Successful axonal regeneration is controlled by both
intrinsic factors (i.e. endogenous capacity of adult neu-
rons to extend axons when injured) and extrinsic fac-
tors, such as support or inhibition of regeneration by
glial cells, scar formation, and vascular supply, among
others (Hoke et al., 2013). The role of mTOR and STAT3
pathways in modulating the intrinsic capacity of neu-
rons to regenerate has been proposed by Hoke (2006).
Deletion of the PTEN gene in retinal ganglion neurons
was shown to activate the mTOR pathway and
enhanced regeneration after optic nerve crush (Park
et al., 2008). More recently, another molecular pathway
controlled by STAT3 has been found to provide a paral-
lel mechanism to further enhance regeneration of retinal
ganglion neuron axons after optic nerve crush (Smith
et al., 2009). Notably, manipulation of both pathways
leads to a synergistic increase in axonal regeneration in
the optic nerve crush model (Sun et al., 2011). In the
adult animal, regeneration has to occur over very long
distances. As the rate of axonal elongation is deter-
mined by the rate of “slow” axonal transport, this is a
very slow process leading to chronic denervation
changes in the Schwann cells in the distal nerve and tar-
get tissues such as muscle (Hoke et al., 2013). A poten-
tial approach to overcome this challenge is to “speed
up” the rate of regeneration by overexpressing a heat
shock protein (hsp27) that plays a key role in axon out-
growth in injured neurons. There are several genes
(Table 10.3) identified as playing significant roles in
neurogenesis in the PNS (SABiosciences, 2013).
Biomarkers of axonal injury
Axon degeneration is a hallmark consequence of
chemical neurotoxicant exposure (e.g. acrylamide),
mechanical trauma (e.g. nerve transection, spinal cord
contusion), deficient perfusion (e.g. ischemia,
 PNS BIOMARKER METHODOLOGY
hypoxia), and inherited neuropathies (e.g. infantile
neuroaxonal dystrophy). Regardless of the initiating
event, degeneration in the PNS and CNS progresses
according to a characteristic sequence of morphologi-
cal changes. These shared neuropathologic features
suggest that subsequent degeneration, although
induced by different injury modalities, might evolve
via a common mechanism. Studies conducted over the
past two decades indicate that Ca21 accumulation in
injured axons has significant neuropathic implications
and is a potentially unifying mechanistic event. It was
proposed that diverse injury processes (e.g. axotomy,
ischemia, and trauma) which culminate in axon
degeneration cause an increase in intraaxonal Na1 in
conjunction with a loss of K1 and axolemmal depolar-
ization. These conditions favor a reverse Na1/Ca21
179
FIGURE 10.8 Principle component
analysis (PCA) of gene expression was pre-
pared on a three-dimensional platform and is
presented here as a graph providing a global
view of the differences among the control
(C1, C2, and C3) and treatment (T1, T2, and
T3) groups. There is a clear separation among
the controls and treatment groups, indicating
differences in their total gene expression pro-
files (Damodaran et al., 2006b).
exchange operation which promotes damaging extra
axonal Ca21/entry and subsequent Ca21/-mediated
axon degeneration (Lopachin and Lehning, 1997). A
spontaneous mutation called WldS (slow Wallerian
degeneration), described by Lyon et al. (1993), taught
us that Wallerian degeneration is not a passive disin-
tegration of axon when the axon is severed from the
cell body but an active process mediated by Nmnat
protein (Mack et al., 2001). More recently, using a
powerful forward genetics screen, a new pathway
that involves Sarm protein has been found to prevent
axonal degeneration after traumatic injury (Osterloh
et al., 2012). It is not clear whether this pathway is
related to Nmnat protein or plays a role in distal axo-
nal degeneration as seen in human peripheral
neuropathies.
180 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS

TABLE 10.1 Protein markers of Schwann cells

Myelin-Forming Schwann Cell Non-Myelin Forming Schwann Cell Common Markers

P0 NCAM S100
P2 GFAP Vimentin
Myelin basic NGF receptor Laminin
protein L1 Galactocerebroside
CNPase A5E3 Seminolipid
Proteolipid protein Ran-2
Myelin-associated glycoprotein

Proteins/Enzymes Involved in Myelination, Demyelination and other Functions of Myelin

Peripheral myelin protein 22

Connexin 32 (Cx32/GJB1)
Periaxin
GDAP1 (Ganglioside-induced differentiation associated protein 1)
NF-L (Neurofilament light chain)
Dyamin-2 (DNM2)
GARS (Glycyl-tRNA synthetase)
Myelin-specific enzyme 2’,3’-cyclic nucleotide 3’-phosphohydrolase

Transcriptional Regulators of Myelin

EGR2 (Early growth response 2)

Sox10 (Sry-Related high-mobility group box-containing gene 10)

Other PNS Proteins

Gamma-enolase; creatine kinase-B; beta-S100 protein

TABLE 10.2 Nervous system related channels as

biomarkers

Calcium Channels: TABLE 10.3 Candidate biomarker genes involved in neuro-

Voltage-Gated: CACNA1A, CACNA1B, CACNA1C, CACNA1D,
CACNA1G, CACNA1I, CACNB1, CACNB2, CACNB3, CACNG2,
CACNG4
Ligand-Gated: RYR3
Transient Receptor Potential Channels: TRPA1, TRPC1, TRPC3,
TRPC6, TRPM1, TRPM2, TRPM6, TRPM8, TRPV1, TRPV2, TRPV3,
TRPV4
Potassium Channels
Delayed Rectifier: KCNA1, KCNA2, KCNA5 (KV1.5), KCNA6,
KCNB1, KCNB2, KCNH1, KCNH2, KCNQ1, KCNQ2, KCNQ3
Inward Rectifier: KCNH2, KCNH6, KCNH7, KCNJ1, KCNJ11,
KCNJ12, KCNJ13, KCNJ14, KCNJ15, KCNJ16, KCNJ2, KCNJ3,
KCNJ4, KCNJ5, KCNJ6, KCNJ9, KCNK1
Calcium-Activated: KCNMA1, KCNMB4, KCNN1, KCNN2,
KCNN3
Other Voltage-Gated Potassium Channels: HCN1, HCN2,
KCNC1, KCNC2, KCND2, KCND3, KCNH3
Modifier Subunits: KCNAB1, KCNAB2, KCNAB3, KCNS1
Sodium Channels
Amiloride Sensitive: ACCN1, ACCN2, ACCN3
Voltage-Gated: SCN10A, SCN11A, SCN1A, SCN1B, SCN2A,
SCN2B, SCN3A, SCN8A, SCN9A
Chloride Channels: BEST1, CLCN2, CLCN3, CLCN7
Sodium/Chloride Transport: SLC12A5
trophic signaling, neuropeptides functions, and neurogenesis in
the PNS
Neurotrophins and Receptors: ADCYAP1R1, ARTN, BDNF, CD40
(TNFRSF5), CNTF, CNTFR, CRHBP, CRHR1, CRHR2, FRS2, FRS3,
FUS, GDNF, GFRA1, GFRA2, GFRA3, GMFB, GMFG, MAGED1,
MT3, NF1, NGF, NGFR, NGFRAP1, NR1I2, NRG1, NRG2, NTF3,
NTF4, NTRK1, NTRK2, PSPN, PTGER2, TFG, TRO, VGF
Neuropeptides and Receptors
Neuropeptide Hormone: CRH, NPFF, NPY, PNOC, UCN
Bombesin Receptors: GRPR
Cholecystokinin Receptors: CCKAR
Galanin Receptors: GALR1, GALR2
Neuropeptide Y Receptors: NPY1R, NPY2R, PPYR1
Neurotensin Receptors: NTSR1
Tachykinin and Receptors: TACR1
Other Neuropeptides and Receptors: NPFFR2 (GPR74), HCRT,
MC2R
Neurogenesis
Peripheral Nervous System Development: GFRA3
Negative Regulation of Neurogenesis: MT3
Other Neurogenesis Related Genes: ARTN, BDNF, CBLN1,
CNTF, CNTFR, FGF2, GDNF, MEF2C, NELL1, NGFR, NRG1,
NTF3, NTRK1, NTRK2, STAT3, TFG
Transmission of Nerve Impulse: CBLN1, CNTF, CRH, GALR1,
GALR2, HCRT, NPFF, NPY, NTSR1, PNOC
 PNS BIOMARKER METHODOLOGY 181

TABLE 10.4 Potential biomarker genes related to pain and related disorders
Conduction of Pain

Ion Channels: TRPA1, TRPV1, TRPV3

Sodium Channels: SCN10A, SCN11A, SCN3A, SCN9A, SLC6A2
Potassium Channels: KCNIP3, KCNJ6, KCNQ2, KCNQ3
Purinergic Receptors: ADORA1, P2RX3, P2RX4, P2RX7, P2RY1
Opioid Receptors: OPRD1, OPRK1, OPRM1
Cannabinoid Receptors: CNR1, CNR2

Synaptic Transmission

Glutamate Receptors: GRIN1, GRIN2B, GRM1, GRM5

Serotonin Receptors: HTR1A, HTR2A
Calcium Channel: CACNA1B

Modulation of Pain Responses

Eicosanoid Metabolism: PLA2G1B, PTGER1, PTGER3, PTGER4, PTGES, PTGES2, PTGES3, PTGS1, PTGS2
Inflammation: ACE, ALOX5, BDKRB1, CALCA, CCK, CCKBR, CCL2, CCR2, CD200, CD4, CHRNA4, CSF1, CX3CR1, DBH, EDN1, EDNRA,
FAAH, GCH1, IL10, IL18, IL1A, IL1B, IL2, IL6, ITGAM, ITGB2, MAPK1, MAPK14, MAPK3, MAPK8, PENK, PNOC, PROK2, TAC1, TACR1,
TLR2, TLR4, TNF
Neurotransmitters: ADRB2, COMT, DBH, MAOB, PDYN, PENK, PNOC
Neurotrophins: BDNF, GDNF, NGF, NTRK1
Parkin Complex: HSPA4 (HSP70), PARK7, STUB1
Parkin Substrate: ATXN2, ATXN3, GPR37, SYT11
Cell Adhesion: APC, APP, CDH8, FN1, NFASC, NRXN3, PTEN, TPBG
Ubiquitination: CDC27, CUL2, FBXO9, LRRK2, PAN2, PARK2, PINK1, SKP1, STUB1, UBB, UBA1, UBE2I, UBE2K, UBE2L3, UCHL1, USP34
Inflammation: FN1, PRDX2, YWHAZ

Apoptosis

Pro-Apoptosis: APC, APP, CASP1 (ICE), CASP3, CASP8 (FLICE), CASP9, CUL2, MAPK9 (JNK2), PSEN2, PTEN
Anti-Apoptosis: APC, BDNF, CASP3, CASP9, NEFL, NR4A2 (NUR77), OPA1, PPID, PRDX2, PSEN2, PTEN, SLC25A4, SNCA, TCF7L2,
UBB, YWHAZ
Mitochondria: CASP3, CASP7, CASP8 (FLICE), HSPA4 (HSP70), LRRK2, NEFL, OPA1, PARK7, PINK1, PTEN, SLC25A4, SNCA, TH,
UCHL1, VAMP1, VDAC3, YWHAZ
Synaptic Vesicles: LRRK2, SEPT5, SV2B, SYNGR3, SYT1, SYT11, TH

Signal Transduction

Dopaminergic: D4S234E, DDC, DRD2, HTR2A, NR4A2 (NUR77), PARK2, PARK7, PINK1, SEPT5, SLC6A3, SNCA, TH
GABAergic: DRD2, GABBR2
MAP Kinase: APC, FGF13, MAPK9 (JNK2), PRDX2, RGS4
Notch: APP, PSEN2, SPEN
Cytoskeletal Organization: APC, CDC42, MAPT, NEFL, PARK2
Ion Transport: ATP2B2, CADPS, CXXC1, DRD2, EGLN1, GBE1, GRIA3, HTR2A, KCNJ6, NSF, PSEN2, S100B, SRSF7, SLIT1, SNCA, VDAC3
Transporters: ATP2B2, GRIA3, SLC18A2, SLC6A3, SLC25A4, SV2B, SYT1, SYT11, VDAC3
Others: ALDH1A1, BASP1, CHGB, DLK1, NCOA1, NTRK2, RTN1

Potential biomarkers of pain
Neuropathic or neurogenic pain is defined as pain initi-
ated or caused by a primary lesion or dysfunction of the
nervous system. Pain is the outcome of noxious envi-
ronmental stimuli, mechanical tissue damage, and infec-
tious or other pathological disease processes. This can
be the result of (a) spinal cord injury (SCI), (b) periph-
eral nerve damage resulting from diabetes or other
autoimmune diseases, (c) treatment with anti-cancer
drugs that affect axon integrity, or (d) post-herpetic
neuralgia (PHN) (Goins et al., 2012). Pain provides a
large therapeutic target for biomarker development, as
a good proportion of humans and other animal
kingdom members have either transient or chronic pain
disorders, thus making it a useful route to understand-
ing the molecular mechanisms of nervous system func-
tion. There are two types of pain: (i) neuropathic pain and
(ii) inflammatory pain. While neuropathic pain often
results from damage to the PNS and/or CNS, peripheral
tissue damage and/or inflammation generally initiates
inflammatory pain. Neuropathic and inflammatory pain
both cause activation of nociceptors (damage-sensing neu-
rons) that innervate the skin, muscle, and viscera and ter-
minate in the laminae of the spinal cord dorsal horn.
Nociceptors conduct information to the CNS via neuro-
transmission and action potentials generated by a variety
of ion channels and receptors (purinergic, opioid, and
182 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
cannabinoid) leading to secondary activation of neuronal
pathways. This stage is followed by synaptic transmission
(via glutamate, serotonin, and dopamine systems).
Inflammatory mediators released by immune cells and
damaged neurons can modulate the transduction nocicep-
tion process. Consequently, the excitability of spinal neu-
rons is modulated via activation of resident microglia that
release growth factors (such as BDNF), chemokines, and
cytokines. Endogenous opioid peptides and arachidonic
acid metabolites acting through G-protein coupled recep-
tors also modulate neuronal excitability. A number of these
pathways are currently being evaluated as potential phar-
macological targets and biomarkers for analgesic develop-
ment for pain management. Table 10.4 provides the list of
genes, identified to be playing important roles in human
neuropathic pain and inflammation (SABiosciences, 2013).
They are involved in the transduction, maintenance, and
modulation of pain responses. A good proportion of these
genes have been validated as biomarkers, while several
others are potential biomarkers for studying the specific
sub pathways within the complex networks. There are
observations of mitochondrial abnormality associated with
pain in the absence of axonal degeneration, suggesting
a link between pain and impaired axonal physiology
(Flatters and Bennett, 2006).
NGF acts as a peripheral pain mediator. It is upregu-
lated in inflammatory states; high-affinity NGF recep-
tors (trkA receptors) are expressed by many
nociceptors; NGF sensitizes peripheral nociceptive
terminals; NGF is retrogradely transported to sensory
neuron somata and regulates genes involved in pain
processing; and NGF neutralization alleviates many
sensory abnormalities in models of persistent pain.
There is also evidence that a second neurotrophin,
BDNF, acts as a central modulator of pain: BDNF is
expressed by nociceptors, and its expression is upregu-
lated in inflammatory conditions by peripherally pro-
duced NGF. BDNF acts through postsynaptic trkB
receptors that trigger intracellular signaling cascades
(Pezet and McMahon, 2006). Many of the common tar-
gets for drug actions in the pain pathways have been
identified. Some of the candidate targets (which
become useful biomarkers) are as follows: (a) TRPV1
receptor, which has been targeted by many drugs,
including capsaicin; (b) NMDA receptor; drugs target-
ing this receptor has not been found to be effective in
the treatment of post-herpetic neuralgia (PHN);
(c) cannabinoids have been tested in studies;
(d) calcium channels are targeted by pregabalin and
gabapentin; (e) sodium channels are the targets for
many antiepileptic drugs, some of which have been
tested in the PHN with only minimal efficacy; (f) the
norepinephrine (NE) transporter is a new target; (g)
drugs that target the aminopeptidase N (APN) and
neutral endopeptidase (NEP) (SABiosciences, 2013).
Potential biomarkers of psychiatric diseases,
neurodegenerative diseases, and insomnia
Dysregulation of GABAergic or glutamatergic synaptic
transmission results in a wide variety of nervous system
disorders, including chronic pain, psychiatric diseases,
neurodegenerative diseases, and insomnia. Of the great
variety of neuronal receptors in the brain, the major
excitatory receptors recognize the ligand glutamate, and
the major inhibitory receptors respond to the ligand
GABA. The GABA neurotransmitter system includes
the GABAA and GABAC classes of ligand gated ion
channels. The glutamate neurotransmitter system
includes NMDA, AMPA, and kainate ligand-gated ion
channels. Key enzymes synthesize GABA or glutamate
as necessary, which are then transported into synaptic
vesicles. Release of GABA or glutamate from vesicles
activates postsynaptic GABA-responsive or glutamate-
responsive ion channels, respectively, initiating
downstream G protein signaling to propagate neuro-
transmission. There are a many drugs that are agonists
or antagonists of the GABA and glutamate neurotrans-
mitter systems. There are a number of genes essential
for the synthesis and transport of GABA and glutamate,
as well as responsive ion channels and downstream sig-
naling. Studying and monitoring these genes at all
levels may yield insights into the interaction of these
excitatory and inhibitory neuronal systems during
essential cognitive functions, thereby facilitating the
process of biomarker development (Table 10.5:
SABiosciences, 2013).
TABLE 10.5 Potential biomarker genes related to glutama-
tergic and GABAergic synapses
Glutamatergic Synapse
Receptors: GRIA1, GRIA2, GRIA3, GRIA4, GRIK1, GRIK2, GRIK4,
GRIK5, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRM1, GRM2,
GRM3, GRM4, GRM5, GRM6, GRM7, GRM8
Downstream Signaling: ADCY7, APP, CACNA1A, CDK5R1,
CLN3, DLG4 (PSD95), GNAI1, GNAQ, HOMER1, HOMER2,
ITPR1, MAPK1 (ERK2), PLA2G6, PLCB1, SHANK2
Transport & Secretion: ADORA1, ADORA2A, AVP, BDNF, IL1B,
P2RX7, SLC17A6, SLC17A8, SLC1A1, SLC1A2, SLC1A3, SLC1A6,
SLC17A6, SLC17A7, SLC17A8, SLC38A1, SLC7A11, SNCA
Metabolism: ALDH5A1, GAD1, GLS, GLUL, PRODH, SLC1A3,
SRR
GABAergic Synapse
Receptors: GABBR1, GABBR2, GABRA1, GABRA2, GABRA4,
GABRA5, GABRA6, GABRB1, GABRB3, GABRD, GABRE,
GABRG1, GABRG2, GABRG3, GABRQ, GABRR1, GABRR2
Downstream Signaling: ADCY7, ADORA1, ADORA2A,
CACNA1A, CACNA1B, GNAI1, GNAQ, GPHN, SNCA
Transport & Secretion: CACNA1A, NSF, P2RX7, SLC1A3,
SLC32A1, SLC38A1, SLC6A1, SLC6A11, SLC6A12, SLC6A13
Metabolism: ABAT, ALDH5A1, GAD1, GLS, GLUL, PHGDH,
SLC1A3
 PNS BIOMARKER METHODOLOGY
Potential biomarkers of motor function,
emotional behavior, temperature regulation,
sensory perception, locomotion, and psychosis
Two of the major neurotransmitter systems such as
dopamine and serotonin play a major role in the previ-
ously mentioned aspects. Dopamine affects brain pro-
cesses that control both motor and emotional behavior
and plays a role in the brain’s reward mechanism.
Serotonin is critical in temperature regulation, sensory
perception, locomotion, sleep, and psychosis.
Pharmacological agents targeting dopaminergic/seroto-
ninergic neurotransmission have been clinically used to
manage several neurological and psychiatric disorders
including Parkinson’s disease, schizophrenia, bipolar
disorder, depression, attention deficit and hyperactivity
disorder (ADHD), and addiction. Besides significant
progress in understanding their structural, genetic and
pharmacological properties, recent studies have uncov-
ered the complexity, intricacy, and plasticity of intracel-
lular signaling mechanisms involved in dopamine and
serotonin receptor function. These receptors act through
diverse G-protein coupled and G-protein independent
mechanisms that trigger downstream intracellular sig-
nal transduction events involving the cAMP/PKA, PI-
3Kinase/AKT, phospholipase A2 (PLA2), and phospho-
lipase C (PLC) pathways. These pathways in turn regu-
late various functions including synthesis, transport,
TABLE 10.6 Potential biomarker genes related to dopamine
and serotonin
Receptors:
Dopamine: DRD1, DRD2, DRD3, DRD4, DRD5
Serotonin: HTR1A, HTR1B, HTR1D, HTR1E, HTR1F, HTR2A,
HTR2B, HTR2C, HTR3A, HTR3B, HTR4, HTR5A, HTR6, HTR7
Synthesis & Degradation
Dopamine: COMT, DBH, DDC, MAOA, TH
Serotonin: MAOA, MAOB, TDO2, TPH1, TPH2
Dopamine & Serotonin Transporters: SLC6A3 (DAT), SLC6A4
(SERT)
Signal Transduction Pathways
cAMP/PKA Pathway: ADCY1, ADCY2, ADCY3, ADCY5, CASP3,
CDK5, CREB1, DUSP1, FOS, PRKACA, MAPK1, PPP1R1B
(DARPP32)
PI3K/AKT Pathway: PIK3CA, PIK3CG, AKT1, AKT2, AKT3,
GSK3A, GSK3B
PLA2 Pathway: ALOX12, CYP2D6, PDE4A, PDE4B, PDE4C,
PDE4D, PDE10A, PLA2G5
PLC Pathway: ITPR1, PLCB1, PLCB2, PLCB3
G-Protein Coupled Receptor Regulation: ADRB1, ADRB2,
ADRBK1, ADRBK2, APP, ARRB1, ARRB2, GRK4, GRK5, GRK6,
SNCA, SNCAIP
Dopamine & Serotonin Gene Targets: BDNF, EPHB1, GDNF,
GFAP, NR4A1 (NUR77), NR4A3 (NOR1), PDYN, PTGS2,
SLC18A1, SLC18A2 (VMAT2), SYN2
183
and degradation of dopamine and serotonin as well
as the transcriptional regulation key genes linked to
multiple neuropathological conditions (Table 10.6:
SABiosciences, 2013).
Potential biomarkers of sleeping disorders
(apnea, insomnia, and desynchronosis)
Synchronization, or “entrainment,” of the circadian
clock occurs via light stimulus of the hypothalamic
suprachiasmatic nucleus (SCN) in the brain and via
hormone signaling from the SCN in peripheral tis-
sues. Interacting positive and negative circadian gene
feedback loops at the transcriptional and post-
translational level sets up the circadian “oscillator”
and ensures tight control over transcription factors
regulating expression of the appropriate genes
required during circadian days or nights. Genes regu-
lated by circadian rhythms are involved in a diverse
range of biological processes that affect physiology,
metabolism, and behavior. Although the circadian
rhythm target genes in its “output” pathways vary
widely from tissue to tissue, the transcription factors
encoded by central clock and clock-controlled genes
are mostly shared across all cell types. Sleeping disor-
ders (such as apnea, insomnia, and desynchronosis)
disrupt the timing of the circadian clock, requiring
re-entrainment and causing fatigue. The list of genes
identified to be involved in these processes is given
in the Table 10.7 (SABiosciences, 2013).
TABLE 10.7 Potential biomarker genes of sleep/wake cycle
Circadian Clock: ARNTL (BMAL1), ARNTL2, BHLHE40 (DEC1),
BHLHE41, CLOCK, CSNK1E, CRY1, CRY2, NR1D1, NR1D2 (REV-
ERB), PER1, PER2, PER3, RORA, TIMELESS
Casein Kinases: CSNK1A1, CSNK1D, CSNK1E, CSNK2A1,
CSNK2A2
CREB Signaling: AANAT, CAMK2A, CAMK2B, CAMK2D,
CAMK2G, CHRNB2, CREB1, CREB3, KCNMA1, HTR7, MAPK1,
MAPK14, MAPK3, MAT2A, PRKACB, PRKACG, PRKAR1A,
PRKAR1B, PRKAR2A, PRKAR2B, PRKCA, PRKCB, PROKR2
Light Sensing Proteins
Melatonin Receptors: MTNR1A, MTNR1B
Opsins: OPN3, OPN4
Others: CHRNB2, CRX
Circadian Regulated Transcription Factors: ALAS1, EGR1, EGR3,
EPO, ESRRA, HLF, IRF1, MYOD1, NFIL3, NKX2-5, PAX4,
POU2F1, RORB, RORC, SMAD4, SP1, SREBF1, STAT5A, TEF,
TFAP2A, TGFB1, WEE1, DBP, PPARA
Other Common Circadian Regulated Genes: CARTPT, CCRN4L,
FBXL21, FBXL3, HEBP1, NCOA3, NMS, NPAS2, NR2F6,
PPARGC1A, PRF1, PTGDS, SLC9A3
184 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
Biomarkers of PNS infection
Interestingly, sensory neurons share many of the same
pathogen and danger molecular recognition receptor path-
ways as innate immune cells, which enable them also to
detect pathogens. In the immune system, microbial patho-
gens are detected by germline encoded pattern recognition
receptors (PRRs), which recognize broadly conserved exog-
enous pathogen-associated molecular patterns (PAMPs).
The first PRRs to be identified were members of the toll-
like receptor (TLR) family, which bind to yeast, bacterial
derived cell-wall components, and viral RNA. Following
PRR activation, downstream signaling pathways are
turned on that induce cytokine production and activation
of adaptive immunity. In addition to TLRs, innate immune
cells are activated during tissue injury by endogenous
derived danger signals, also known as damage-associated
molecular patterns (DAMPs) or alarmins. These danger
signals include uric acid, and heat shock proteins released
by dying cells during necrosis, activating immune cells
during noninfectious inflammatory responses. PRRs
including TLRs 3, 4, 7, and 9 are expressed by nociceptor
neurons, and stimulation by TLR ligands leads to induc-
tion of inward currents and sensitization of nociceptors to
other pain stimuli. Furthermore, activation of sensory neu-
rons by the TLR7 ligand imiquimod leads to activation of
an itch-specific sensory pathway. These results indicate
that infection-associated pain and itch may be partly due
to direct activation of neurons by pathogen-derived factors,
which in turn activate immune cells through peripheral
release of neuronal signaling molecules. A major DAMP/
alarmin released during cellular injury is ATP, which is
recognized by purinergic receptors on both nociceptor neu-
rons and immune cells. Purinergic receptors are made up
of two families: P2X receptors, ligand-gated cation chan-
nels, and P2Y receptors, G-protein coupled receptors. In
nociceptor neurons, recognition of ATP occurs through
P2 3 3, leading to rapidly desensitizing cation currents and
pain, while P2Y receptors contribute to nociceptor activa-
tion by sensitization of TRP and voltage-gated sodium
channels. In macrophages, ATP binding to P2 3 7 receptors
leads to hyperpolarization, and downstream activation of
the inflammasome, a molecular complex important in gen-
eration of IL-1beta and IL. Therefore, ATP is a potent dan-
ger signal that activates both peripheral neurons and
innate immunity during injury, and some evidence even
suggests that neurons express parts of the inflammasome
molecular machinery. The flip side of danger signals in
nociceptors is the role of TRP channels in immune cell acti-
vation. TRPV2, a homologue of TRPV1 activated by nox-
ious heat, is expressed at high levels in innate immune
cells. Genetic ablation of TRPV2 led to defects in macro-
phage phagocytosis and clearance of bacterial infections.
Mast cells also express TRPV channels, which may directly
mediate their degranulation. It remains to be determined
whether endogenous danger signals activate immune cells
in a similar manner as nociceptors.
A key means of communication between immune
cells and nociceptor neurons is through cytokines. Upon
activation of cytokine receptors, signal transduction
pathways are activated in sensory neurons leading to
downstream phosphorylation of membrane proteins
including TRP and voltage-gated channels. The result-
ing sensitization of nociceptors means that normally
innocuous mechanical and heat stimuli can now acti-
vate nociceptors. Interleukin 1 beta and TNF-alpha are
two important cytokines released by innate immune
cells during inflammation. IL-1beta and TNF-alpha are
directly sensed by nociceptors which express the cog-
nate receptors, induce activation of p38 map kinases
leading to increased membrane excitability. Nerve
growth factor (NGF) and prostaglandin E(2) are also
major inflammatory mediators released from immune
cells that act directly on peripheral sensory neurons to
cause sensitization (Chiu et al., 2013)
MicroRNAs as potential biomarkers of
PNS injury
The microRNAs (miRNAs) are a recently identified
class of small regulatory RNA molecules found in most
organisms. They are encoded by the genomes and pro-
cessed into 22 nucleotide products, which play impor-
tant roles in the post-transcriptional regulation of
important cellular pathways in diverse biological pro-
cesses, including cell proliferation, apoptosis, tissue
morphogenesis, tumorigenesis, and heart disease
(Dugas and Notterpek, 2011). Recently, changes in
microRNA expression profiles have been detected in
different injury models, and emerging evidence strongly
indicates that these changes promote neurons to survive
by shifting their physiology from maintaining structure
and synaptic transmission (Wu and Murashov, 2013).
The list of miRNA that showed altered expression is
presented in Table 10.8.
TABLE 10.8 mi(RNAs) whose expression changed the most
in the referenced studies
(Bremer et al. (2010), Yun et al. (2010), Verrier et al. (2010))
miR-138, miR-138, miR-129
miR-140, miR-338, miR-145
miR-146b, miR-193
miR-195, miR-222
miR-204, miR-29a
miR-27b
miR-30a
miR-338-3p
miR-34a
Highly upregulated: miR-21, miR-221
 PNS BIOMARKER METHODOLOGY 185

TABLE 10.9 Differential expression of microRNAs after peripheral denervation and renervation

Condition Denervation (4 mo) Reinnervation (4 mo)

Upregulated miRNAs rim-miR-1 rno-miR-133a rno-miR-1 rno-miR-133a rno-miR-206

Ampd1 LOC49S749 Akap9 G0s2 Cit Hnrpu
Atp2c1 LOC680782 Elf2 Hnrpu Gdnf Lsamp
Cacnb2 MGC108776 Gas6 Hspb1 Mcf2l MGC108776
Cenpc1 Mns1 Gp1bb MGC108776 Ptprd Mef2
Chac2 Npy I12rg Npy RT1-A2 Npy
Cltc Rasa1 Klhl24 Sgk Stau1 Ppfibp2
Potential target genes Ddx5 Serpina3k LOC500110 Sox6 Sv2b
Echdc1 Ubc Nexn Wif1
G6pdx Usp33 Nr3c1
Havcr2 Pdk4
Hnrpu Plp
Hspd1 Ptprd
LOC292666 Rnf103
LOC299282 Zfp131

Source: Jeng et al., 2009.

Yu et al. (2011) noted altered microRNA expression
following sciatic nerve resection in dorsal root ganglia of
rats. The expression pattern of miR-206 was different
from that of miR-1 and miR-133a. Notably, two genes
(Hnrpu and Npy) and one gene (Ptprd) were potentially
regulated both in the denervated and reinnervated mus-
cle by miR-1 and miR-133a, respectively. There were six
potential target genes (Hnrpu, Lsamp, MGC108776,
Mef2, Npy, and Ppfibp2) of the upregulated miR-206 in
the reinnervated muscle. Among these, three (Hnrpu,
Npy, and MGC108776) were potentially regulated by
both miR-1 andmiR-206. Because theMef2 transcription
factor was reported to promote the transformation of
type II fast glycolytic fibers into type I slow oxidative
fibers, the upregulation of miR-206 with decreased
expression of the Mef2 transcript in the 4-month reinner-
vated muscle, which presented type II fiber predomi-
nance 4 months after nerve microanastomosis, might
indicate the role of miR-206 in determining the fibertype
after peripheral nerve regeneration (Tables 10.9 and
10.10 provide the details of target genes for these altered
miRNAs and their functional role) (Jeng et al., 2009).
Chemical-induced peripheral neuropathy
Because of the need for complete eradication of cancer
cells, a higher dose of drugs are used in cancer chemo-
therapy (Table 10.11) which very often results in
chemical-induced peripheral neuropathy (CIPN), a dose-
limiting neurotoxic effect of chemotherapy, that can lead
to early cessation of cancer treatment (Wang et al., 2012).
This can result in an increased risk of relapses and
decreased survival rate. Biomarkers to assess the suscep-
tibility or tolerance level of such treatment approaches
can be very useful in making decisions about the choice,
dose, and duration of the drugs that can help those can-
cer patients. Inflammatory cascade activation, proinflam-
matory cytokine, upregulation, and neuroimmune
communication pathways play essential roles in the initi-
ation and progression of CIPN. Most notably, TNF-a, IL-
1b, IL-6, and CCL2 are involved in neuropathic pain.
These cytokines could be used as biomarkers for predict-
ing the onset of painful peripheral neuropathy and early
axonal damage (Wang et al., 2012). Reactive oxygen spe-
cies (ROS) have been shown to play a causal role in the
development and maintenance of paclitaxel-induced
pain (Fidanboylu et al., 2011) and hence the levels of ROS
can be used as a biomarker for certain drugs. Xiao et al.
(2012) demonstrated highly similar data on mechano-
allodynia, mechano-hyperalgesia, and cold-allodynia
that have a delayed onset, gradually increasing severity,
a distinct delay to peak severity, and significant increase
in the incidence of swollen and vacuolated mitochondria
in peripheral nerve axons, but not in their Schwann cells
in oxaliplatin and paclitaxel treatments. They attributed
this commonality to their mitotoxic effect on primary
afferent neurons.
Biomarkers of altered axonal transport
Axon transport mechanisms play a major role in trans-
porting nutrients, organelles and other molecules
towards the presynaptic terminals by a process called
anterograde transport, while the retrograde transport is
186 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS

TABLE 10.10 Target genes of modulated microRNAs by peripheral denervation and reinnervation

Gene Symbol Accession no. Gene Description

Akap9 NM_001037093 A kinase (PRKA) anchor protein (yotiao) 9
Ampd1 NM_138876 Adenosine monophosphate deaminase 1 (isoform M)
Atp2c1 NM_131907 ATPase, Ca11 transporting, type 2C, member 1
Cacnb2 NM_053851 Calcium channel, voltage-dependent, beta 2 subunit
Cenpc1 NM_001004098 Centromere protein Cl
ChaC2 NM_001025016 Cation transport regulator homolog 2
Cit NM_001029911 Citron (rho-interacting, serine/threonine kinase 21)
Cltc NM_19299 Clathrin, heavy chain (He)
Ddx5 NM_001007613 Ddx5 gene
Echdc1 NM_001007734 Enoyl coenzyme A hydratase domain containing 1
Elf2 NM_001012181 E74-like factor 2
G0s2 NM_001009632 G0/G1 switch gene 2
G6pdx NM_017006 Glucose-6-phosphate dehydrogenase X-linked
Gas6 NM_057100 Growth arrest specific 6
Gdnf NM_019139 Glial cell line derived neurotrophic factor
Gp1bb NM_053930 Glycoprotein lb, beta polypeptide
Havcr2 NM_001100762 Hepatitis A virus cellular receptor 2 homolog
Hnrpu NM_057139 Heterogeneous nuclear ribonucleoprotein U
Hspb1 NM_031970 Heat shock protein 1
Hspd1 NM_022229 Heat shock protein 1 (chaperonin)
Il2rg NM_080889 Interleukin 2 receptor, gamma
Klhl24 NM_181473 Kelch-like 24
LOC292666 NM_001025641 Similar to pregnancy-specific beta 1-glycoprotein
LOC299282 NM_182474 Similar to serine protease inhibitor
LOC498749 NM_001034947 Similar to putative TRAP and TNF receptor associated protein
LOC500110 NM_001024327 Similar to zinc finger protein EZI
LOC680782 XM_001058844 Similar to signal peptidase complex subunit 3 homolog
Lsamp NM_017242 Limbic system-associated membrane protein
Mcf21 NM_053951 MCF.2 cell line derived transforming sequence-like
Mef2 NM_001014035 Myocyte enhancer factor 2
MGC108776 NM_001017466 Similar to Snf7 homolog associated with Alix 3
Mns1 NM_001007752 Meiosis-specific nuclear structural protein 1
Nexn NM_139230 Nexilin (Nexn), transcript variant s
Npy NM_012614 Neuropeptide Y
Nr3c1 NM_012576 Nuclear receptor subfamily 3, group C, member 1
Pdk4 NM_053551 Pyruvate dehydrogenase kinase, isoenzyme 4
Plp NM_030990 Proteolipid protein
Ppfibp2 NM_0011005S2 Protein tyrosine phosphatase, receptor-type, F interacting protein,
binding protein 2 (liprin beta 2)
Ptprd NM_019140 Protein tyrosine phosphatase, receptor type, D
Rasa1 NM_013135 RAS p21 protein activator 1
Rnf103 NM_053438 Ring finger protein 103
RT1-A2 NM_001008829 RT1 class 1a, locus A2
Serpina3k NM_012657 Serine (or cysteine) peptidase inhibitor, clade A, member 3K
Sgk NM_019232 Serum/glucocorticoid regulated kinase
Sox6 NM_001024751 SRY-box containing gene 6
Stau1 NM_053436 Staufen RNA binding protein homolog 1
Sv2b NM_057207 Synaptic vesicle glycoprotein 2b
Ubc NM_017314 Ubiquitin C
Usp33 NM_001017379 Ubiquitin specific peptidase 33
Wif1 NM_053738 Wnt inhibitory factor 1
Zfp131 NM_001100698 Zinc finger protein 131

Source: Jeng et al., 2009.

a process by which damaged organelles and recycled
plasma membrane (packed in endocytotic vesicles)
are transported back to the neuron cell body. There
are many major human neurodegenerative pathologi-
cal disorders, such as Alzheimer’s disease,
Parkinson’s disease, and amyotrophic lateral sclerosis
(ALS), that display axonal pathologies including
abnormal accumulations of proteins and organelles,
highlighting that disruption of axonal transport is an
early and perhaps causative event in many of these
 PNS BIOMARKER METHODOLOGY 187

TABLE 10.12 Genes involved in hereditary peripheral

TABLE 10.11 Chemotherapeutic agents causing peripheral
neuropathy
neuropathy
CMT2E
Platinum Compounds CMT2A
Cisplatin NF-L
Oxaliplatin Gigaxonin
Carboplatin Mitofusin
Taxanes MAP8
Paclitaxel MAP1B LC
Docetaxel G-protein Rab7
Vinca alkaloids RILP
Vincristine Dynein
Vinblastine Myotubularin-related protein 2 (MTMR2)
Vinorelbine MTMR13/set-binding factor 2
Other agents GDAP1
Bortezomib DNM2
Thalidomide Apolipoprotein C-I precursor (Diabetic PNS)
Lenalidomide
Colistin

Anti-alcohol Drugs Epigenetic biomarkers of axonal regeneration

Disulfiram
Anticonvulsants
Phenytoin (Dilantins)
Heart or Blood Pressure Medications
Amiodarone
Hydralazine
Perhexiline
Infection Fighting Drugs
Metronidazole (Flagyls)
Nitrofurantoin
Thalidomide
INH (Isoniazid)
Skin Condition Treatment Drugs
Dapsone
diseases. Several studies on neurodegenerative disor-
ders have shown that defects in axonal transport are
early pathogenic events in a number of human neuro-
degenerative diseases (De Vos et al., 2008). Similarly,
defective axonal transport has been shown to be
involved in organophosphate-induced delayed neuro-
toxicity (OPIDN) (Damodaran et al., 2011).
Disruption to axonal transport can occur via a num-
ber of routes (De Vos et al., 2008). These disruptions
include damage to (a) molecular motors, (b) microtu-
bules, (c) cargoes (such as inhibiting their attachment
to motors), and (d) mitochondria, which supply
energy for molecular motors (De Vos et al., 2008). At
cellular and tissue level axonal degeneration accom-
panied by synergistic neuronal and glial cell death
pathways (e.g. apoptosis, necrosis, autophagy) have
been implicated as major factors in neurotoxicity
(Damodaran et al., 2011).
Gadd45 alpha, one of the key regulators of epigenetic
modification of gene expression, is highly upregulated
after axonal injury. In parallel, many genes involved in
axonal elongation and synaptic activity are demethy-
lated upon axonal injury. Further studies are required
to verify whether it is possible to use epigenetic manip-
ulation to bring the adult neurons to a more “imma-
ture” state to promote better regeneration (Hoke et al.,
2013).
Biomarkers from studies on inherited
peripheral neuropathies
A number of peripheral neuropathies are inherited, of
which a number of them are associated with documen-
ted defects in axonal transport (De Vos et al., 2008).
Table 10.12 lists genes involved in hereditary peripheral
neuropathy, discussed in this section. In Charcot-Marie-
Tooth disease (CMT) type 2E, caused by mutations in
the neurofilament light gene (NF-L), mutant protein
aggregates perturb the cytoskeletal network (particu-
larly microtubules), and disrupt the normal localization
of mitochondria (Perez-Olle et al., 2004). The overex-
pression of CMT2E-linked NF-L leads to impaired axo-
nal transport in vitro (Brownlees et al., 2002) and in vivo
(Dequen et al., 2010). Furthermore, in a mouse model of
CMT2A caused by mutations in mitofusin, axonal trans-
port of mitochondria was compromised (Baloh, 2007),
and in giant axonal neuropathy, caused by mutations in
gigaxonin, deletion of gigaxonin in mouse led to
impaired retrograde axonal transport (Ding et al., 2006).
This impairment of axonal transport appears not only
true for axonal but also for demyelinating hereditary
neuropathies. Indeed, in an animal model of X-linked
demyelinating/type I Charcot-Marie-Tooth neuropathy
188 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
(CMT1X), an inherited peripheral neuropathy caused by
mutations in the gap junction protein connexin32,
Vavlitou et al. (2010) observed reduced axonal transport
and axonal pathology before demyelination occurs.
Dynein dysfunction appears directly involved in at least
a subset of these diseases. First, the deletion in gigaxo-
nin in mouse leads to increased MAP8 and MAP1B LC
levels. This, in turn, traps dynein and impairs axonal
retrograde transport. More directly, mutations in the
G-protein Rab7 trigger CMT2B, leading to constitutive
activity of mutant proteins. Rab7 controls dynein/
dynactin recruitment to lysosomes and late endosomes
through its effector protein RILP and was demonstrated
to be essential for axonal retrograde transport
(Deinhardt et al., 2006). It is thus probable that CMT2B
mutations affect, in a still incompletely understood
manner, dynein dependent transport.
Some proteins implicated in demyelinating CMT,
peripheral myelin protein 22, protein zero (P0), and con-
nexin32 (Cx32/GJB1), are crucial components of myelin
(Shames et al., 2003). Periaxin is involved in connecting
myelin to the surrounding basal lamina. Early growth
response 2 (EGR2) and Sox10 are transcriptional regula-
tors of myelin genes. Mutations in the small integral
membrane protein of lysosome/late endosome, the
myotubularin-related protein 2 (MTMR2), and
MTMR13/set-binding factor 2, are involved in vesicle
and membrane transport and the regulation of protein
degradation. Pathomechanisms related to alterations of
these processes are a widespread phenomenon in demy-
elinating neuropathies because mutations of myelin
components may also affect protein biosynthesis, trans-
port, and/or degradation. Related disease mechanisms
are also involved in axonal neuropathies although there
is considerably more functional heterogeneity. Some
mutations, most notably in P0, GJB1, ganglioside-
induced differentiation-associated protein 1 (GDAP1),
neurofilament light chain (NF-L), and dynamin 2
(DNM2), can result in demyelinating or axonal neuropa-
thies introducing additional complexity in the patho-
genesis. Often, this relates to the intimate connection
between Schwann cells and neurons/axons leading to
axonal damage even if the mutation-caused defect is
Schwann-cell-autonomous. This mechanism is likely for
P0 and Cx32 mutations and provides the basis for the
unifying hypothesis that, also, demyelinating neuropa-
thies develop into functional axonopathies. In GDAP1
and DNM2 mutants, both Schwann cells and axons/
neurons might be directly affected. NF-L mutants have
a primary neuronal defect but also cause demyelination.
The major challenge ahead lies in determining the indi-
vidual contributions by neurons and Schwann cells to
the pathology over time and to delineate the detailed
molecular functions of the proteins associated with
CMT in health and disease.
The hereditary sensory and autonomic neuropathies
(HSAN) encompass a number of inherited disorders
that are associated with sensory dysfunction (depressed
reflexes, altered pain, and temperature perception) and
varying degrees of autonomic dysfunction (gastroesoph-
ageal reflux, postural hypotension, and excessive sweat-
ing). Numerical classification of four distinct forms of
HSAN has been proposed. Each HSAN disorder is
likely caused by different genetic errors that affect spe-
cific aspects of small fiber neurodevelopment, which
result in variable phenotypic expression (Axelrod and
Gold-von Simson, 2007). Types I to V have been shown
to have mutations in the following genes: SPTLC1,
HSN2, IKBKAP, NTRK1 (TRKA), and NTRK1.
Wallerian degeneration (WD) is one of the most ele-
mentary reactions of the peripheral nervous system. It
occurs when nerve fiber continuity is interrupted
through traumatic, toxic, ischemic, or metabolic events.
Axonal injury triggers a cascade of events including,
for example, breakdown of the blood nerve barrier
(Mellick and Cavanagh,1968), proliferation of Schwann
cells and recruitment of circulating macrophages
(Taskinen and Röyttä, 1997), and reorganization of the
endoneurial space and changes in the endoneurial
extracellular matrix (ECM) components, as well as ele-
vation of neurotrophin and cytokine production.
Neurodegenerative diseases characterized by brain, spi-
nal cord, and PNS involvement often show widespread
accumulations of tau aggregates. Leroy et al. (2007) gener-
ated a transgenic mouse line (Tg30tau) expressing a human
tau protein bearing two pathogenic mutations (P301S and
G272V) in the forebrain and the spinal cord. These mice
developed age-dependent brain and hippocampal atrophy,
central and peripheral axonopathy, progressive motor
impairment with neurogenic muscle atrophy, and neurofi-
brillary tangles and had decreased survival. Axonal spher-
oids and axonal atrophy developed early before
neurofibrillary tangles. Neurofibrillary inclusions devel-
oped in neurons at 3 months and were of two types, sug-
gestive of a selective vulnerability of neurons to form
different types of fibrillary aggregates. A first type of tau-
positive neurofibrillary tangles, more abundant in the fore-
brain, were composed of ribbon-like 19-nm-wide filaments
and twisted paired helical filaments. A second type of tau
and neurofilament-positive neurofibrillary tangles, more
abundant in the spinal cord and the brainstem, were com-
posed of 10-nm-wide neurofilaments and straight 19-nm
filaments. This Tg30tau model thus provides evidence that
axonopathy precedes tangle formation and that both
lesions can be dissociated from overt neuronal loss in
selected brain areas but not from neuronal dysfunction.
This study also established that the size, shape and width
of the tangles and the biochemical component can be dif-
ferent in various parts of the nervous system (Leroy et al.,
2007). Thus, these aspects could be useful as biomarkers
 PNS BIOMARKER METHODOLOGY
that define the pathology and disease progression. They
have also shown the appearance and disappearance of var-
ious types of tau epitopes (phosphorylation independent
and dependent as well as conformational epitopes) as a
function of aging (1, 3, and 12 months), thus establishing
the potential of using phosphorylation and conformational
alternates as potential biomarkers to evaluate disease pro-
gression in both CNS and PNS.
Cytoplasmic dynein 1 is the major molecular motor
moving cargoes such as mitochondria, organelles, and
proteins towards the minus end of microtubules.
Dynein is involved in multiple basic cellular functions,
such as mitosis, autophagy, and structure of endoplas-
mic reticulum and Golgi, but also in neuron-specific
functions, in particular retrograde axonal transport.
Dynein is regulated by a number of protein complexes,
notably by dynactin. Several studies have supported
indirectly the involvement of dynein in neurodegenera-
tion associated with Alzheimer’s disease, Parkinson’s
disease, Huntington’s disease, and motor neuron dis-
eases (Eschbach and Dupuis, 2011). First, axonal trans-
port disruption represents a common feature occurring
in neurodegenerative diseases. Second, a number of
dynein-dependent processes, including autophagy or
clearance of aggregation-prone proteins, are found
defective in most of these diseases. Third, a number of
mutant genes in various neurodegenerative diseases are
involved in the regulation of dynein transport. This
includes, notably, mutations in the P150Glued subunit
of dynactin that are found in Perry syndrome and
motor neuron diseases. Interestingly, gene products that
are mutant in Huntington’s disease, Parkinson’s disease,
motor neuron disease, or spinocerebellar ataxia are also
involved in the regulation of dynein motor activity or of
cargo binding (Eschbach and Dupuis, 2011).
Biomarkers of demyelination injuries
Axonal degeneration contributes to permanent neurolog-
ical disability in inherited and acquired diseases of mye-
lin affecting PNS and CNS (Kiryu-Seo et al., 2010). The
role of mitochondrial dysfunction in axonal degeneration
through the cyclic events of myelination, demyelination,
or remyelination has been proposed. Demyelination has
been shown to induce ATFm 3 (activating transcription
factor 3) in DRG neurons. Knockdown of neuronal ATF3
by shRNA abolished the demyelination-induced increase
in axonal mitochondrial transport and increased nitro-
tyrosine immunoreactivity in axonal mitochondria, sug-
gesting that neuronal ATF3 expression and increased
mitochondrial transport protect demyelinated axons
from oxidative damage. In response to insufficient ATP
production, demyelinated axons increase the size of sta-
tionary mitochondrial sites and thereby balance ATP
189
production with the increased energy needs of nerve
conduction. The induction of ATF3 supports an axonally
initiated/derived signal that is transported to the neuro-
nal cell body. In this regard, ATF3 expression is modu-
lated by retrograde axonal transport of JNK (Lindwall
and Kanje, 2005). Axonally-derived signaling pathways
can also be initiated by neurotrophins and the myelin
proteins, NOGO, MAG and oligodendrocyte-myelin gly-
coprotein, and involve activation of cAMP and protein
kinase A (Hannila and Filbin, 2008). Similar pathways
and additional transcription factors may be modulated
following demyelination in both PNS and CNS.
Mitochondrial function in Schwann cells has been shown
to be important in maintenance of axons, and disruption
of mitochondria in Schwann cells also can lead to axonal
degeneration (Viader et al., 2011). Hence, gene products
involved in the mitochondrial structure and function
may qualify for biomarker status.
Biomarkers of neuromuscular junction injuries
Neurotoxicants that act at the neuromuscular junction can
be divided into two classes: Those that alter the pheno-
typic synthesis, storage, or release of acetylcholine (ACh)
and those that disrupt the postsynaptic function of the
effector cells, usually associated with the recognition of
ACh by its target receptor, activation of conductances
through the receptor-associated ion channel, or impaired
hydrolysis of ACh (Atchison and Spitsbergen, 1994).
There are two specific steps in the neurotransmission
at the neuromuscular junction such as: (a) presynaptic
processes that are associated directly with the synthesis
and release of neurotransmitter, which occur in the pre-
synaptic nerve terminal, and (b) post-synaptic processes
that are associated with the binding of transmitter to its
receptor sites in the postsynaptic membrane, resulting
in opening of ion channels, thereby leading to postsyn-
aptic polarization. The neuromuscular junction has been
the specific target for neurotoxins such as botulinum
toxin or many of the cholinesterase inhibitors.
TABLE 10.13 Surrogate tissue markers
Blood:
Erythrocyte acetyl cholinesterase (AChE) for organophosphates
Free erythrocyte protoporphyrin (FEP) for lead
Lymphocyte neurotoxicity target enzyme (NTE) for
organophosphates
Blood aminolevulinic acid dehydratase (ALA-D) for intermittent
porphyria
Carboxyhemoglobin (CO-Hb) for carbon monoxide
190 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS

TABLE 10.14 Candidate biomarker genes connected with inherited PNS disorders
The Candidates that were Genotyped for PNS Disorders:

ACCN2, ACE, ACTB, ACTGl, ACTRlA, ACTRlB, AD0RA2A, ADRA2B, AGT, AGTRl, AKRlBl, AKTl, AKT2, APC, ARPI l, AXINl, BMF,
CACNAlA, CACNAlB, CAPZAl, CAPZA2, CAPZA3, CAPZB, CD86, COMT, CTLA4, CTNNBl, CTSS, CYP3A4, CYP3A5, DCTNl, DCTN2,
DCTN3, DCTN4, DCTN6, DNCLl, DNCL2A, DNM2, DVLl, DVL2, DVL3, DYNClHl, DYNClIl, DYNC1I2, DYNClLIl, DYNC1LI2, DYNC2H1,
DYNC2LI1, DYNLL2, DYNLRB2, ECGFl, EGR2, FGD4, FIG4, GARS, GCHl, GDAPl, GJBl, GJB2, GJB3, GJEl, GLRA3, GLS2, GLUL, GSK3A,
GSK3B, HAPl, HSN2, HSPBl, HSPB8, HTRlB, IKBKAP, IL6, KIFlA, KIFlB, KIF3A, KIF3B, KIF5A, KIF5B, KIF5C, LITAF, LMNA, MAPKl,
MAPKlO, MAPKI l, MAPK 12, MAPKl 3, MAPK14, MAPK3, MAPK4, MAPK6, MAPK7, MAPK8, MAPK9, MClR, MFN2

Other Genes Connected with Inherited PNS Disorders:

MPZ, MTMR2, NDRGl, NEFL, NFE2L2, NGFB, NPY, NR 112, NTRKl, OPRDl, OPRKl, OPRLl, OPRMl, PLPl, PMP22, PNOC, POLG, POLG2,
PONl, PRPSl, PRX, PSMBl, PSMBlO, PSMB2, PSMB3, PSMB4, PSMB5, PSMB6, PSMB7, PSMB8, PSMB9, PTGERl, PTGER2, PTGER3,
PTGER4, PTGSl, PTGS2, SBF2, SCN3A, SCN9A, SH3TC2, SLC12A6, SPTBNl, SPTBN2, SPTBN4, SPTBN5, SPTLCl, SURFl, TCFl, TCF4, TH,
TNF, TRAK2, TRPVl, TRPV4, TTR, VIP, WNTl, WNTlOA, WNTlOB, WNTI l, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A,
WNT5B, WNT6, WNT7A, WNT8A, WNT8B, WNT9A, WNT9B and YARS

Biomarkers from surrogate tissue analysis for
peripheral nervous system disorders
Surrogate tissue analysis can be very useful for monitoring
toxicant exposure and effect, monitoring disease develop-
ment and progression, and drug efficacy testing (Tang
et al., 2006). There are many accessible tissues (e.g. blood,
urine, cerebrospinal fluid, cord blood, buccal cells, or milk)
that can be used as a surrogate tissue (Table 10.13). Tang
et al. (2001) showed the specific global gene expression pro-
files of blood samples from animals that had various neu-
rological disorders such as ischemic stroke, hemorrhagic
stroke, kainite-induced seizures, hypoxia, and insulin-
induced hypoglycemia. Most of the components of the
PNS and CNS may not be accessible for tissue or cell pro-
curement. Blood has the genetic complement of the PNS
and CNS structures; in addition, signaling and other path-
ways of PNS require abundant receptors and other special-
ized molecules and cells that are very often represented in
the peripheral blood. Furthermore, recent studies from
human disease conditions such as neurofibromatosis type
1 (NF1), Tourette syndrome, and anticonvulsant drugs in
pediatric epilepsy indicated the existence of quantifiable
markers while highlighting the need for more studies to
optimize the methodology for peripheral tissue biomarker
development (Tang et al., 2006). Global gene expression
studies using whole blood from patients with post-
traumatic stress, stroke, and migraine yielded valuable
data on candidate biomarkers (Segman et al., 2005; Moore
et al., 2005). Measuring viral load as a surrogate marker for
HIV infection and development of antiretroviral therapies
has been proposed and practiced (Hoke et al., 2013).
Another potential surrogate marker is the density of intrae-
pidermal nerve fibers, which correlates with neuropathy
severity as gauged by the total neuropathy score, sural sen-
sory nerve action potential amplitudes, and quantitative
sensory testing using toe cooling and vibration detection
thresholds, and with neuropathic pain as measured by the
Gracely visual analog scale (Ebenezer et al., 2007).
DNA biomarkers of toxicity in the peripheral
nervous system
Recently, Johnson et al. (2011) showed that an indivi-
dual’s risk of developing a peripheral neuropathy after
thalidomide treatment can be mediated by polymorph-
isms in genes governing repair mechanisms and inflam-
mation in the PNS. They found an association between
TrPN (thalidomide related peripheral neuropathy) with
SNPs (single nucleotide polymorphisms) ABCA1
(rs363717), ICAM1 (rs179999), PPARD (rs2076169),
SERPINB2 (rs6103), and SLC12A6 (rs7164902). They
analyzed DNA samples from 1495 patients and selected
SNPs from functional regions of 964 genes spanning 67
molecular pathways thought to be involved in the path-
ogenesis, treatment response, and other adverse effects.
They have conducted similar studies with vincristine to
verify the specificity of the data obtained for TrPN,
thereby establishing the usefulness of SNP screening for
personalized therapy approaches for victims of toxicant
exposure. Table 10.14 provides the list of candidate
genes genotyped for mutations in PNS disorders.
Biomarkers of cholinergic system and its
relevance to the peripheral nervous system
Acetylcholine (ACh) is widely distributed in the nervous
system and has been implicated as playing a critical role
in cerebral cortical development, cortical activity, control-
ling of cerebral blood flow, and the sleep wake cycle as
well as in modulating cognitive performances and learn-
ing and memory processes (Schliebs and Arendt, 2011).
Changes in the cholinergic system during aging and in
AD have been documented by assessing the major func-
tional components of cholinergic cells and signaling; the
ACh synthesizing and degrading enzymes choline acetyl-
transferase (ChAT) and acetylcholinesterase (AChE),
respectively; the vesicular ACh transporter (VAChT) that
 PNS BIOMARKER METHODOLOGY
transports ACh into the vesicles; the cholinergic musca-
rinic receptors (mAChRs) and nicotinic ACh receptors
(nAChRs) for synaptic signaling; as well as the require-
ment of cholinergic neurons to receive neurotrophic sup-
port by NGF mediated through high- (trkA) and low-
affinity (p75NTR) receptors for survival (Schliebs and
Arendt, 2011).
Peripheral nervous system biomarkers for
exposure to organophosphates
Organophosphate insecticides share a common mecha-
nism of toxicity, through inhibitory effects on cholines-
terase enzymes in the nervous system.
Organophosphates undergo chemical changes in the
environment as well as in the human body. It is neces-
sary to understand these changes in order to measure
and interpret biomarkers of exposure to organopho-
sphates. One chemical reaction that occurs in humans is
a transformation at the double bond of the central phos-
phorus atom from sulfur to oxygen. This metabolic reac-
tion takes place in the liver and results in activation of
the organophosphate to a more potent inhibitor of cho-
linesterase enzymes (CDC, 2009). Hydrolysis of the
organophosphates yields a dialkylphosphate and the
leaving group, which do not inhibit cholinesterase
enzymes and can be measured as biomarkers of expo-
sure to organophosphates, as the metabolites are subse-
quently eliminated from the body in the urine (CDC,
2009). An individual with acute symptomatic overexpo-
sure to organophosphates will usually have an abnor-
mally low level of activity of cholinesterase enzymes
measured in the serum (as butyrylcholinesterase, also
known as pseudocholinesterase) or in red blood cells
(as RBC cholinesterase, which is more closely correlated
with acetylcholinesterase activity in the nervous sys-
tem). Paraoxonase (or PON1) enzyme is responsible for
the hydrolysis and deactivation of organophosphates.
Polymorphisms in the PON1 gene exist in humans add-
ing variability in OP-exposure response. In a recent
study on the associations between OP pesticide use and
PNS function, by administering PNS tests to 701 male
pesticide applicators in the Agricultural Health Study
(AHS), it was shown that long-term exposure to OP pes-
ticides is associated with signs of impaired PNS func-
tion among pesticide applicators (Starks et al., 2012).
Biomarkers of organophosphorus-ester induced
delayed neurotoxicity
Organophosphorus-ester induced delayed neurotoxicity
(OPIDN) is a neurodegenerative disorder characterized
by ataxia progressing to paralysis with a concomitant
central and peripheral distal axonopathy (Damodaran
191
et al., 2011). Excessive accumulation of aggregated micro-
tubules and neurofilaments in the distal part of the large
myelinated axons is a major pathological hallmark of
OPIDN (Figure 10.3). As the disorder progresses, neuro-
filaments are partially matted and comparatively rari-
fied, but microtubules are better preserved and visible
for a long time in OPIDN (Damodaran, 2009). Although
the exact mechanism of OPIDN development is yet to be
determined, there have been suggestions that OPIDN is
related to specific inhibition of a particular protein
known as neuropathy target esterase (NTE). To add to
the complexity, other suggested pathways include cho-
linergic inhibition of AChE and differential alterations of
several upstream and downstream molecules, and their
associated biochemical machinery significantly contri-
butes to the pathophysiology of OPIDN (Damodaran,
2009). AChE inhibitors (e.g. sarin, DFP) exposure may
alter the regulation of the cholinergic system differen-
tially, depending on the dose, duration, and mode of
exposure as evidenced by numerous studies, thus affect-
ing various functions such as muscle function, cognition,
and sleep (Damodaran, et al., 2003; Damodaran et al.,
2006a,b; Damodaran, 2009).
Various hormonal or chemical stimuli cause increased
concentration of cAMP in the cell, but the majority of
cellular responses resulting from increased level of
cAMP are mediated by PKA. It is generally recognized
that phosphorylation/dephosphorylation of target pro-
teins is involved in early cellular functions such as pro-
liferation, differentiation, apoptosis, or degeneration by
toxic chemicals (Gupta et al., 1997, 2000). The cAMP
response element binding protein, CREB, is a transcrip-
tion factor. CREB is phosphorylated by PKA and initi-
ates the synthesis of target proteins. Increased
phosphorylation of several cytoskeletal proteins and
alterations in the levels of several enzymes and proteins
were reported (Abou-Donia and Nomeir, 1986).
Alterations in the levels of mRNA of CamKinase II
alpha sub-unit (Gupta et al., 1997), neural filament trip-
let proteins (Gupta et al., 1997), GFAP, vimentin
(Damodaran et al., 2001), alpha tubulin (Damodaran
et al., 2001), beta tubulin subtypes (Damodaran et al.,
2009), and GAPDH (Damodaran et al., 2002b) have been
shown. Immediate early induction of c-fos (Gupta et al.,
2000) and c-jun (Damodaran and Abou-Donia, 2000b)
has been demonstrated. Furthermore, differential induc-
tion of PKA, CREB, and p-CREB (Gupta and
Abou-Donia, 1995; Damodaran, 2009) in DFP-treated
hen brain at some time points has been found. The pro-
tein levels of protein kinase C (PKC), CaM kinase II,
and several phosphatases (i.e. phosphatase 1 (PP1), and
phosphatase 2A (PP2A), phosphatase 2B (PP2B)) in the
spinal cord of DFP-treated hens after 1, 5, 10, and 20
days were estimated and their potential roles discussed
(Gupta and Abou-Donia, 1995).
192 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
In a comparatively recent study, sarin (molecular
structural analogue of DFP) exposure initiated several
validated and hitherto unreported pathways immedi-
ately (within 15 minutes of exposure) and a significant
number of important genes (pathways) showed persis-
tently modified levels at 2 hours and 3 months post
treatment (Damodaran et al., 2006a,b). Furthermore, val-
idation and candidate gene studies showed a similar
overall pattern for both DFP (exposure to hens) and
sarin (exposure to rats), in spite of the differences in
species tested and structure of the analogues used
(Damodaran et al., 2000a; Damodaran and Abou-Donia,
2000b; Damodaran et al., 2002a,b,c,d). Thus, there are
common and evolutionarily conserved pathways (gene
clusters) that are sensitive to AChE and NTE inhibition
and probably other esterases’ inhibition, irrespective of
the test organism and differences in the structures of
OPs tested. These organophosphorus esters-exposure
related physiological genomic and nongenomic effects
(such as hormonal, inflammatory, and other types) very
often persist for a long time, if appropriate remedial
measures are not taken (Damodaran, 2009).
AUTONOMIC NERVOUS SYSTEM
Autonomous nervous system (ANS) activity may be cat-
egorized into nine measurable physiological systems or
pathways, activated through sympathetic and parasym-
pathetic mechanisms. These pathways provide a frame-
work for a comprehensive review of stress reactivity
measures.
Measuring autonomic nervous system activity
Approximately 50 tools identified according to the auto-
nomic nervous system (ANS) pathway are listed in the
ANS Measures Table in Appendix A of the monograph
published by Defense Centers of Excellence for
Psychological Health and Traumatic Brain Injury (web
site: www.dcoe.health.mil) (Brierley-Bowers et al., 2011).
Some of these tools, such as the electrocardiogram
(EKG), are established and widely used. The utility of
each tool is dependent upon its purpose of use, target
environment, and the sensitivity of measurement
needed. The three ANS pathways most often used to
measure the impact of mind-body skills, as seen in
Table 2 on page 9 of the above mentioned monograph,
are cardiac, vascular, and respiratory (Brierley-Bowers
et al., 2011). These are the ANS pathways that physi-
cians monitor via the vital signs (blood pressure, heart
rate, temperature, and respiratory rate).
Cardiac measurement
Cardiac activity, one of the most commonly assessed
ANS pathways, is among the most sensitive areas to
ANS changes. Cardiac activity is measured via heart
rate, with increased rate signifying activation of the
SNS, and via heart rate variability. One widely used
measure is manually taking the pulse; it offers a nonin-
vasive, nonpainful, relatively quick measure of heart
rate. An EKG is a standard measure of the electrical
activity, i.e. the depolarization of the heart during each
beat over time. Sensors placed on the skin using two,
three, five, or twelve leads capture electrical activity
(Brierley-Bowers et al., 2011).
Vascular measurement
The vascular or circulatory system is measured via tem-
perature and blood pressure. As part of the stress
response when the SNS is activated, blood vessels in
the limbs constrict, causing a decrease in temperature in
the extremities while increasing the blood flow to the
muscles, brain, lungs, and heart, increasing core temper-
ature. While thermometers can accurately measure
body temperature, tools which may measure the
decreased temperature of the extremities (i.e. stress)
include auxiliary sensors such as those used on the
intelligent garment and biodots. Blood pressure, the
measure of the pressure of the blood within the arteries,
is a product of heart rate, intravascular volume status,
cardiac muscle contractility, and systemic vascular resis-
tance. As part of a response to a stressor arterial blood
pressure increases. Blood pressure can be measured
through the following three methods: (i) the ausculta-
tory method, (ii) oscillometric method, and (iii) photo-
plethysmographic method (Brierley-Bowers et al., 2011).
Respiratory measurement
The respiratory system includes the inhalation of air,
which is then channeled into the alveoli of the lungs to
deliver oxygen to the circulatory system, and the exha-
lation of carbon dioxide. When the SNS is activated due
to exposure to a stressor, breath rate increases and
breaths become shallower. There are two methods of
monitoring the respiratory system: direct and indirect.
One form of direct measurement includes spirometers.
Spirometers measure the amount and rate of airflow
over a period of time by having the individual breathe
into a device, which visually illustrates the volume of
air displaced, much like a breathalyzer. Indirect mea-
sures of respiration include the use of a plethysmo-
graph. A pulmonary plethysmograph measures the
total lung capacity and functional residual capacity of
the lungs (Brierley-Bowers et al., 2011).
 AUTONOMIC NERVOUS SYSTEM
Galvanic skin response measurement
The galvanic skin response (GSR), one of the earliest
tools used in psychological research, offers a method of
measuring the electrical resistance of the skin. In
response to a stressor, GSR nonspecific increase and
basal resistance decreases. GSR is obtained by attaching
two leads to the skin and acquiring a base measure. As
an activity is performed, recordings are made from the
leads. Active GSR is when a current is passed through
the body and the resistance is thereby measured. GSR is
sensitive to immediate emotional arousal as well as gen-
eral mood or acute stress responses. The e-meter and
polygraph are also versions of a GSR which are widely
used (Brierley-Bowers et al., 2011)
Catecholamine measurement
Catecholamines, such as norepinephrine, epinephrine, and
cortisol, are hormones released during a stress response. In
response to a stressor, an endocrine signal cascade is initi-
ated primarily by the release of corticotropin-release factor
(CRF) from the paraventricular nucleus of the hypothala-
mus, resulting in increased secretion of glucocorticoids (i.e.
cortisol), mineralocorticoids, and androgens. As a result,
catecholamine metabolites, specifically vanillylmandelic
acid (VMA), can be used to gauge sympathetic activity.
Concentrations of catecholamine metabolites can be deter-
mined by measuring the following: VMA concentration in
urine samples; dihydroxyphenylglycol (DHPG),
3-Methoxy-4-hydroxyphenylglycol (MHPG), and VMA
levels in serum; and catecholamine and related metabolites
in saliva. Radioimmunoassay (RIA) with High
Performance, Enzyme-Linked Immunosorbent Assay
(ELISA), and Liquid Chromatography-Mass Spectrometry
(LC-MS) can be used (Brierley-Bowers et al., 2011).
Cortisol measurement
Cortisol, commonly referred to as the stress hormone, is
secreted by the adrenal glands and impacts the sympa-
thetic and parasympathetic nervous systems. Cortisol is
the most potent glucocorticoid produced and is secreted
by the adrenal cortex. Its release is activated through
the hypothalamus pituitary adrenal (HPA) axis, also
known as the limbic-hypothalamic-pituitary-adrenal
axis (LHPA axis). In response to a perceived threat,
CRF is released from the hypothalamus and binds to
receptors on the anterior pituitary lobe. This activates
release of adrenocorticotropic hormone (ACTH) from
the anterior pituitary. Cortisol is released when ACTH
binds to receptors in the adrenal medulla. In addition to
stress or trauma, many studies link cortisol levels to cir-
cadian rhythms, temperature, infection, exercise, and
obesity. Several tests are available to monitor cortisol
193
levels: 24-hour urine collection, blood testing, and saliva
sampling (Brierley-Bowers et al., 2011).
Pupillary response measurement
The pupillary response may occur for several reasons,
such as exposure to light, sexual stimulation, before sleep,
or in response to a stressor. Pupillary response varies the
size of the pupil of the eye via the iris dilator muscle,
which dilates in response to a stressor. Three widely
accepted and used measures of pupillary response are the
Colvard Pupillometer, Dynamic Binocular Infrared
Pupillometer, and the NeurOptics VIPt-200 Pupillometer.
The pupillometer is widely used to diagnose narcotic
influence, head injury, Parkinson’s disease, rheumatoid
arthritis, or lupus. It may also be used to assess degenera-
tion of eye tissue in people with severe diabetes. All the
tools have been shown to be satisfactory and reliable
(Brierley-Bowers et al., 2011).
Salivary amylase measurement
Most measures of salivary amylase, the enzyme that initi-
ates the chemical breakdown in the mouth, and gastroin-
testinal activity, have limited use. Acinar cells, which
produce salivary amylase, are innervated by sympathetic
and parasympathetic pathways. Some studies have found
sympathetic activity increases amylase synthesis, which
increases amylase concentration in the saliva, and para-
sympathetic activity increases saliva flow rate with no or
little effect on amylase synthesis. As these effects produce
an overall increase in absolute salivary amylase output,
they should be considered in relating salivary amylase to
stress reactivity (Brierley-Bowers et al., 2011).
Sweat analysis
One noninvasive and nonstressful approach to evaluat-
ing neural and immune systems is through collection of
sweat. The cutaneous sweat patch is commercially avail-
able and FDA approved. It is easily applied at any time
of day and can be worn for an extended period of time
with minimal discomfort. After collection, plasma and
sweat neuroimmune biomarkers are detected by recy-
cling immunoaffinity chromatography (RIC) coupled
with laser-induced fluorescence (Marques et al., 2010).
Preliminary data in a population of women with major
depressive disorder (MDD) showed elevated sweat
levels of pro-inflammatory cytokines, sympathetic neu-
ropeptides (neuropeptide-Y [NPY]) and pain-related
neuropeptides (substance P [SP], calcitonin gene-related
peptide [CGRP]), but decreased parasympathetic (vaso-
active intestinal peptide [VIP]) neuropeptides relative to
controls, which strongly correlated with plasma levels
(Marques et al., 2010).
194 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
ROLE OF PERIPHERAL NERVOUS
SYSTEM IN PARKINSON’S DISEASE
AND LEWY BODY DISEASE
Autonomic nervous system involvement occurs at early
stages in both Parkinson’s disease (PD) and incidental
Lewy body disease (ILBD), and affects the sympathetic,
parasympathetic, and enteric nervous systems (ENS)
(Cersosimo and Benarroch, 2012). It has been proposed
that alpha-synuclein (α-SYN) pathology in PD has a dis-
tal to proximal progression along autonomic pathways.
The ENS is affected before the dorsal motor nucleus of
the vagus (DMV), and distal axons of cardiac sympa-
thetic nerves degenerate before there is loss of paraver-
tebral sympathetic ganglion neurons. Consistent with
neuropathological findings, some autonomic manifesta-
tions such as constipation or impaired cardiac uptake of
norepinephrine precursors occur at early stages of the
disease even before the onset of motor symptoms.
Biopsy of peripheral tissues may constitute a promising
approach to detect α-SYN neuropathology in autonomic
nerves and a useful early biomarker of PD. Parkinson’s
disease (PD) is among the most frequent neurodegener-
ative disorders associated with aging. Over the past
decade, advances in genetics and neuropathology have
provided further insight into the etiopathogenesis of
PD. Several nonmotor manifestations, such as auto-
nomic, sleep, and olfactory dysfunction, may occur at
early stages of disease and precede the onset of motor
symptoms. Strong evidence indicates that autonomic
involvement in PD starts in the periphery. Both its early
involvement and the accessibility of peripheral tissues
make the peripheral autonomic system an attractive tar-
get for detection of early biomarkers of the disease.
Cerebrospinal-fluid-based kinetic biomarkers of axonal
transport in monitoring neurodegeneration of PD
patients and animal model systems have been shown to
be valuable for this purpose (Fanar et al., 2012).
Neural stress system of PNS
The two main arms of the stress system include the
autonomic nervous system (ANS) and the
hypothalamic-pituitary-adrenal (HPA) axis. These two
neural stress systems coordinate the response of many
other physiological systems to a stressor, including the
immune and cardiovascular systems, bringing the body
back to homeostasis. The nervous and immune systems
communicate with each other in a bidirectional manner.
Collection of sweat and saliva, and measurement of
heart rate variability are noninvasive methods that can
be applied to evaluate neuroimmune interactions.
Levels of neuropeptides, such as NPY, VIP, SP, and
CGRP, were shown to have tight correlation with levels
in plasma in patients with a history of depression
(Marques-Deak et al., 2006). The sympathetic and para-
sympathetic (vagus nerve, mediated by ACh) branches
of the ANS play an important role in the regulation of
the cardiovascular system and the immune system,
where the parasympathetic nervous system has been
shown to have an anti-inflammatory influence on the
immune system. A prominent circadian variation in
HRV, with significant increases during the night and
decreases during the day, is observed in healthy indivi-
duals. Previous studies have shown that this increase in
nighttime HRV is blunted by acute stress, as well as
conditions such as chronic alcoholism. Decreased HRV,
indicative of reduced parasympathetic vagal tone, is an
independent risk factor for morbidity and mortality.
BIOMARKERS OF ENTERIC NERVOUS
SYSTEM
Glutamate is the major excitatory neurotransmitter in the
enteric nervous system (ENS). Prolonged stimulation of
enteric ganglia by glutamate caused necrosis and apo-
ptosis in enteric neurons. Acute and delayed cell deaths
were observed. Excitotoxicity was more pronounced in
cultured enteric ganglia than in intact preparations of
bowel, presumably because of a reduction in glutamate
uptake. Glutamate immunoreactive neurons were found
in cultured myenteric ganglia, and a subset of enteric
neurons expressed NMDA (NR1, NR2A/B), AMPA
(GluR1, GluR2/3), and kainate (GluR5/6/7) receptor
subunits. Glutamate receptors were clustered on enteric
neurites. Stimulation of cultured enteric neurons by kai-
nic acid led to the swelling of somas and the growth of
varicosities (“blebs”) on neurites. Kainic acid also pro-
duced a loss of mitochondrial membrane potential in
cultured enteric neurons at sites where blebs tended to
form. These observations demonstrate, for the first time,
excitotoxicity in the ENS and suggest that overactivation
of enteric glutamate receptors may contribute to the
intestinal damage produced by anoxia, ischemia, and
excitotoxins present in food (Kirchgessner et al., 1997).
EMERGING AVENUES OF PNS
BIOMARKER DEVELOPMENTS:
EXOSOMES
Most, if not all, types of mammalian cells release small
membranous vesicles known as exosomes. In addition
to their protein content these vesicles have recently
 CONCLUDING REMARKS AND FUTURE DIRECTIONS
been shown to contain messenger RNA (mRNA) and
microRNA (miRNA) species. Roles for these vesicles
include cell cell signaling, removal of unwanted pro-
teins, and transfer of pathogens, such as prions,
between cells. As exosomes can be isolated from circu-
lating fluids such as serum, urine, and cerebrospinal
fluid, they provide a potential source of biomarkers for
neurological conditions. Exosomes have been proposed
as being a means of intercellular communication in the
normal physiology of the nervous system; several
reports have demonstrated the release of exosomes by
different cell types such as astrocytes or microglial neu-
rons (Corrado et al., 2013). Recently, Lachenal et al.
(2011) demonstrated that exosomes released by differen-
tiated neurons are regulated by synaptic glutamatergic
activity, and might thus be part of normal synaptic
physiology. Exosomes may also play a key role in neu-
ronal communication during neurodegenerative dis-
eases, such as Alzheimer’s, Parkinson’s or prion
diseases. For example, Alzheimer’s and Parkinson’s dis-
ease are also characterized by the accumulation of mis-
folded proteins; recent studies have shown that the
misfolded protein incorporation into exosomes protects
them from degradation and also facilitates their delivery
over long distances. Parkinson’s disease is characterized
by intracellular aggregates of α-synuclein, the Lewy
bodies, in dopaminergic neurons. Alvarez-Erviti et al.
(2011) showed that α-synuclein released from cells over-
expressing the protein is efficiently transferred to recipi-
ent normal cells through exosomes. Moreover,
Surgucheva et al. (2012) showed that another member of
the synuclein family, γ-synuclein, secreted from neuro-
nal cells into exosomes, can be transmitted to glial cells,
thus promoting the aggregation of intracellular proteins.
In Alzheimer’s disease, exosomal proteins were found
to accumulate in the plaques of AD patient brains, thus
suggesting that exosome-associated-amyloid peptides
may be involved in plaque formation. Another charac-
teristic of Alzheimer’s disease is the intraneuronal
aggregation of abnormally modified microtubule-
associated tau proteins. Saman et al. (2012) have recently
shown that tau proteins are mainly secreted through
exosomes in vitro and in vivo.
PERSONALIZED MEDICINE
The convergence of advancing research using the latest
“Omics” technology in biomarker development and
drug-testing approaches has made it feasible to have an
accurate, individual specific targeted therapy for PNS
toxicity disorders.
195
CONCLUDING REMARKS AND FUTURE
DIRECTIONS
In this chapter, the latest evolving concepts about PNS
pathology and efforts to identify biomarkers regarding
the following have been discussed: (a) mechanisms of
PNS injury; (b) inherited forms of PNS disorders,
(c) molecular and cellular mechanisms of the demyelin-
ation and remyelination process, (d) nature of neuro-
muscular junction injuries and recovery, and
(e) surrogate tissue analysis. Brief summaries of newer
approaches of methodologies currently applied in bio-
marker development were presented. Extensive lists of
proven biomarkers and candidate biomarkers identified
so far were also presented. Biomarkers that are involved
in ANS functions as well as general categories such as
ion channel functions, pathways of pain, neurodegener-
ative disorders, motor function, emotion, sleep disor-
ders, infection response, axonal transport, chemically
induced PNS disorders, OPIDN, neural stress systems,
miRNA, exosomes, and other categories involving PNS
structure and function have been presented and dis-
cussed. The following future directions will be of great
help.
With the recent announcements about the human
brain mapping initiatives by NIH promoting great
enthusiasm in functional mapping of the human brain,
it will be a productive effort to extend the efforts to the
PNS, in addition to the CNS, to promote better under-
standing about PNS structure and function in health
and disease. The following specific areas require imme-
diate attention and action:
1. In spite of a reasonable amount of knowledge about
well-defined reflex reactions in the human body,
which take care of both voluntary and involuntary
functions, more biomarkers are needed to identify
deviant pathways in PNS disorders of various
types to identify the cause and the response to any
form of therapy.
2. The role of post-translational modifications such as
phosphorylation and conformational alternates
(resulting from pathological mechanisms) as poten-
tial biomarkers to evaluate disease initiation and
progression in PNS disorders and their effect on
the CNS, need further investigation.
3. Despite a constellation of indirect evidence, direct
links between the motor (Dyenin) itself and neuro-
degeneration in inherited and acquired PNS disor-
ders are few, and further research on its ability to
serve as biomarker of PNS disorders are warranted.
4. The role of mitochondria in Schwann cell pathology
and myelination requires more extensive work,
which can lead to identification of novel markers.
196 10. PERIPHERAL NERVOUS SYSTEM TOXICITY BIOMARKERS
5. Research on exosomes could be rewarding, to iden-
tify mechanisms that may explain variations in clin-
ical severity of various neurological disorders
where the boundaries of CNS and PNS involve-
ment become vague due to a paucity of reliable
information in vesicle transport.
6. Studies on miRNA can be of very special signifi-
cance, as they are seen in exosomes and elsewhere,
playing important roles in stimulus response in
effector tissues.
7. More studies on inherited disorders of PNS are
required as they have been such a good source of
identifying the mutant macromolecules involved in
pathogenesis of PNS disorders.
8. Even though studies on chemical-induced PNS dis-
orders seen in patients receiving chemotherapy for
cancer as well as other chronic disease conditions
such as diabetes and high blood pressure are cur-
rently underway, the efforts need to be stepped up,
keeping in mind the large number of individuals
affected around the globe.
9. Epigenetic marker studies are still in their infancy,
and there are only very few studies published so
far. More studies are required in this regard.
10. Global approaches have yielded several sets of use-
ful data on potential biomarkers. These biomarkers
need to be tested using multiplex assay approaches
for diagnosis and therapy response, so the best
ones can become a part of routine clinical testing
and reflex testing modalities.
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