Transcriptional Profiling in Rat Hair Follicles following Simulated Blast Insult: A New Diagnostic Tool for Traumatic Brain Injury
et al. (2014) Transcriptional Profiling in Rat Hair Follicles following Simulated Blast Insult: A New
Diagnostic Tool for Traumatic Brain Injury. PLoS ONE 9(8): e104518. doi:10.1371/journal.pone.0104518
Transcriptional Profiling in Rat Hair Follicles following Simulated Blast Insult: A New Diagnostic Tool for Traumatic Brain Injury
Jing Zhang 0
Lisa Carnduff 0
Grant Norman 0
Tyson Josey 0
Yushan Wang 0
Thomas W. Sawyer 0
Christopher J. Martyniuk 0
Valerie S. Langlois 0
Richard Jay Smeyne, St. Jude Children's Research Hospital, United States of America
0 1 Chemistry and Chemical Engineering Department, Royal Military College of Canada , Kingston, Ontario , Canada , 2 Defence Research and Development Canada - Suffield, Medicine Hat , Alberta , Canada , 3 University of New Brunswick and Canadian River Institute , Fredericton, New Brunswick , Canada
With wide adoption of explosive-dependent weaponry during military activities, Blast-induced neurotrauma (BINT)-induced traumatic brain injury (TBI) has become a significant medical issue. Therefore, a robust and accessible biomarker system is in demand for effective and efficient TBI diagnosis. Such systems will also be beneficial to studies of TBI pathology. Here we propose the mammalian hair follicles as a potential candidate. An Advanced Blast Simulator (ABS) was developed to generate shock waves simulating traumatic conditions on brains of rat model. Microarray analysis was performed in hair follicles to identify the gene expression profiles that are associated with shock waves. Gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) were used to identify cell processes and molecular signaling cascades affected by simulated bomb blasts. Enrichment analyses indicated that genes with altered expression levels were involved in central nervous system (CNS)/peripheral nervous system (PNS) responses as well as signal transduction including Ca2+, K+transportation-dependent signaling, Toll-Like Receptor (TLR) signaling and Mitogen Activated Protein Kinase (MAPK) signaling cascades. Many of the pathways identified as affected by shock waves in the hair follicles have been previously reported to be TBI responsive in other organs such as brain and blood. The results suggest that the hair follicle has some common TBI responsive molecular signatures to other tissues. Moreover, various TBI-associated diseases were identified as preferentially affected using a gene network approach, indicating that the hair follicle may be capable of reflecting comprehensive responses to TBI conditions. Accordingly, the present study demonstrates that the hair follicle is a potentially viable system for rapid and non-invasive TBI diagnosis.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files. The microarray data for this experiment can also be accessed through NCBI Gene Expression Omnibus (GEO) database (Accession ID:
Funding: This research was supported by the Defence Research and Development Canada (DRDC) Technology Investment Fund (TIF), http://www.drdc-rddc.gc.
ca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Blast-Induced Neurotrauma (BINT) has become an issue for
soldiers since high explosives were introduced to warfare in the
mid-19th century. BINT is considered a source of traumatic brain
injury, or TBI . TBI describes any brain injury caused by the
mechanical impact to the cranium. It has been considered to be a
major cause of war casualties due to the wide adoption of
blastorientated modern weaponry reported first in 1916 by British
medical director Major Fredrick Mott, based on his post-mortem
examinations on two WWI soldiers succumbed to blast injury .
In fact, soldiers experiencing damage from explosive devices
composed 74% of total war casualties from Operation Enduring
Freedom (OEF) and Operation Iraqi Freedom (OIF) .
Increased number of war casualties from those recent military
activities has placed the matter of TBI under the spotlight of news
media and the research community . Impacts of TBI to the
brain can be categorized into either primary or secondary
damages. Primary damages are the acute injury consequences to
the brain that usually occur immediately after exposure to
traumatic conditions (e.g. penetrating brain injury); whereas
secondary damages refer to the prolonged effects of post initial
trauma, which are usually reflected on a cellular or subcellular
level . Thanks to the advanced modern operational medicine
and effective implementation of body armour during combat, the
acute consequences (primary damage) caused by
explosiveinduced TBI has been greatly reduced. That is, the initial
exposure to traumatic condition may not accompany immediate
or acute damage to the subject. As a result, most battlefield blast
related TBIs are considered as mild TBI (mTBI). It was reported
that of 266,810 documented TBI cases in US service member,
approximately 75% were classified as mTBIs . Therefore,
secondary damage of mTBI is particularly vital in this case. The
clinical symptoms of blast associated mTBI may include
headaches, confusion, short-term memory loss, changes in
cognition; changes in personality, vertigo, tinnitus and seizures
Current TBI/mTBI research has focused on two
interconnected streams: investigating molecular pathology and exploring
efficient diagnostic targets. A good understanding of the molecular
mechanisms behind mTBI symptoms would lead to the discovery
of specific biomarkers that can be used for diagnostic purposes
. At present, the primary sources for TBI research are blood
and brain tissues from either TBI patients or laboratory rodent
models . Although it is crucial for a thorough
understanding of TBI (especially the primary injuries) by directly examining
the locales of injury, it would be beneficial to utilize an equally
robust system that accurately reflects the molecular consequences
from TBI exposure and, at the same time, be conveniently
translated into a biomarker system. It is also well established that
different cells house key information that is both specific to their
type as well as communication between them under a given
environmental and/or medical condition. Therefore, a system
containing multiple cell types would provide a comprehensive
molecular signature network under TBI conditions, especially for
the secondary injuries. Such a system can be selected based on (a)
level of exposure to traumatic conditions that may induce TBI, (b)
versatility of molecular responses during or after impact, and (c)
accessibility. With the current prevalence of blast-induced TBI in
veterans returning from recent conflicts, a non-invasive diagnostic
methods or biomarker system that can be used to determine the
severity of TBI both efficiently and effectively is in demand.
The mammalian hair follicle system represents a well-coordinated
and highly sophisticated process that requires crosstalk between
specialized cells, ranging from follicular stem cell populations to
fully differentiated cells such as keratinocytes . Hair
morphogenesis and hair cycle progression are governed by
underlying cellular signal transduction as well as the subsequent
regulation of gene expression. Therefore, the mammalian hair
follicle system has the potential to sense environmental stress and
produce corresponding physical and/or molecular responses. In
fact, it has been reported that the hair follicle is capable of
responding to various stressors such as ultrasound and sensitive to
the neurohormones, neurotransmitters as well as cytokines whose
release is triggered by conventional stress responses . In
addition, hair follicles can be easily obtained by simple hair
plucking, which requires no professional skills. Accordingly, the
mammalian hair follicle system may be an optimal candidate for
TBI research and a potential source for biomarker exploration for
At its core, secondary damage of mTBI is a consequence of
altered biological signal transduction and gene expression under
traumatic conditions. Stress responsive signal pathways and gene
expression subsequently lead to adjustment to metabolism and
cellular processes. Studies have shown the involvement of
pathways including G Protein-coupled Receptors (GPRs),
TollLike Receptors (TLRs), Janus Kinase (JAK)/Signal Transducer
and Activator of Transcription (STAT) as well as
MitogenActivated Protein Kinase (MAPK) signaling cascades in TBI
responses . It is also not a surprise that mTBI interrupts
calcium and potassium homeostasis, leading to release of the
neurotransmitter glutamate, which subsequently initiates cell
death cascades . Other key secondary injuries after the
initial trauma include inflammatory responses, synaptic central
nervous system (CNS) responses and cell survival/proliferation,
among others .
The current study utilizes hair follicles from a blast exposed rat
model (Rattus norvegicus) to characterize gene expression patterns
in the hair follicle. An Advanced Blast Simulator (ABS) was
developed and deployed to deliver a single pulse shock wave, and
head-only exposures of rats were carried out in a manner that
simulated primary blast, and included elements of concussive and
whiplash forces. Studies have demonstrated that the amount and
quality of hair follicle RNA extract is sufficient for microarray
hybridization . Therefore, a 60K rat microarray was used
to identify the TBI responsive gene expression pattern in rat hair
follicles following blast exposure. We hypothesized that hair follicle
houses the said molecular signatures identified in other TBI
diagnostic and model systems and is able to respond similarly to
TBI. We provide proof of principle that the hair follicle can be a
useful tissue for assessing and monitoring TBI.
Materials and Methods
2.1. Animal experiment
A custom built advanced blast simulator (ABS; approx. 30.5 cm
in diameter and 5.79 m in length) located at DRDC (Defence
Research and Development Canada), Suffield Research Centre
(Medicine Hat, Alberta, Canada) was used for producing
simulated blast waves . Four transparent polycarbonate
windows ports were equipped on the shock tube to visually
observe the response of rat to shock waves. Two high-speed
cameras were also used to conduct high speed photography.
Male adult Sprague-Dawley (SD) rats (350400 g, 9 to 10 week
of age, Charles River, Montrean, QC) were used in the current
study. The animals were acclimated to the experimental
environment for at least one week before use. For exposure, rats were
stabilized in plastic sleeves and 3% isoflurane (in oxygen)
anaesthetic was conducted for minimum 8 min. The sleeves were
then placed in the ABS with the rat head positioned in the test
area for shock wave exposure (20, 25 and 30 psi for approximately
4 msec positive duration). Fig. S1 is a representative of a single
shockwave at 25 psi. Other detailed information with regard to
blast simulation procedure can be found in supplementary
After exposure, rats were closely observed for signs of stress and
general health either 1 or 7 days before being sacrificed. At the end
of the observation period, rat whisker follicles (n = 48) were
quickly pulled and placed in RNAlater (Qiagen, Toronto, ON,
CA). Samples were then stored at 280uC until further analysis. In
conducting this research, the authors adhered to the Guide to the
Care and Use of Experimental Animals and The Ethics of
Animal Experimentation published by the Canadian Council on
Animal Care. All experimental procedures were approved by the
Animal Care Committee at Defence Research and Development
2.2. Sample Preparation
RNA was isolated from a pooled sample of 68 rat hair follicles
using a Qiagen microkit (Qiagen, Toronto, ON, CA). Briefly,
follicle samples were homogenized with a sonicator (Ultrasonic
Dismembrator-150T, Thermofisher, Ottawa, ON, CA) and a
HiBind RNA Spin Column was then used to purify the RNA.
Concentrations and integrity of total RNA were determined with a
NanoDrop-2000 spectrophotometer and an Agilent Bioanalyzer
2000 . The mean RNA integrity values (6 SD) for all samples
used in the microarray analysis was 7.32 (61.09).
2.3. Microarray Analysis
A commercially available SurePrint G3 Rat GE 8660K
(G4853A) was purchased from Agilent (Mississauga, ON, CA).
Microarray analysis was performed using hair follicles from three
sham control rats and five rats that were exposed to simulated
blast. Microarray hybridizations were performed according to the
Agilent One-Color Microarray-Based Gene Expression Analysis
protocol using Cyanine 3 (Cy3) and followed the protocol outlined
in Langlois and Martyniuk (2013). Briefly, 200 ng total RNA/
sample was used to produce cRNA (Agilent Low RNA Input
Fluorescent Amplification Kit). Fragmentation of the cRNA
(Agilent, Gene Expression Hybridization Kit) was followed by
17 h hybridization at 60uC. An ozone barrier slide was used to
cover rat microarrays before being scanned using an Agilent High
Resolution DNA Microarray Scanner. Expression data from .tif
images were extracted by the Feature Extraction Software
(v10.7.3.1). The quality of microarray data was evaluated by
manual inspection of the quality control reports provided from the
Agilent software and each microarray was deemed to be of high
quality. Raw expression data was imported into JMP Genomics
v5.1. Raw intensity data for each microarray was normalized using
Quantile normalization and differentially regulated transcripts
were identified, as determined using an one-way analysis of
variance (ANOVA) followed by a False Discovery Rate (FDR) set
at 5%. All raw microarray data for this experiment have been
deposited into the NCBI Gene Expression Omnibus (GEO)
A subset of genes was selected and subjected to qPCR analysis
for validating microarray results. The genes were angiotensin I
converting enzyme 2 (Ace2), Tlr2, phosphoprotein enriched in
astrocytes 15a (Pea15a), AHNAK nucleoprotein (Ahnak), tp53
regulated inhibitor of apoptosis 1 (Triap1), actin related protein
2/3 complex subunit 4 (Arpc4), Stat5a, spindle and kinetochore
associated complex subunit 2 (Ska2), serpin peptidase inhibitor
(Sperini), serpin peptidase inhibitor clade A (Serpina1) and
hemoglobin beta (Hbb). All the qPCR gene targets were involved
in key cellular processes that were reported to be TBI responsive
(see the discussion section for details). In addition, elongation
factor-1 alpha (Ef1a) used as housekeeping genes for
standardization. Detailed procedure is described in supplementary materials.
Primer set and optimized qPCR conditions of each gene can be
found in Table S1.
2.4. Microarray data analysis
Intensities were first filtered in JMP Genomics using the average
negative control intensity, the lowest two points on the standard
curve (Agilent spike in controls) and the dark intensity signal.
Using these metrics, the limit of detection was estimated to be an
intensity of 3.0 and all spots measured below this intensity were
assigned a value of 3.0. Based upon the quality control report,
Feature Extraction, no probe on the rat microarray was saturated.
Functional enrichment for gene ontology was performed in JMP
Genomics. A p-value of 0.05 was the binary cut-off for the Fishers
Gene set enrichment analysis (GSEA) was conducted using
Pathway Studio 9.0 (Ariadne, Rockville, MD, USA) and ResNet
9.0. The number of gene probes mapped in Pathway Studio was
39, 422. For duplicate probes, the probe that showed the lowest
pvalue (i.e. most significant) was used for gene set ranking and the
background list for GSEA was the rat genome. Genes were
permutated 1,000 times using the Kolmogorov-Smirnov classic
approach as an enrichment algorithm. To broaden the analysis, all
pathways were expanded to include cell processes and functional
classes in target gene seeds. Gene set categories examined for
enrichment within the microarray data included the curated
Ariadne cell signaling and metabolic pathways as well as Gene
Ontology (GO) terms. Sub-network enrichment analysis (SNEA)
for proteins and chemicals regulating cell processes was also
performed to identify gene networks regulated in hair follicles
following blasting (p-value was set at p,0.05). Additional
information on SNEA can be found in Martyniuk and Langlois
and Chishti et al. . Positional GSEA was used to analyze
chromosome and chromosomal region enrichment.
3.1. Animal experiments
No obvious signs of injury were exhibited in animals
immediately after shock wave exposure. Rats from experimental groups
also showed no appreciable difference from sham control animals
regarding the time to revive from anaesthetic as well as the overall
mobility after revival. Prolonged observation (seven days post
exposure) also showed no visible injury in the animals experiencing
shock wave or weight change comparing between test and sham
control rats. Position and movement of the rat head upon shock
wave challenge was analyzed using high-speed cameras and
Tracker computer software (www.cabrillo.edu/,dbrown/
tracker/) (Fig. S2, S3). The kinematic results showed that the
head of animals displaced from its initial position according to the
strength of the shock wave. The results also suggested that the
position of the metal pin used for stabilizing animals greatly
influenced the path it took to return to the initial head position.
The detailed description of the results upon shock wave exposure
can be found in supplementary materials.
3.2. Microarray analysis
There were 1,396 gene probes that were differentially expressed
at a p-value,0.05. There were 646 transcripts that had a
61.5fold change (p,0.05). Transcripts that showed a 10-fold decrease
in the follicles included tyrosine phosphatase-like A domain
containing 2 (Ptplad2), arachidonate 12-lipoxygenase epidermal
(Alox12e) and coiled-coil domain containing 68 (Ccdc68) while
transcripts showing a 10-fold increase included basic helix-loop-helix
family member e23 (Bhlhe23), hemoglobin alpha (Hba), hemoglobin
adult chain 2 (Hba-a2) and hemoglobin beta (Hbb). The complete
list of differentially expressed genes is found in Appendix S1.
Appendix S5 is an abbreviation list containing all the genes
mentioned throughout the study. A subset of differentially expressed
genes was selected from the list to conduct data validation via
realtime RT-PCR (qPCR). Microarray data was significantly correlated
to qPCR results (R2 = 0.66, p = 0.004) (Fig. S5). Detailed description
of qPCR results can be found in supplementary material.
3.3. Gene ontology (GO) term analysis
Gene ontology functional analysis provided a first glimpse of the
biological relevance of the potential TBI responses in mammalian
hair follicle. Raw data were analyzed and enriched independently
into three main domains: biological processes, molecular function
and cellular component (the complete GO term list can be viewed
in Appendix S2). For biological processes, 112 GO terms were
enriched with p#0.01 upon blast exposure in rat hair follicles. As
shown in Table S2A, major themes include cell signal
transduction, CNS response (synaptic), stress responsive cell survival and
proliferation, and inflammatory response. The TLR signaling
pathway represents a major theme under the cell signaling
category as multiple related GO terms were enriched. Genes
related to 42 GO terms under this category were over-represented
with p#0.01 when comparing control rats with the animals after
blast exposure (Table S2B). Enriched molecular functions based
on the microarray analysis were those support metabolism, signal
transduction, hair follicle specific processes, etc. Table S2C shows
a selection of enriched GO terms under cellular component (p#
0.01). Enriched genes were primarily associated with extracellular
spaces (extracellular space, extracellular region, extracellular
matrix, proteinaceous extracellular matrix, fibrinogen complex,
hemoglobin complex), plasma membrane (plasma membrane,
bleb, stored secretory granule), reticulum (sarcoplasmic reticulum,
endoplasmic reticulum lumen) and nuclear chromosome. Visual
representations of gene networks involved in the inflammatory
responses and Ca2+ homeostasis can be found in Fig. 1 and Fig.
3.4. Gene set enrichment analysis (GSEA)
Following GSEA, a total of 21 pathways were enriched with
statistical significance (p,0.05) in hair follicles in rats upon blast
simulation. Pathways were further grouped into three categories:
receptor signaling (five pathways), cell signaling (seven pathways)
and metabolism (nine pathways) (Table 1).Our results suggest that
TLR/Activator protein 1 (AP1) and Growth hormone receptor
(GHR)/STAT pathways were the two receptor signaling pathways
that exhibited the highest statistical significance (p#0.01)
compared to other differentially enriched pathways (p,0.05),
reasoning the responses of these two pathway are more likely to happen.
Genes connected to the TLR pathway (Tlr2, Tlr4, Tlr5, and Tlr6)
showed a decrease in steady state mRNA levels in the rat hair
follicles (Fig. 2A). In addition, AP1 was also reported to be
responsive to transforming growth factor beta receptor (TGFBR)
upon shockwave exposure (Table 1). Beside the GHR, other
receptors including C-X-C chemokine receptor type 4 (CXCR4),
interferon-gamma receptor (IFNGR) were connected to STAT
signaling (Fig. 2B). To be more stringent, we filtered genes based
on intensity that were below the detection limit of the microarray.
Cell Signaling pathway enrichment contained 4 processes with p#
0.01. The results also suggested that there was an enrichment of
genes involved in the guanylate cyclase pathway as well as
Adherens Junction Regulation (p,0.05) (Table 1). Under the
metabolism category, there were 5 pathways with p#0.01
3.5. Positional GSEA
Chromosomal positional GSEA was carried out to identify
specific chromosomal regions that were significantly enriched in
the rat genome upon blast exposure in rat hair follicles. In
response to blast, differentially expressed genes (p,0.05) were
located preferentially on a couple of rat chromosomes (Table 2).
Chromosomes 1, 6 to 10, 12, 13, 15 and 17 exhibited multiple
enriched regions responsive to blast exposure, among which
chromosomes 1 (1p13, 1q32, 1q43, 1p12), 7 (7q22, 7q11, 7q13)
Expanded # of Entities
# of Measured Entities
Cell signaling pathways
Skeletal Myogenesis Control
Gap Junction Regulation
Guanylate Cyclase Pathway
Adherens Junction Regulation
Gonadotrope Cell Activation
Receptor signaling pathways
Arachidonic acid metabolism
Metabolism of estrogens and androgens
Vitamin B6 (pyridoxine) metabolism
omega-6-fatty acid metabolism
omega-3-fatty acid metabolism
and 13 (7q22, 7q11, 7q13) encompassed the chromosomal regions
that contained genes most affected by blast simulation.
A number of serine protease inhibitor genes (e.g., Serpinb3, -5,
- 11 and -12) on the chromosome 13 position 13p13 were
preferentially down-regulated (,40%) following blast (Fig. 3). Six
members of this gene family showed a decrease in expression while
three others were increased in mRNA abundance in rat hair
follicle. Other genes within this position with decreased transcript
levels included B-cell CLL/lymphoma 2 (Bcl2), vacuolar protein
sorting 4 homolog B (Vps4b), and phosphatidylinositol glycan
anchor biosynthesis (Piga), and phosphatidylinositol-glycan
biosynthesis class n (Pign). Interestingly, a region on the
Xchromosome was identified as enriched and included
gastrinreleasing peptide receptor (Grpr), Gpr 64, -143 and -173.
GSEA was conducted on the genes that were differentially
enriched on chromosome 1, 7 and 13, in order to explore the
functional significance of those chromosomes in rat hair follicle
upon blast exposure. Considering the number of pathways
enriched on those chromosomes, it is beyond the scope of the
current paper to discuss all the processes. Hence, we focus on the
pathways that were also enriched in microarray GSEA analysis
described in section 3.4. The complete GSEA results for all three
chromosomes can be found in Appendix S3. For the receptor
signaling category, our GSEA results on positional enrichment
data suggest that genes associated with TLR signaling were
enriched on chromosome 1 and 7 while those related to STAT
pathways were found on all three chromosomes. Genes involved in
TGFBR/AP1 signaling were enriched on chromosome 1. Similar
to TLR pathway, GHR signaling was also a represented enriched
group on chromosomes 1 and 7. The categories of cell signaling
and metabolic pathway also showed overlap between microarray
GSEA and GSEA on chromosome 1, 7 and 13. These data
support those data that were collected for the microarray GSEA
and localizes groups of genes with specific functions to
3.6. Sub-network enrichment analysis (SNEA)
SNEA was conducted to build blast responsive sub-networks in
rat hair follicle based on regulatory networks inferred from the
literature. Potential upstream regulators, cell processes and related
diseases were analyzed. Our results showed a total of 147 potential
cell processes were responsive to blast exposure in rat hair follicles
with p#0.01 (the complete process list is provided in Appendix
S4A). Here we focus on the processes that are closely associated
with pathways enriched in GO term and GSEA analysis. Table 3
lists the candidates grouped to that structure provided for the GO
term analysis. Similar to GO terms and GSEA analysis, some of
the major themes affected by shock wave blasting were signal
transduction, metabolism, cell survival/proliferation, brain injury
responsive cellular events as well as hair follicle specific responses.
There was excellent coverage of many of the annotated gene
networks in the rat (.90%) (Appendix S4A). Networks related to
cell signaling were, in general, overwhelmingly decreased (213%).
Genes with up to 45% decrease in transcript levels are shown in
Fig. 4A. However, up-regulations were observed for the genes
connected to Ca2+ export and Ca2+-dependent signal transduction
Figure 2. Selected pathways enriched by GSEA analysis. Red indicates that the gene is increased and green indicates that the gene is
decreased in mRNA levels. (A) Factors involved in TLR/MAPK pathway; (B) factors involved in JAK/STAT pathway pathways for GH, prolactin, and
Table 2. Chromosome enrichment identified regions of the rat genome that contain groups of genes that preferentially increased
or decreased in mRNA levels in the hair follicle.
(Fig. 4B). In addition, a few of the processes showed potential
positive responses reflected by up-regulations on transcript levels of
the related genes (Fig. 4C). It is worth noting that a major process
identified in the SNEA analysis was synaptic CNS (Table 3). For
example, networks related to dopaminergic and cholinergic
pathways, neurotransmitter secretion and uptake, and transcripts
involved in nervous system physiology and action potential
generation were affected in rat hair follicles. Fig. 4D shows
differentially expressed genes connected to cholinergic synaptic
Other themes that were identified using SNEA included
reproduction, behavioural regulations, hormonal control (Table 3)
and potassium homeostasis (Fig. 4E). Reproductive gene networks
such as copulation, sperm entry, estrus, ovary function, and proestrus
were also affected and the majority of sub-networks related to
reproduction were decreased in hair follicles. Behavioral-related
gene networks were all decreased in median fold change. In terms of
hormonal control, vasopressin and adrenocortical secretion were
SNEA analysis is also capable of mapping differentially
expressed genes that are related to diseases. A total of 260 diseases
were identified as enriched in the hair follicles from blast exposed
rats (p,0.05, the complete list can be viewed in Appendix S4B), of
which 94 conditions were significant at p#0.01. Major themes
include ion homeostasis (e.g., hyponatremia and hypokalemia),
heart disease (e.g., arteriolopathy, ventricular fibrillation, aortic
valve stenosis, etc.), blood circulation disorders (e.g., artery
stenosis, hemodynaic abnormalities, etc.), and inflammation (e.g.,
granuloma and inflammatory lesions), among others.
To test the hypothesis that hair follicle gene expression is
sensitive to blast exposure, we analyzed hair follicles of whiskers
harvested from rats exposed to simulated blast in a shock tube
specially designed to produce single pulse shock waves. Although
this simulator produced shock waves that simulated all the key
characteristics of classical free-field blast-wave flow conditions,
including the negative phase and secondary shock, the head
constraint utilized introduced confounding variables .
Kinematic analysis using high speed photography showed that
significant concussive and whiplash forces, in addition to the
primary overpressure insult, were also produced during the
simulated blast exposures. In order to explore the possibility of
hair follicle used as a research and diagnostic system for TBI, the
current study examines responses of the well-studied brain trauma
responsive biological processes such as nervous system response,
ion exchange and GPR dependent signaling cascades,
inflammatory response and apoptosis regulations. High-throughput
gene expression profiling presents a robust way of exploring
molecular mechanisms behind TBI responses in mammalian hair
follicle. The consistent advancement in bioinformatics throughout
years has enabled various enrichment algorithms that can be used to
analyze the complexity of signal transduction and the related
downstream events through multiple types of interaction networks
. Thus the current study makes use of the
microarrayenrichment analysis approach to depict the potential similarity of
the aforementioned molecular signatures between hair follicle and
other established systems including brain and blood.
First of all, the enrichment analyses suggested that synaptic
CNS response as a major theme enriched in the rat hair follicles
following blast exposure: related GO terms under include
regulation of synaptic transmission and synaptic transmission. In
fact, synaptic homeostasis and transport are tightly associated to
TBI . In particular, the cholinergic neurotransmission
impairment was reported in rat models experiencing TBI .
It has been revealed that CNS response is a main theme upon TBI
. The SNEA analysis revealed even more synaptic related
cellular events, including neurotransmitter uptake, ganglion
neurotransmission, neuromuscular synaptic transmission, synaptic
transmission, cholinergic synaptic transmission, synaptogenesis,
neuronal activity, transmission of nerve impulse, strongly
indicating that synaptic homeostasis-related nervous system responses
play critical roles in rat hair follicles upon blast. Our results further
suggest the genes encoding receptors involved in cholinergic
neurotransmission were up-regulated in their transcript levels,
including histamine h3 receptor (Hrh3), cholinergic receptor,
muscarinic 3 (Chrmm3), opioid receptor mu 1 (Oprm1), solute
carrier family 18 member 3 (Slc18a3), adenosine a1 receptor
(Adora1), 5-hydroxytryptamine (serotonin) receptor 1a (Htr1a),
adrenoceptor beta 2 (adrb2), dopamine receptor d3 (Drd3) and
formyl peptide receptor 1(Fpr1). Given its critical roles in nervous
system, changes to cholinergic neurotransmission machinery can
lead to responses in both CNS and PNS . In addition, TBI
responsive synaptic transmission was proposed to be
glutamaterelated in mammalian brain . In the current study, such
regulation was supported by the over-representation of the
differentially expressed genes associated to glutamate metabolism.
The microarray results suggested regulation of genes encoding
related receptors, including glutamate receptor 4 (Gria4) and
glutamate receptor ionotropic n-methyl d-aspartate 2a (Grin2a).
The GO term domain cellular component confirmed the potential
ligand-receptor interactions with enriched subcellular locales of
extracellular environment and plasma membrane that house most
of the related protein products.
Various types of signal transduction responses were also
identified, including ion exchange and GPR-dependent
pathways. Firstly, our microarray and enrichment analyses suggest
changes in expression profiles of genes encoding Ca2+, K+ and Cl2
cellular transport and localization. It has been established that the
release of excitotoxic amino acids upon TBI triggers alteration in
Ca2+ transport across cellular membranes as a major secondary
injury that is usually associated with cellular damage . The
microarray results suggest that expression levels of genes involved
in stimulating Ca2+ transport were significantly up-regulated,
including Platelet-derived growth factor receptor beta polypeptide
(PDGFRB) and Transient receptor potential cation channel
subfamily v member 2 (TRPV2) . SNEA analysis indicated
that blast exposed rat hair follicle triggered regulation of genes
responsible for sarcoplasmic reticulum calcium release, confirmed
by the enriched intracellular locale sarcoplasmic reticulum by GO
term analysis. A sustained influx of Ca2+ into cells and
compartments such as mitochondria and nucleus can initiate
apoptotic signaling cascades as it leads a disruption in metabolic
processes . Genes involved in K+ transport were also enriched
by GO term (positive regulation of potassium ion transport,
potassium channel inhibitor activity) and SNEA (potassium ion
import/homeostasis) analyses. Such regulation was also proposed
by Reinert et al. as another ion transmission related secondary
injury following severe primary injuries of TBI . It was
previously reported that the transmembrane events of chloride ion
was regulated by the corresponding adaptations of proteins
responsible for capillary permeability upon TBI , which was
also enriched by SNEA.
Receptor signaling pathway was another enriched signal
transduction theme upon blast simulation by the enrichment
analyses, as indicated by the GO term receptor clustering. Such
pathways include GPR-dependent signaling (GO term
enrichment) and its major regulator of the guanylate cyclase pathway
(GSEA) . It is well documented that GPRs are able to trigger
signal transduction through key pathways and are crucial for
mediating cellular responses to medical disorders .
Specifically, it was reported that GPRs were regulated in response
to TBI in rat brain . One example of GPR-mediated signal
transduction is MAPK pathway , which was also enriched by
GO term analysis. MAPK signaling cascades consist of three main
kinase pathways: extracellular signal regulated kinase (ERK), c-Jun
N-terminal kinase (JNK) and p38 MAPK, which are sensitive to
various environmental change and stress conditions . It was
proposed that all three MAPK pathways were TBI sensitive .
GPR also facilitates JAK/STAT signaling, which is also
TBIresponsive [12,55]. GPR-based activation of JAK/STAT pathway
requires Rho GTPase activity . Both GO term and GSEA
analyses showed that genes involved in JAK/STAT pathway and
regulations on Rho GTPase were over-represented, indicating the
Rho GTPase-dependent GPR/JAK/STAT a potential in rat hair
follicles upon blast simulation.
TLR pathway is another signaling pathway affected at
transcriptomic level in hair follicles following blast (GO term
and GSEA analyses). It was previously demonstrated that TLR
pathways were responsive to TBI in the brain of the mice and
proposed as a biomarker for stroke in blood [22,57]. Many of the
TLRs mapped had a decrease in transcript level following blast,
namely: Tlr2, Tlr4, Tlr5 and Tlr6. TLRs are signaling molecules
that assist in the regulation of the immune response to tissue
damage. The qPCR analysis also showed consistent decrease in
transcript level of Tlr2, further confirming the results observed in
microarray analysis. TLRs also function as upstream receptors to
MAPK cascades . Our results suggest potential
TLRdependent inhibition of MAPK signal transduction associated
with down-regulated transcript levels of Tlrs and genes involved in
MAPK pathways. Therefore, it appears that rat hair follicles are
capable of responding to TBI conditions similar to mammalian
brain in terms of regulation on TLR pathways.
As mentioned above, some of the enriched signaling pathways
upon blast exposure are directly linked to inflammatory responses,
which is another well-known TBI response . The enriched
JAK/STAT and TLR/NFkB pathways are major upstream
signaling cascades that are able to trigger inflammatory response
[12,59]. The JAK/STAT-mediated inflammatory responses are
cytokines-dependent (e.g. interleukins (IL) . Our GO term
analysis revealed multiple biological processes and molecular
functions enriched towards regulations on IL, indicating potential
connections between JAK/STAT/IL-dependent inflammatory
responses. The GSEA analysis also suggested additional receptor
STAT interactions that could lead to JAK/STAT-dependent
inflammation, such as GHR and IFNGR . GHR interacts
with JAK/STAT signaling through AP1/FUN/FOS pathway
. The microarray results suggested decreased transcript
levels of Ifngr2, Jak2 and all the downstream Stat genes, suggesting
that the IFNGR2/JAK/STAT pathway may be inhibited in hair
follicle upon shock wave exposure. Our qPCR analysis also
suggested a decrease of Stat5a transcript level, consistent with the
result from microarray. In terms of TLR/NFkB, the results from
blast exposed rat hair follicles showed decrease in transcript levels
of the related genes. Since TLR/NFkB pathway stimulates
immune response, while inhibiting inflammation , the results
suggested a potential pro-inflammation regulation. Along with the
observed decrease of Tlr4 transcript level, the GO term results also
showed a decrease in the adaptor molecule myeloid differentiation
factor 88 (MYD88). TLR4 is able to exacerbate cell damage in the
brain and trigger inflammatory responses following trauma .
Activation of TLR4 stimulates NFkB, which in turn affects genes
that encode pro-inflammatory molecules. MYD88 is part of the
TLR2-MyD88-NFkB pathway that is related to the release of
IL1b, a key mediator in the inflammatory response.
As a result of pro-inflammation signal transduction, multiple
inflammatory responses were reported following TBI exposure,
including elevation of intracranial pressure, accumulation of
polymorphonuclear leukocytes as well as increased proliferation
of Natural Killer (NK) cells . Genes involved in such processes
were preferentially regulated in the rat hair follicles, as
demonstrated in GO term analysis. As shown from the pathway analyses,
genes involved in inflammatory response with altered expression
profile were significantly enriched. Therefore, shock wave
simulated blast exposure led to a full spectrum of inflammatory
responses ranging from signal transduction to specific cellular
processes, suggesting rat hair follicle is capable of reflecting the
complete molecular complexity of brain trauma-induced
Furthermore, genes involved in defense response, cellular
defense response, response to mechanical stimulus and protein
refolding upon were enriched in GO term analysis upon blast
exposure. Biological processes related to cell survival and
proliferation secondary injuries that are commonly associated
with the initial trauma to the brain . Therefore, it is not a
surprise that multiple programmed cell death related GO terms
and GSEA pathways were enriched. It is worth noting that
glutamate metabolism also plays a critical role in neuronal
injuryrelated cell death via gap junction regulation . Consistent with
the over-represented genes involved in glutamate metabolism in
GO term analysis, gap junction regulation was enriched in GSEA.
TBI-associated cell death occurs in both and secondary injuries
over prolonged post-traumatic periods [28,68]. Apoptosis is
controlled by both pro- and anti-apoptotic factors responsive to
environmental and/or cellular conditions . The anti-apoptotic
B-cell CLL/lymphoma 2 (Bcl2) is a critical factor in regulating
apoptosis. Altered expression of Bcl-2 gene may lead to an
imbalance in the homeostatic equilibrium between cell survival
and death . Our microarray data showed that the transcript
levels of Bcl2 were significantly down-regulated, suggesting
potential apoptosis events in hair follicles upon blast. Studies have
shown regulated Bcl2 expressions following brain trauma .
Furthermore, the previously discussed signaling pathways are also
involved in cell survival. For example, AP1 transcription factor has
also been shown to regulate a host of target genes that are involved
in programmed cell death following brain ischemia .
SNEA disease network analysis also suggests that hair follicle is
able to reflect comprehensive TBI consequences beyond direct
responses. The results suggest that the rat hair follicle system is
able to reflect potential changes in blood pressure and related
cardiovascular responses upon blast exposure. Cardiovascular
homeostasis is a major theme enriched in rat hair follicle and
essential to the health of both body and brain. Following moderate
to severe TBI, this equilibrium is often disrupted and can result in
the loss of blood pressure autoregulation and attenuation of the
normal bradycardic response to injury . The gene
subnetworks showed significant trends in pathways related to brain
blood flow, artery baroreflex and cardiovascular reflex. Many of
these pathways showed significantly lower gene expression levels
than control (up to a 45% reduction). Magnetic resonance imaging
(MRI) assessments of cerebral blood flow performed following
experimental TBI in mice have shown that hemorrhagic shock
and systemic hypotension resulting from blood loss exacerbate cell
death in the hippocampus . Moreover, internal blood surges
induced by blast over-pressure have been hypothesized to induce
increases in cerebral perfusion pressure and micro-tears in both
cerebral blood vessels and blood-brain-barrier (BBB) . The
transfer of kinetic energy from the blast causes a rippling effect
that oscillates blood vessels to the brain causing morphological
alterations . These cerebrovascular insults result in BBB
breakdown as seen in several animal studies [13,7778] and
may be the reason for the up-regulation in the SNEA of blood
vessel morphogenesis pathway seen in our results.
Additionally, positional enrichment analysis suggested that
chromosome 1, 7, 13 and corresponding regions on them were
preferentially affected by blast simulation. Rat chromosome 1 (the
largest rat chromosome) houses the highest number of genes that
are responsive to neurological disorders . Chromosome 1 was
also considered related to cardiovascular disorders . Previous
study has demonstrated a locus on chromosome 7 is involved in
blood pressure disorders while chromosome 13 is linked to
hypertension , consistent with the SNEA disease
enrichment. In addition, a region on the X-chromosome (Xq21) was
preferentially responsive to blast simulation, suggesting sex
differences in the response to shockwave exposure may exist at
the level of the transcriptome.
Accordingly, the current study demonstrated that gene
expression was responsive to blast exposure in rat hair follicles. Multiple
gene set enrichment analyses suggested that several types of cell
signal transduction cascades, cell survival and nervous system
responses. The results suggested that, on a molecular level, rat hair
follicle responds to traumatic conditions similarly to brain and
blood of both human patients and lab rodent models (e.g., name
with pathways are the same). In addition, transcriptomics
responses in the rat hair follicle corresponded to brain trauma
related medical disorders as well as potential TBI-responsive
chromosome positional enrichment. These data indicate that hair
follicle may be a robust system with similar TBI molecular
signatures that have been characterized in other systems.
Furthermore, hair follicles can be easily obtained via simple
plucking without professional skills. These features make hair
follicle an optimal biomarker system for TBI in the context of
military activities as well as other traumatic conditions that could
lead to brain injury. In conclusion, the present study demonstrated
for the first time that mammalian hair follicle is a potentially
optimal system for both TBI pathology study and biomarker
Figure S1 Representative of a single shock wave. This
shock wave is produced with a 25 psi target pressure.
Figure S2 Displacement of rat nose in XY plane. Origin is
initial resting position of nose. Origin is initial resting position of
nose; the path of movement was tracked using high speed video
and tracking the tip of the nose.
Figure S3 Radial distance displacement of rat nose
from origin. Origin is initial resting position of nose.
Figure S4 Factors involved in Ca2+ homeostasis; red
indicates that the gene is increased in transcript level.
Figure S5 (A) Linear regression analysis showing correlation
between microarray and qPCR; fold change to control was plotted
for both microarray and qPCR. (B) Comparison of transcript level
change in rat hair follicle upon shock wave exposure between
microarray and qPCR.
Table S2 Table S2A: Enriched GO terms in rat hair follicles
after blast exposure under the domain of Biological Processes.
Table S2B: Enriched GO terms in rat hair follicles after blast
exposure under the domain of Molecular Functions. Table S2C:
Gene expression data for the rat
tranAppendix S2 Appendix S2A: GO term enrichment, Biological
Processes domain. Appendix S2B: GO term enrichment,
Molecular Functions domain. Appendix S2C: GO term enrichment,
Cellular Components domain.
This research was supported by the Defence Research and Development
Canada (DRDC) Technology Investment Fund (TIF) to VSL (PI), CJM
and YW. The authors thank the excellent technical assistance by Tracy
Weiss, Peggy Nelson, Grant Hennes, Cory Vair and Julia Barnes at DRDC
during animal exposure to blast.
Conceived and designed the experiments: YW CJM VSL. Performed the
experiments: LC GN TJ CJM. Analyzed the data: JZ GN TJ. Contributed
reagents/materials/analysis tools: YW TWS CJM VSL. Contributed to the
writing of the manuscript: JZ LC TJ.
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