Investigation of blast induced neurotrauma in a rodent models

CHAPTER 1
INTRODUCTION

1


1.1 Overview of Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the foremost causes of disability and death
in both civilian settings and theatres of war. TBI can be simply defined as damage to the
brain tissue resulting from external mechanical forces (Center for Disease
Control)(CDC)). In terms of gross clinical classification, TBI can be further sub-classified
as penetrating or closed head injury. TBI severity ranges from mild to moderate to
severe. TBI severity is clinically determined using the Glasgow Coma Scale (GCS),
which assesses the conscious state of a TBI patient in terms of eye, verbal and motor
responses. The severity of TBI using the GCS is categorised as mild (GCS >13),
moderate (GCS 9 - 12) and severe (GCS <9). The Department of Veteran Affairs (VA)
and Department of Defense (DoD) have also consolidated a classification (Table 1) for
the diagnosis of concussion/TBI in the military (Table 1). This classification table utilises
an array of criteria that includes structural imaging, loss of consciousness (LOC),
altered level of consciousness (ALOC), post-traumatic amnesia (PTA) as well as the
GCS. In this study, the effects of explosive blast waves on the central nervous system
(CNS) and functions of the brain were investigated.

Table 1: VA/DoD Classification of TBI Severity
Criteria
Structural Imaging
Loss of Consciousness
Alteration of Consciousness
/Mental State
Post-Traumatic Amnesia

Glasgow Coma Scale (Best
available score in the first
24 hrs)

Mild

Moderate

Severe

Normal
0-30min
A moment up to 24
hrs
0-1 day

Normal or abnormal
>30min and <24 hrs
> 24 hrs, Severity based
on other criteria
>1 day and <7 days

Normal or abnormal
>24hrs
> 24 hrs, Severity based
on other criteria
>7days

13-15

9-12

<9

The time-course effects of TBI can be defined as a two- stage process: acute
primary brain injury followed by secondary brain injury.

Primary injury cannot be

prevented by pharmacological or surgical intervention and is due to the direct and initial
insult to the brain caused by the application of external mechanical forces. These
mechanical forces include (i) direct and blunt force impact causing rapid acceleration
and deceleration of the head, (ii) explosive blast emitted pressure waves and/or (iii)
2


penetrating forces that cause localised brain tissue damage. These mechanical forces
may cause immediate necrosis of torn and overstretched cells (give reference).

For closed head TBI, the most common form is the coup-contra coup injury which
can be distinguished by either focal or diffused axonal injuries (DAI) (Anderson and
McLean, 2005). Coup-contra coup injury happens when an impact or violent motion
brings the head or skull to a sudden deceleration while the brain is still accelerating,
causing the brain to slam into the inner skull and bounced to in the opposite direction,
thereby

resulting

in

focal

injuries

at

both

sides

of

the

brain.

These

acceleration/deceleration forces can also cause stretching and shearing of the brain
tissue when rotational forces are involved. In addition, relative differences in the tissue
densities moving at different speeds can cause diffused axonal injury (Anderson and
McLean, 2005).

Secondary brain injury is the resultant neurochemical and neuro-inflammatory
complications from arising from the primary mechanical insult (Zhang et al., 2004).
Physiologically, these manifest as tissue ischemia, hypoxic damage, edema, and
increased intracranial pressure (ICP). Secondary brain injury is usually characterized by
resultant neuronal cell death and functional deficits after injury, including slightly
compromised cells and nearby health cells (Borgens RB and P, 2012). Following
primary brain insult and necrosis of injured tissue, a delayed cascade of cytotoxic

apoptosis commonly attributed to NMDA excitotoxicity of the injured neurons occur (Yi
JH and AS., 2006, Mehta A et al., 2012 ), with generation of free radicals (Pun PB et al.,
2009, O'Connell, 2012), mitochondrial dysfunction and disruption (Robertson CL et al.,
2006, Cheng et al., 2012), and irreversible metabolic disturbances (Tsutsui S and PK.,
2012, Xu F et al., 2012). Importantly, secondary brain injury presents a window that
may be responsive to potential therapeutic interventions to improve neurological
outcome after TBI.

3


1.2 Overview of Blast-induced Neurotrauma

Exposure to explosive blast devices like improvised explosive devices (IEDs),
land mines and rocket propelled grenades (RPG) accounts for almost two thirds of the
casualties sustained by the US military in Iraqi and Afghanistan wars (Ling G et al.,
2009, Cernak and Noble-Haeusslein, 2010, Belmont PJ Jr, 2012 ). In the recent wars
however, advancement in protective armour may reduce penetrating and blunt impact
TBIs caused by the blast and protecting from blast lung injury. Nevertheless, with
advanced far-forward medical care, more injured soldiers are being diagnosed with
some form of blast related TBI or blast-induced neurotrauma (BINT) (Courtney and
Courtney, 2011, Ling and Ecklund, 2011a) . A recent study suggests that up to 89% of
studied brain trauma patients from these wars might have sustained some form of BINT
(MacGregor AJ et al., 2011). BINT has become a major area of concern in current
military medicine and has been termed the “signature wound” of the recent conflicts
(Warden., 2006, Ling G et al., 2009, Ling and Ecklund, 2011b). In deployed US military,
from year 2000 to the first quarter of 2012, around 244 217 combat personals suffered
some form of TBI of which mild TBI (mTBI) alone accounts for 76.8% of the total
(www.DVBIC.org).

1.21

Physics of Explosive Blast

A blast wave generated by an explosive detonation begins with a rapid and
instantaneous rise in air pressure that lasts from less than a millisecond (i.e. IEDs), to
less than a hundred millisecond (i.e. air/fuel bombs, nuclear) (Courtney and Courtney,
2011). In an ideal Friedlander blast wave, which is generated by an explosion in an
open field, there is a rapid rise in blast overpressure followed by an exponential decay
leading to a negative pressure phase (IG, 2001 , Cernak and Noble-Haeusslein, 2010)
(Fig 1). The blast wave progresses from the source of the explosion as a sphere of
compressed air (shock front) that is followed by an area of rapidly expanding gases
travelling at supersonic speed (Rossle, 1950). It is the interaction of the blast wave with
the body, travelling at different speeds through the different human tissue densities that

4


results in blast injury (Bowen et al., 1968, IG, 2001 ). Traditionally, these injuries were
thought to occur primarily to the gas-filled organs (auditory, pulmonary and
gastrointestinal systems). Hence, the Bowen’s curves were generated as an estimation
of tolerance of the lung to peak overpressure and the positive phase duration of blast
exposure. The Bowen’s curves (Bowen et al., 1968) and updated estimates by Axelsson
(Axelsson H and JT., 1996), were based on studies of thirteen mammalian species
which allowed for an extrapolated estimate on human blast-induced lung injury criteria
and mortality rate. However, the Bowen’s curves were predominately geared towards
lung injury and do not serve as a good estimate for CNS tolerance or BINT (Geoffrey
Ling et al., 2009).

peak pressure

positive phase
negative phase

Fig 1: Schematic of an ideal Friedlander wave form describing the relationship of pressure wave versus
temporal changes for high explosive detonated in a free field with no surfaces nearby that can interfere.
There is an initial rapid rise in positive blast overpressure, hitting a peak overpressure, followed by an
exponential decay leading to a negative pressure phase. Diagram taken from Wikipedia.

Generally, blast injuries can be described into four different injury categories in
relation to their mechanisms of injury: primary, secondary, tertiary and quaternary.

i.

Primary blast injury is the direct result of the explosive generated supersonic
blast wave's interaction on the body, by inducing rapid changes in atmospheric
pressure.
5


ii.

Secondary blast injury is due to the impact of bomb fragments, debris or objects
put in motion after being accelerated by the blast wind which can lead to
penetrating ballistic or blunt force injuries.

iii.

Tertiary blast injury occurs as a result of victims being thrown by the blast
pressure wave. For example, victims may be thrown onto the ground or propelled

through the air, and striking solid objects resulting in blunt trauma.

iv.

Quaternary blast injury is defined as any explosion-related injury or illness not
due to any of the above, such as burns and inhalational injuries. High
temperatures generated from the explosive gases can also render fatal burns to
victims close to the detonation.

1.22

Experimental Blast Injury Models

With the understanding that the brain is a vulnerable target for blast injury (Kaur
C et al., 1995 , Cernak et al., 2001, Ling G et al., 2009, Cernak and Noble-Haeusslein,
2010), there has been a flurry of research geared towards the understanding of BINT.
However, BINT remains a difficult injury to predict and diagnose (Hoge et al., 2009) and
the mechanisms by which blast exposure results in BINT are still poorly understood. A
wide spectrum of blast injury models have been investigated ranging from non human
primates, large porcine models, rodents shock tubes to in vitro cell cultures (Kane MJ et
al., 2012 ) and even computational modeling, in a bid to better understand blast effects .

Current research groups investigating BINT utilize both large and small-animal
models, and with different methods of generating the blast pressure. Majority of BINT
research however, makes use of small animal blast exposure models that rely on
compressed gas-driven shock tubes to generate blast. (Elsayed, 1997, Cernak et al.,
2011, Chavko et al., 2011, Garman et al., 2011b, Leonardi et al., 2011, Reneer et al.,
2011, Dalle Lucca JJ et al., 2012). These compressed gas-driven shock tubes consist of

6

special membranes rupturing at predetermined pressure thresholds. These shock tubes
generate blast over pressure (BOP) with significantly lower peak threshold pressure but
longer positive overpressure durations. Some groups utilize traditional chemical
explosives as the pressure generator (Kaur C et al., 1995 , Moochhala SM et al., 2004,
Annette Säljö et al., 2009, Risling et al., 2011, Rubovitch V et al., 2011 ). For this study,
we used an explosive model in a bunker using the same blast device as in Risling et al.,
2011. In addition, majority of the blast studies conducted exposed the experimental
animals to full body blasts without any protection (Cernak et al., 2011, Chavko et al.,
2011). Few make use of selective protection to the head or body using Kevlar materials
(Bauman et al., 2009b, Garman et al., 2011a) or custom made acrylic holders (Cernak
et al., 2011, Chavko et al., 2011). Blast studies employing large animals models were
limited. Most use porcine models (Säljö A et al., 2008, Bauman et al., 2009a,
Shridharani et al., 2012) and only one study employed non-human primates (Lu J et al.,
2012). Besides investigating single blast exposure, BINT resulting from multiple blast
exposures have also been recently investigated (Ahlers ST et al., 2012, Balakathiresan
et al., 2012).

Similar to Bowen’s studies which provides an estimate of blast mortality tolerance
based on the peak overpressure and duration of blast exposure, most of the BINT
studies also describe their studies as a result of varying exposures of peak
overpressures. However, the blast overpressures used to induce BINT span over a wide
range. For example, studies using rodents have experimental overpressures ranging
from very low level blast exposure 20 – 60KPa (Moochhala SM et al., 2004, Annette
Säljö et al., 2009, Pun et al., 2011, Rubovitch V et al., 2011 ), to 90 to 170 KPa
(VandeVord et al., 2011, Ahlers ST et al., 2012, Bir et al., 2012, Valiyaveettil et al.,
2012) and the highest at 339 KPa (Cernak et al., 2001, Cheng et al., 2010, Cernak et
al., 2011, Koliatsos VE et al., 2011). Annex A provides a list of experimental BINT
studies conducted and the pressure profiles reported. Unfortunately, most research

groups only indicate the peak pressure and positive pressure duration, without
specifying the impulse data.

7


Recent reviews had proposed three possible mechanisms that may lead to BINT
(Cernak and Noble-Haeusslein, 2010, Courtney and Courtney, 2010, Bolander et al.,
2011). The three mechanisms via which BINT may occur are (i) through direct blast
pressure propagation through the skull, (ii) via indirect transmission through the
vascular system and (iii) through acceleration and/or rotation of the head due to skull
flexure. Although the direct transcranial propagation was deemed most significant for
BINT, (Cernak et al., 2011), selective shielding of the thorax and abdomen showed
reduced mortality and brain injury. This suggests that the blast wave may transfer
kinetic energy through the vasculature and trigger pressure oscillations in blood vessels
leading to brain injury (Cernak et al., 2011). However, many other studies did not
provide evidence to reinforce this mechanism. Both Bauman (Bauman et al., 2009a)
and Garman (Garman et al., 2011a) reported that even with thoracic shielding on
respectively, swine and rodent models, they still recorded high ICP similar to the applied
blast pressure. Hence this suggesting that blast pressure was unlikely to be transferred
through the vascular systems. Other reported ICP by (Chavko et al., 2007) and (Säljö A
et al., 2008); showed similar or even higher ICP recordings than the applied blast
pressure, with no protection, suggesting a mechanism of direct transfer of energy
through a skull/brain interface. Interestingly, multiple studies (Bolander et al., 2011,
Leonardi et al., 2011, VandeVord et al., 2011) indicate an alternative skull flexure
hypotheses, especially the superior rat skull, which presents a very high stain rate to the
brain through the skull/dura interface. However, a swine study by (Shridharani et al.,
2012) demonstrated a lower attenuation ratio (ratio of air pressure versus ICP)
compared to the rodent studies of (Leonardi et al., 2011) and (Säljö A et al., 2008), and
questions the degree of energy transfer to the brain tissue. Interestingly, a study using

human subjects and low level of acoustics wave pressure (80Hz) showed that though
the skull acts as an attenuator of higher frequencies, internal cerebral membranes such
as the falx cerebri can reflect and focus shear waves within the brain (Clayton et al.,
2012).

Due to the complex nature of the blast pressure transmission and the ambiguity
about how the pressure wave affects the brain, the research community is still

8


attempting to identify the mechanism causing TBI and the added complexities
associated with mTBI and its associated neurological presentations.

1.3 Study Objectives

The mechanisms resulting in BINT has not been elucidated. This study aims to
investigate the pathophysiological effects primary blast overpressure induced injury in
the brain using two different rodent blast models: a shocktube and modified open blast
model. Histopathology examinations of the brain after blast injury will be correlated to
systemic biomarker changes and behavioral outcomes, to better understand the
relationship between pressure profiles varying blast overpressure and resultant brain
injury.

9


CHAPTER 2
MATERIALS AND METHODS

10


2.1

Establishment of a Blast Tube Model

2.11 Animal subjects
Male Sprague-Dawley rats (Taconic, Denmark) weighing 300-350 grams were
used in these experiments. After 1 week quarantine, animals were food restricted to
85% of their starting weight to ensure motivation to do the behavioral task. The animals
were allowed a weekly weight increase of 5% and had full access to water. All rats were
housed in pairs, separated by an acrylic divider, under conditions of room temperature
on a 12 hrs regular light/dark cycle. All animal experimentation protocols in this research
project were approved by Karolinska Institutet Institutional Animal Care and Use
Committee (permit number N143/09). Table 2 showed the breakdown of animals usage.
Table 2: Number of animals used for each injury and behavioural test and the time of sacrifice
Test Condition

Sacrifice Time Point
3hrs
24hrs
72hrs
2g explosive

RAM trained
5 Choice Trained

Total
2 weeks

9
8
Subtotal

9
8
17

5g explosive with body armour
Sham
Untrained
RAM trained
5 Choice Serial Reaction Task

2.12

3
8

8

8
10
10
Subtotal
Total animals used

3
24
10

10
47
64

Blast tube model and explosive protocols
The blast tube (Fig 2) used for this study was designed at the Swedish Defence

Research Agency (FOI) and described by Clemedson and Criborn (Clemedson CJ and
Criborn CO, 1955). The tube was initially used for rabbits and in vitro experiments on
muscular tissues (Clemedson CJ. et al., 1956, Clemedson CJ. and Pettersson H, 1956).
Subsequently, the tube was modified by Suneson for experiments with rats and used by
Saljo with an electric igniter for her thesis work (Säljö A et al., 2000). After further
modification, the blast tube was used with a non‐electric NONEL interval igniter (Risling
M et al., 2002a, Risling M. et al., 2002b).

11


Figure 2: Schematic diagram and picture of blast tube at Södersjukhuset, Sweden.

The Swedish army explosive M/46 SPRANGDEG46 PTR-NSP 71 PETN
(pentaerythritol tetranitrate) was used as the blast source. The shocktube had
previously been certified for up to 6 g of PETN. The PETN was mounted with tape on a
non-steel NONEL branch tube igniter at the closed end of the blast tube (Fig 2). The
NONEL tubing was connected to a control box in an adjacent room. Male adult rats,
weighing 300 to 380g, were deeply anaesthetized by an intra-peritoneal injection of 2.4
ml/kg of a mixture of 1 ml Dormicum® (5 mg/ml Midazolam, Roche), 1 ml Hypnorm®
(Janssen) and 2 ml of distilled water. The anesthetized rat was mounted at a distance of
1 m from the charge on either a wire mesh holder or with a specially fabricated metal
body armour (BA) that protected the animal’s body along the horizontal length of the

tube (Fig. 3).
3a

3b

Figure 3a) The anesthetized animal was mounted in a meshed metallic net to reduce acceleration
movement but not BOP exposure; b) Anesthetized animal was mounted with full body armour protection
to prevent lung injury, but with the head exposed to full BOP transmission.

12


For safety, all personnel were counted before the experiment and were held in an
adjacent room separated by a steel door during the blast. After the steel door was
closed and secured, accelerated ventilation in the blast tube lab was initiated and the
experiment supervisor ignited the charge. In the event of ignition malfunction, the
charge would be removed and destroyed after 15 min. After ignition of the charge, the
animal was rapidly removed from the blast tube setup and examined for airway
obstruction or bleeding. Body weight was monitoredbefore and after injury (up to 3
days) for any changes.

2.13

Optimizing explosives based on mortality
Pilot studies were carried out initially to determine the level of BOP that will result

in brain injury. The amount of explosives used ranged from 2 g to 5 g PETN, without
BA. The studies showed that without armour BA protection, all animals did not survive
more than 3 g PETN (~350 KPa). There was 50% mortality at 2.5 g explosives (2 of 4
animals died) and 100% survival at 2 g explosive (~240kPa). Post-mortem examination

of animals that did not survive 2.5 g and 3 g explosives were determined to have died
predominantly due to pulmonary haemorrhage. Behavioural assessment was conducted
on animals exposed to 2 g explosive without BA for up to 2 weeks to determine if there
was any degree of cognitive injury. Our results showed that 2 g explosive BOP (around
240kPa) was not enough to induce any significant behavioural changes in the test
animals and gross post mortem observations did not detect any obvious lung injury.

After understanding the lower limits of blast pressure that did not induce injury,
animals were exposed to a higher amount of explosive but with protective BA
subsequently (Fig. 3b). This BA used was designed to mimic the body armour worn by
the soldiers in the deployed scenario. The fabricated metal BA protects the animals
from possible lung or bodily injury but still allows full blast pressure transmission to the
cranial. For the main study, the effects of BOP to cause CNS injury was examined
using 5 g explosive on animals with full BA in comparison to 2 g explosive on animals
without any protection (blast control).

13


2.2 Modified Open Field Primary Blast Overpressure Model

To further understand the CNS injury profiles at different BOPs, an open field
blast injury model was conducted. This model allows us to examine a more specific
blast overpressure spectrum by varying distance from blast source and pressure
characteristics that can result in blast induced brain injury. This model was established
at ATREC Pte Ltd, Singapore. Explosives tests were conducted in a 10 by 10m
concrete and steel reinforced room.

2.21

Animal subjects

Male Sprague-Dawley rats (NUS CARE (Singapore) and ARC (Australia)
weighing 300-350 grams were used in these experiments. For this set of studies, the
animals used were housed in two different locations to explore the possibilities of
carrying out non-invasive magnetic resonance imaging at Biopolis, A*STAR. Hence, for
the pilot testing and first two sets of blast experiments, animals used were housed in
DSO and thereafter, at A*STAR, Singapore.

Experimental procedures were separated into 2 sections; the biomarker group
and the behavioral group. For the behavioral group, the animals were trained in the
selected tasks (except for BWT) till a stable baseline before subjected to the injury
procedure. Briefly, after 1 week quarantine after arrival at housing facilities, animals
were food restricted to 85% of their starting weight to ensure motivation to do the
behavioral task. The animals were allowed a weekly weight increase of 5% and had full
access to water. All rats were housed in pairs, separated by a perforated metal divider,
under conditions of room temperature on a 12 hrs regular light/dark cycle. Animals were
trained daily and rested for the weekend until reaching stable baseline. Baseline
performance was established prior to injury exposure and behavioral testing was carried
out after blast exposure until sacrifice at up to 1 month after injury. For the biomarker
group, animals were not trained in the behavioral task but were allowed to acclimatize to

14


the housing for 1 week before injury exposure. All animal experimentation protocols in
this research project were approved by the DSO Institutional Animal Care and Use
Committee (protocol number DSO/IACUC/09/74). A schematic of the experiment
procedure is shown below (Fig 4).

Fig 4: Schematic of Open Field experimental blast model for Biomarker group (left) and Behavioural
group (right).

15


2.22

Experimental Setup

Computer stimulations of the different amount of explosives and the resultant
pressure intensity was carried out to estimate and achieve the BOP required as below
and the amount of explosive to be used.
Injury
Mild
Moderate

Distance (m)

Pressure (KPa)

Positive duration (ms)

3

350

3

2.25

600

2.7

The required blast intensity and duration was an extension of the BOPs tested in
the shocktube model. The modified open field blast model was finalized using 5kg of
TNT (with PETN core) at a fixed distance of 2m and 3m away from the explosives. The
layout of the blast model is shown in Fig 5 and Fig 6. The explosive was placed in the
center of the room and was elevated one meter above the ground using a specially
fabricated wooden frame (Fig 5). This was done to reduce possible blast pressure
reflections from the ground and to permit equal radiation of pressure outwards to the
animals. Animals were placed in a metal rectangular wire mashed cages (1.5m by 0.8m
by 0.5m LWH) (Fig 6) at predetermined distances of either 2m or 3m from the explosive.
The wire mesh cages were placed in four quadrants relative to the explosive and
diagonally opposite to each other for each distance. The four cages were elevated
similarly at 1 m above the ground by metal rack stands and were connected in series
with a connecting beam secured to the rack stand. This arrangement not only prevented
the cages from moving during the blast and also ensured consistency between each
blast test.

16


Animal cages
Explosive

Connecting beam

Fig 5

Fig 5: Photograph of the experimental setup. The 4 animal cages were placed in the four quadrants
facing the 5kg TNT (with PETN booster) explosive at the epicentre. The cages were secured to a 1 m
high supporting rack and the 4 racks were linked up by a connecting beam to improve stability and
structural integrity.

Animal cages

Pressure
sensor

Fig 6

Supporting Rack

Fig 6: A close-up view of the wire mesh animal cage. The pressure sensor was placed at the same height
and distance with the animals for accurate blast pressure measurements

17


2.23

Pilot test

A pilot study was conducted to establish the experimental protocols of rodent
blast injury model and the resultant mortality of the animals when exposed to the
predetermined blast intensity. Animals with body armour will survive the blast exposure
at both mild and moderate pressure. Animals at mild blast pressure had a 90% survival
rate and none of animals suffered any adverse effects or injury or burns. With the
establishment of the mortality rate and calibration of the required blast distances,

subsequent blast experiments were then conducted to investigate the degree of BINT in
animals model with and without body protection.

2.24

Injury induction - Blast exposure

Under continuing anesthesia (ketamine/xylazine 75mg/10mg/kg at 0.2ml/100g
given ip), rats were subjected to explosive BOP with/without full body protection with
just head exposure. Full body protection consists of a customized acrylic holder that
covers the entire thoracic and lower body of the rodent whilst leaving the head exposed
(Fig 7a-b). The customized acrylic body armour consists mainly of a cylindrical holder to
contain the body (75mm in diameter, separated in two pieces) with a cone shape head
opening (with a 40mm diameter opening, 10mm length). An additional piece of cone
shape acrylic prevents head rotation. The anaesthetized animal was allowed to lie
comfortably inside the holder and the head opening is wide enough for the neck region
to prevent any suffocation. Soft sponges were added around the neck region prevent
any contact injury to the neck during blast exposure. The ears of the animals were
taped down with surgical micropore tape to prevent direct blast pressure to the tympanic
membrane. In addition, aqueous gel (Aquasonic® 100 Ultrasound Gel) was spread onto
the facial region, including the whiskers and eye and any other parts of the body that
was exposed to prevent any instances of burn injury. When the animal were prepared
and secured inside the armour, cable ties were used to fully secure the armour casing
as well as for the animal cages (Fig 8). A total of 5 blast experiments were carried out
(not inclusive of pilot testing). The numbers used for the study is shown in Table 4.

18


Fig 7b

Fig 7a

Fig 7a-b: Custom-made acrylic “body armour” to shield and protect thoracic region of the animals.

Fig 8: Picture showing the animals secured inside the body armour and tied down to the wire mesh
cages. The exposed heads were covered with adequate amount of aqueous gel to prevent burn injury.

Table 3
2m BH
Blast

3m BL
26

53
Total

Blast Sham
21
100

Table 3: Number of animals used for each injury and behavioural test and the time of sacrifice. BH: blast
high, BL: blast low.

19


2.3

General Experimental Procedures

2.31

Behavioural Testing

A series of neurobehavioral tests that were reported to be sensitive behavioral
measures for TBI were carried out to assess the recovery of the animals from the injury
exposure in the aspects of memory, motor coordination and sustained attention. The
neurobehavioral tasks, RAM (test memory), beam-walking test (test motor coordination)
and 5CSRTT (test sustained attention) were carried out as detailed below. Baseline
performance was established prior to injury exposure and behavioral testing was carried
out after blast exposure until sacrifice at up to 1 month after injury.

2.311 Radial Arm Maze
The radial arm maze (RAM, Fig 9) is a common tool used to investigate and
measure specific aspects of spatial working and reference memory of rodents. This task
is based upon the principle that the animals have evolved an optimal strategy to explore
their environment using their spatial memory abilities and obtain food with the minimum
amount of effort. Notably, damage to temporal lobe structures, particularly the
hippocampus, normal aging, and a variety of pharmacological agents would cause
impairment in spatial memory that could lead to decreased performance on the radial
arm maze.

Reward
dish

Central
Arena

Fig 9: Overhead view of the Radial Arm Maze and MazeSoft software.
20


The RAM (Panlab, Spain) consists of an octagonal central platform with eight
automated sliding guillotine doors giving access to eight radiating arms of equal lengths
(Wx1690mm, Lx1250mm, Hx1450mm)(Fig. 9). The apparatus is made of black
plexiglas and mounted on a tripod with adjustable height. Each arm has lateral walls
higher on the proximal side of the arm than on the distal side. In the distal extremity of
each arm contains a detachable recessed cup for baiting with food pellet (45mg,
raspberry flavored, Test diet). Each arm contains 2 sets of photoelectrical cell mounted
on proximal and distal end of the arm to differentiate between arm entries and visits.
Automation of the RAM by the MazeSoft software (Panlab, Spain) allowed; (i) the
location of the rat detected by the photoelectrical cells and visualized on the computer
screen, (ii) controlling the opening and closing of the guillotine doors to each radial
arms. Prominent extra-maze visual cues are present to allow spatial recognition of arm
position.
The experimental protocol of RAM was conducted in three main phases; namely:
(i) habituation, (ii) training, (iii) actual trials. Three days before habituation, animals were
deprived of food until their body weight was reduced to 85% of the initial free-feeding
weight. During habituation phase (span of 2-3 days) the food restricted rats were
familiarized to the maze. All the maze doors were kept opened and food rewards were
scattered around the maze to entice the rats to explore. After the rats were able to freely
explore the maze and consumed the food rewards at the food disc at the distal arm, the
protocol continued into the training phase (span of 10 or more days). The rats were
trained to retrieve a food pellet from each selected baited arm only once. Here, the rats
were placed in the center arena with all doors closed and after 30 s, all eight doors to
the radial arms will open and the rats allowed to freely explore until the entrance into
one arm was detected. Then all doors would close, except corresponding to the arm
being visited. After the animal turned back to the central area, the open door would then

be closed; all doors would remain closed until the confinement time (5s) had elapsed
and then reopened again for a new choice. This cycle of events was repeated
automatically until the rats visited all four baited arms or after 10 min had elapsed. For
each trial, the arm choice, latency to obtain all the pellets (i.e. response latency),
21


number of visits to each arm were automatically recorded by the MazeSoft software.
After each run, the maze was cleaned with absorbing paper to prevent a bias due to
olfactory intra-maze cues. Over time, the rats would also learn that certain arms were
not baited (i.e. reference memory) and avoid them accordingly. Once the rats achieved
a stable baseline performance (>85% accuracy) over a three-day period, they would be
induced with injury or sham-operated, then tested again at 72 h after injury to evaluate
spatial memory changes.

The confinement time was a critical feature of the maze in that they restricted the
rat to the central platform area between choices for a short period, hence preventing the
animal from developing a biased response habit. For example, without temporary
confinement between each arm choice, the rat could successfully solve this task by
simply always turning right/left after each choice and entering the first arm away from
the previously chosen one. This simple strategy does not require an accurate
knowledge of the spatial environment or spatial memory, and would give a biased
response pattern. Unless the investigator is primarily interested in studying response
patterns, it is best to have the rat confined to the central platform prior to making each
arm choice. The current protocol defined a five seconds delay during confinement of the
rats to central arena. This would increase the difficulty of the rats to remember which
previous arms it had visit (hence working memory errors) and increase the duration
required for training.

From the experimental trials, the following data may be obtained for each rat for

data analysis and interpretation:
i.

Number of revisit to baited arms: A working memory error used to indicate spatial
working memory performance.

ii.

Number of visit to non-baited arms: A reference memory error used to indicate
spatial reference memory performance.

iii.

Latency to retrieve all baited arms: A measure of the level of motivation of the rat
on the RAM task.

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2.312 Beam-walking Test (BWT)

The beam-walking task (BWT) is a commonly used test procedure for the
assessment of balance and coordination. The traversing of the narrow beam by the
rodent subject involves both central and peripheral neural processes for proper
integration of sensory inputs (i.e. proprioception & balance) and the subsequent
elicitation of motor adjustment (i.e. muscle tone & limb movement). As compared to the
rotarod (also used for measurement of motor coordination & balance), the BWT may be
potentially less stressful for the rodent subject since negative reinforcement (i.e. foot
shock) is not actively used during task training. Subjects are also generally able to
master the beam walking task in a shorter time (i.e. within a day), as compared to the

long training period required to achieve baseline performance on the rotarod.
1m
0.5m
Goalbox

A

B

C

~30cm

Foam cushion
Fig 10: Laboratory set-up of Beam Walk Apparatus

The apparatus used in the beam-walking test is very basic. It consists mainly of a
narrow flat beam (2.5cm wide) leading to a brightly decorated goal-box. Foam cushions
are placed below the beam to cushion any accidental fall. The conduct of the beam
walking test consists of three phases; namely: 1) Habituation, 2) Progressive beam
training, and 3) Actual trials. In the habituation phase, the food-restricted rat is first
placed into the goal-box to feed on the rat chow provided. Subsequently, progressive
beam training is initiated, starting from the beam-end closest to the goal-box (Point A)
and progressing gradually to the opposite end (Point C) (Fig. 10). A neurologically intact
rat will traverse the beam with no difficulty.

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2.313 Five-Choice Serial Reaction Time Task

The Five-Choice Serial Reaction Time Task (5CSRTT) was originally developed
for use with rats by Trevor Robbins’ group in Cambridge to specify the psychology and
underlying neurobiology of attentional processes. More recently, it has also been used
to study impulse control. The task requires the detection of brief flashes of light
presented pseudo-randomly across five spatial locations. Detection is signaled by a
nose-poke response from the rat. Stimuli are presented rapidly in multiple trials, and
hence the task does have some degree of analogy with continuous performance tests
used in humans (Leonard, 1959; Wilkinson, 1963). As has been discussed in many
reviews (Bushnell, 1998; Robbins, 2002), manipulations of the basic task parameters
provide relatively independent behavioral indices of dissociable aspects of attention,
impulse control, and even, compulsive behavior.

The apparatus used for the 5CSRTT consists of an operant chamber placed
within a sound-attenuating box. Within the chamber, five niches are found on one side
and each of these niches contains a light emitting diode (i.e. for stimulus presentation)
and an infrared motion sensor. On the opposite side of the chamber, a food magazine
linked to a food pellet dispenser is installed for implementation of positive reinforcement
to correct responses (Fig. 11).

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Food Magazine
(ie. for reward)

5 niches with
LED & infrared
sensors

Fig 11: Interior view of the five-choice chamber.

The conduct of the 5CSRTT for assessment of sustained attention consists of
three main phases; namely: 1) Response Training, 2) Progressive 5CSRTT Training,
and 3) Actual trials. A food restriction protocol is also in place to ensure motivation on
the task and appropriate nutrition of the rats. Briefly, in the “Response Training” phase,
the food-restricted subjects are placed into the chamber for habituation to the context
and food pellets, and, subsequently, the shaping of the nose-poke and pellet retrieval
behavior. The subjects are then moved on to the “Progressive 5CSRTT Training” in
which a progressive training protocol trains the subjects to detect and nose-poke the
appropriate niche for food reward with shortening time window over sessions. Upon
satisfaction of prescribed performance criteria (>60% correct; less than 20% missed;
over 3 consecutive days) at stipulated test parameters (1s stimulus duration; 5s limited
hold), the subjects may be used to evaluate the test substances. Retest reliability is
superb for this test but residual effect (i.e. crossover effect) of agent used should be
considered.

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