The Effects of Mild Traumatic Brain Injury on the Activation of a Diffuse Noxious Inhibitory Control System in Rats

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The Effects of Mild Traumatic Brain Injury on the Activation of a Diffuse Noxious Inhibitory Control System in Rats
Baker, Brandi
Yezierski, Robert ( Mentor )
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Gainesville, Fla.
University of Florida
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The Effects of Mild Traumatic Brain Injury on the Activation of a
Diffuse Noxious Inhibitory Control System in Rats

Brandi Baker


The sensation of pain involves the transmission of nerve impulses from the source of tissue damage or injury,

up through the spinal cord, and through multiple pain-processing pathways in the brain. These pathways have

a protective function as they help detect sources of injury or potentially tissue damaging stimuli before it is too

late. Take for example people with Riley-Day syndrome. Fifty percent of individuals with this condition only live to

the age of 20 because they are not equipped with a functioning pain-processing system vital for the recognition

of harmful or injurious stimuli.1 At the other end of the pain continuum are individuals suffering from chronic

pain conditions such as Post Concussive Syndrome (PCS). These patients present symptoms and impairments such

as poor concentration, dizziness, along with head and neck pain.2 One theory as to how this chronic pain

condition develops involves a cascade of secondary events associated with mild traumatic brain injury

(MTBI). However, the effects of MTBI on pain are not understood because most of the research related to

MTBI focuses on cognitive function and recovery, thereby making studies related to altered sensation, including

pain, a novel area of research in the field of brain injury.

MTBIs are often due to vehicular accidents, falls, sports injuries, and acts of violence, and most commonly

affect young men between the ages of 15 and 24.3 MTBI, like other types of brain injury, involve both primary

and secondary events. The primary event is the direct, mechanical disruption of brain tissue that occurs at the

time of injury. The secondary phase encompasses a wide range of chemical and anatomical events in the

central nervous system (CNS) that occur after brain injury. Secondary injury in the field of central nervous

system trauma is of great interest because it has the potential of being, at least in part, prevented. However,

despite significant scientific strides in this direction, there exists no effective treatment strategy. Treating brain

injury is extremely challenging, in part, due to the wide range of physiological changes associated with brain

trauma. A well-characterized effect of TBI is diffuse axonal injury. Trauma to the head causes a shearing

and stretching of axons in the brain. This physical stress on nerve processes causes a variety of secondary

events including axonal swelling, structural damage, compromises in axonal transport, changes in

phosphorylation states, mitochondrial damage, and, in some cases, axonal disconnection from post-synaptic targets.4

The widespread diffuse fiber damage that is typical for MTBI can affect the processing of sensory

information, including that related to the sensation of pain. The reason for this is that processing of

nociceptive signals occurs in a decentralized manner and involves many areas of the brain and brainstem.5

The intensity of the pain experience, for example, is represented in the somatosensory and anterior cingulate

cortex, and in the periventricular area.5 The S1 somatosensory cortex is believed to be responsible for

discriminating between nociceptive and non-nociceptive stimuli, the coding of stimulus intensity, and the

localization of sensory events on the body.6 At cortical levels, the temporally integrated response to repetitive

or prolonged nociceptive stimuli is encoded in Brodman's area 3a. A bi-directional inhibitory interaction has also

been proposed between area 3a and adjacent areas of the somatosensory cortex, based on cortical activity

mapping studies.7 It is therefore possible that MTBI could lead to abnormal sensory states by disturbing

the functional balance existing between different parts of the somatosensory cortex as well as connections

with subcortical structures. Therefore, the goal of the present study was to investigate how recent MTBI

interferes with these circuits to cause changes in the processing of sensory and affective aspects of pain perception.


Traumatic Brain Injury

Twelve female Sprague-Dawley rats, weighing between 280 and 300 grams, were housed in standard cages with

ad libitum food and water. All animals were treated in accordance with the guidelines set forth by the University

of Florida IACUC. Six rats were initially anesthetized in a large glass container using a gauze pad saturated with 3-

5 ml of liquid halothane (2-Bromo-2-chloro-1,1,1-triluoroethane) that was taped to the top of the container. Once

the animal was sedated, a subcutaneous injection of a Ketamine, Xylazine, and Acepromazine mixture (Rat

Cocktail) was administered. The dosages of each drug per animal were 27.8 mg/kg of Ketamine, 5.57 mg/kg

of Xylazine and 0.91 mg/kg of Acepromazine (10 mg/ml). Once the animal was deeply anesthetized, the scalp

was shaved to allow for a midline incision in the scalp. The skin was reflected and the periosteum covering the

bone was removed. The skull was scraped to ensure adequate fixation of a 10 mm stainless-steel disc to the skull

on the coronal suture between lambda and bregma with dental acrylic. The animal was placed in a prone position

on a foam bed of known spring constant within a Plexiglas frame. A 450 gram brass weight was dropped onto

the disk from a height of 1 meter through a Plexiglas tube held in place with a ring stand onto the disc (Figure

1). The weight impacted the skull with a velocity of 4-6 m/s, causing a maximum skull compression of 0.2 mm

as calculated by Marmarou et al.8 After injury, the head incision was closed with staples and the animal was placed

in an enclosed circular area for 6 hours to monitor recovery from the above procedures. In addition to the six

animals undergoing TBI, a second group of six animals received the same anesthesia and scalp incision as the

MTBI group; however, they did not receive the head injury. Behavioral testing of all 12 animals resumed the

day after surgery. The staples were removed one week post-incision. Post-injury data was collected for six weeks.

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Behavioral Assessment

Two behavioral tasks were run each week. The first test assessed the escape behavior of animals

in response to a thermal stimulus delivered to the feet (either 36EC or 45EC). The shuttle-box

apparatus used in this test consisted of an aluminum thermal plate measuring 18 X 29 X 2 cm and

an escape platform measuring 12.5 X 15.3 cm, tilted at an angle of 12E toward the dark enclosure

(Figure 2A).9 The plate and platform were separated by a swinging divider, which served to isolate

an aversive 50 W halogen light on the side of the escape platform from the thermal plate.

Both compartments were enclosed within a heat-absorbing, tinted, Plexiglas box. Rats were

first acclimated to the box using 36EC in the absence of the light. Gradually the plate temperature

was raised and the light implemented to allow the rats to learn the difference between the

two chambers. After training, baseline data was collected four days per week for three weeks prior

to MTBI. The trial time for each session was 10 minutes, during which the time spent on the

platform (escape duration) was measured. Each rat was tested at both temperatures each day,

varying the order of temperature. In testing escape behavior, the 45EC temperature was chosen

because it is slightly higher than a temperature required to activate thermal nociceptors and because

it did not cause tissue damage. The 36EC stimulus was a neutral temperature used as a control

for learned avoidance of the thermal plate.

Figure 2A. Operant escape testing appartus.

Figure 2B. Dark Box testing appartus.

The second outcome measure, assessed once per week, used a two-sided box separated by a

small passageway that was accessible over a raised platform. This test, referred to as the Dark Box

test, served as a motor control for the operant escape test (Figure 2B).9 Three walls of both

chambers and the translucent floor were equipped with florescent lights that served to elicit escape

from one side of the box to the other. A computer program automatically conducted a test consisting

of 10, 140-second-long trials. After an initial 10-second lights out period, both chambers

were illuminated. When the animal moved from one compartment to the other, the lights in the

occupied compartment shut off, but the other side stayed lit for a total of 70 seconds. The

computer recorded the animal's escape latency, which was the time between the presentation of

light and its movement to the other side. After 70 seconds, the light in the original compartment shut

off and a 70-second time-out with no light followed. This process was repeated for a total of ten trials.


Performance data on the two behavioral tasks during post-injury testing were compared to

the respective baselines of each group. Data collected on the operant escape task revealed a

significant difference in escape behavior between the incision-only group and the incision + MTBI

group during the first week post-injury (Fig 3). No significant differences between the groups were

seen after the first week. The platform duration of the incision-only group, using a plate temperature

of 45EC, decreased compared to baseline (Fig 3), indicating a decrease in sensitivity to the

thermal stimulus. The amount of time spent on the platform by the incision + MTBI group at 45EC did

not significantly differ from that of the pre-injury period suggesting no change in sensitivity to

the thermal stimulus. Differences between the respective baseline and post-injury platform times for

the two groups were calculated to be approximately 60 seconds. The results of the Dark Box test for

both groups revealed no differences in escape latencies from pre- to post-injury. These results show

that the experimental conditions did not affect the motor performance of the animals.

Escape Platform Time Difference Between Incision-Only
Group and MTBI+Incision Group

CL MT BI +miias mIup

Figure 3. Diagram showing the longer platform time of the MTBI+ incision group than

the incision-only group during week 1. Escape behavior returned to normal by week 2.


The results from the escape test showed that the incision-only group spent less time on the

platform, meaning they spent a longer duration on the thermal plate at 45EC. This demonstrates

a decrease in sensitivity among the incision-only group. In contrast, the platform time of the MTBI

group did not significantly differ from baseline, suggesting their sensitivity to the noxious

thermal stimulus did not decrease as in the incision-only group. The escape latencies in the Dark Box

test were not changed from pre- to post-injury, indicating that any change in behavior noted in

the operant escape task was not due to changes in motor behavior. One possible explanation for

the decrease in escape duration for the incision-only group is that the incision, staples, or stress from

the experimental condition activated an endogenous inhibitory system that decreased

thermal sensitivity. This change was not seen in the incision + MTBI group presumably because the

head trauma interfered with the activation of an intrinsic inhibitory system and, therefore, this group

was incapable of showing the same decrease in thermal sensitivity. The results of this study are the

first to show an effect of MTBI on the processing of sensory information. The results support

the conclusion that diffuse damage to multi-synaptic inhibitory pathways in the brain caused by

the injury resulted in a "disabling" of pain inhibitory systems. MTBI may have damaged

descending inhibitory pathways or circuitry responsible for their activation, thereby decreasing

the influence of these pathways on thermally sensitive neurons in the spinal cord. Regardless of

the exact mechanisms responsible for the behavioral effect, it is clear that there was an absolute

change in the processing of thermal information in the incision + MTBI group.

In the incision-only group, it is believed that a pain inhibitory system was activated by the tissue

damage from the incision conditions, from the stress of having staples in the skin, and/or from

a combination of these conditions. The fact that this difference lasted only one week suggests

that removal of the staples and recovery of the incision no longer provided the stimulus required

for activation of the inhibitory pathways. Further studies that may elucidate the source of the

pain-induced or stress-induced changes are necessary to gain a better understanding of the

neurological changes produced by MTBI. The logical progression from these data will be to study

the gross histology as well as to search for biochemical markers that signify a change in the

normal functioning of the brain after MTBI. Pharmacological studies that would help identify

the mechanism responsible for the effect in the incision-only group would involve trying to block

this effect with the opiate antagonist naloxone. Previous studies support a role of endogenous opiates

in the modulation of pain pathways10'11. In an effort to evaluate the persistence and time course of

the MTBI effect and the reproducibility of the incision effect, it will be important to repeat the

incision protocol in both groups of animals. Biochemical assays at the time-point of maximal effect

should provide further insight into the mechanism responsible for the behavioral differences between

the two groups. Continued research related to the impact of acute head injury on sensory processing

will allow for a more comprehensive assessment and treatment of brain injury patients as well as

a better understanding of the varying severity of head injuries and the functional deficits

experienced clinically.



Hayek S. et al. Spinal deformity in familial dysautonomia. Prevalence and results of bracing. J Bone

Joint Surg Am. 82, 1558-1562 (2000).


Alexander, M.P. Mild traumatic brain injury: Pathophysiology, natural history, and clinical

management. Neurolog


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