    
The Effects of Environmental Cross-Over on Inflammation-Induced Nociception 
 
 
by 
 
 
 
Jillian Kay Armentrout 
 
 
 
 
Submitted in Partial Fulfillment of the Requirements 
 
for the Degree of 
 
Master of Science 
 
in the 
 
Biological Sciences 
 
Program 
 
 
 
 
 
YOUNGSTOWN STATE UNIVERSITY 
 
August, 2014 
 
 
 
 
 
 
 
 
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  ABSTRACT 
 
 
There are many studies in which environmental enrichment (EE) has focused 
on modifying animal behavior leading to improved cognitive functions, such as 
learning and memory. There are also increasing numbers on how EE positively affects 
a subject’s pain tolerance by increasing thresholds. In order to better understand how 
an environment can affect nociceptive behaviors, this study looked at the effects of 
cross-over from one environment to another after induced inflammation. Male 
Sprague-Dawley rats were initially randomly assigned three to a cage; four of the 
cages were standard, only containing bedding and food/water, while the other four 
cages were supplemented with toys (enriched). Baseline behavioral measurements, 
paw thickness, paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) 
were collected. After five weeks, CFA was injected into the left hind paw of all 24 rats 
and the cross-over was conducted. Two of the initial standard cages stayed standard, 
while the remaining two standard cages changed to enriched. Two of the initial 
enriched cages changed to standard, while the remaining two enriched cages stayed 
enriched. The behavioral measurements were then conducted post CFA at 2 hours, 7 
days, 14 days, 21 days, 28 days, and 35 days. A repeated measures ANOVA was 
conducted on paw thickness for all four treatment groups (standard-standard, 
standard-enriched, enriched-standard, enriched-enriched) at baseline, 2 hours, 7 days, 
14 days, 21 days, 28 days, and 35 days post-inflammation. F
10.125,44
=1.403 with  no 
significant difference among home cage treatment groups (p-value = 0.180). There 
was, however, a significant difference across testing times (2h, 7d, 14d, etc.; p-value < 
0.001) and F
3.485,44
=1.403. The repeated measures ANOVA conducted on PWT 
revealed F
3.801,44
=
 
2.520 and no significant difference among the four housing groups 
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(p-value = 0.311). There was a slight significant difference across behavioral testing 
times (p-value = 0.046), and F
11.402,44
=1.169. The repeated measures ANOVA 
conducted on PWL showed no significant difference among home cage housing 
groups (p-value = 0.446) and F
11.244,44
=1.004.There was a significant difference across 
behavioral testing times (p-value < 0.001) and F
3.748,44
=22.405. 
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TABLE OF CONTENTS 
ABSTRACT             iii 
TABLE OF CONTENTS           v 
LIST OF FIGURES                                               vi 
ACKNOWLEDGEMENTS           vii 
 
CHAPTERS           CHAPTER    PAGE 
 ,  INTRODUCTION            I        1 
 
 , ,  MATERIALS AND METHODS           II        26 
 
 , , ,  RESULTS              III      32 
 
IV. DISCUSSION             IV      39 
 
REFERENCES             VI       44 
 
APPENDIX A: Animal Use Approval Form 
 
 
 
 
 
 
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LIST OF FIGURES 
 
FIGURES         PAGE 
1. Anterolateral System           13      
2.  Gate Control Theory of Pain                     14         
3. Housing Conditions           28  
4. Experimental Design           31 
5. Thickness            35 
6. Paw Withdrawal Threshold          36  
7. Paw Withdrawal Latency          37 
     
 
TABLES         PAGE 
      1. C-fiber Nociceptors and A-delta fiber Nociceptors               12 
 
 
 
 
 
 
 
 
 
 
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  ACKNOWLEDGEMENTS 
 
 On this amazing journey that I have taken over the past two years there are 
quite a few people I would like to thank. First, I extend great gratitude and thanks to 
Dr. Tall for everything she has done. Not only has she been there for guidance 
through my research, but also through life. I would also like to thank Dr. Shoup-Knox 
and Dr. Womble for their constant feedback and helpful remarks throughout this 
journey. There is no way I would be where I am today if it were not for the help of 
these two.  
 I would also like to thank my family for their on-going support and love 
through everything. Also, a tremendous thank you to my R.J. If it were not for his 
constant pushing and support I do not know how I would have made it through the 
past two years! 
 
 
 
 
 
 
 
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Chapter 1                                                                                      
 ,  Introduction 
A. Pain: The Sensory Experience 
Historically, pain was treated as a subcategory of somatic sensation (Kandel et 
al., 2000), but studies conducted on this modality have concluded that pain must be 
viewed as a distinct sense. Pain is a highly individualized experience and is 
modifiable (Melzack, 1961). Pain’s variable nature is due to a number of reasons, 
including the capacity of an individual to deal with a stressful situation, both the 
internal (endogenous hormones) and external (exogenous medications) algesic and 
analgesic chemicals, and the individual’s personal characteristics such as sex, age, 
and personality (Ramirez-Maestre et al., 2003).  The most fitting definition for pain is 
that it is a complex phenomenon of unpleasant sensory, emotional, and cognitive 
experiences that are brought about by real, potential, or perceived tissue damage 
(Terman and Bonica, 2003). Physiological pain is known to be a direct result of tissue 
damage and serves as an adaptation, or alert system, to make organisms aware of 
noxious stimuli by triggering negative, affective responses. It is important to 
remember that pain is a heavily modulated sensation and is integrated with many 
other systems of the brain, rather than a separate sense functioning in isolation 
(Shepherd, 1994). 
1. Inflammation and Pain 
Tissue damage causes inflammation, a nonspecific immune response. There 
are four cardinal signs and symptoms of localized inflammation: pain, swelling, 
redness, and heat. Inflammatory pain is caused by a variety of chemical factors 
released by damaged cells, called the “Inflammatory Soup” (e.g., prostaglandins, 
Substance P, hydrogen ions, bradykinin), that leads to neuroplastic changes. These 
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 
changes consist of an increase in sensitivity by  afferent neurons in the peripheral 
nervous system (PNS), and an increase in sensitivity of spinal cord dorsal horn 
neurons as well as other neurons in the central nervous system (CNS; Ji, 2004).  
Upon induction of the inflammatory response, the immune system responds 
with both direct and indirect defenses in order to combat the changes that occur 
during inflammation. Direct defense consists of the phagocytosis of damaged cells, 
while the indirect defense consists of two branches; innate immunity and 
adaptive/acquired immunity. The major cell type that plays a precipitating role in all 
three defense systems is the mast cell. These cells serve to give the body the ability to 
resist toxins/organisms that are capable of causing damage to tissues and organs. Mast 
cells are spread throughout connective tissues of the body, especially beneath the 
surface of the skin, near blood vessels and near lymphatic vessels. These cells 
synthesize and release heparin, a weak anticoagulant, and histamine, a chemical 
mediator of inflammation which increases vascular permeability (Eroschenko, 2005). 
With this increased permeability, excess fluid leaks out of circulation into the 
interstitium and induces edema. Along with increased vasodilation, edema accounts 
for the swelling, redness, and warmth associated with localized inflammation. 
Serotonin, bradykinin, and a variety of prostanoids are also released by mast cells at 
the site of local inflammation (Fearon and Locksley, 1996). 
 Prostanoids are a family of lipid based molecules that are derived from the 
action of cyclooxygenases or prostaglandin synthases on eicosanoids. Prostanoids can 
be divided into three groups, prostaglandins, prostacyclins, and thromboxanes 
(Nicolaou, 2004). Prostanoids are active in processes such as blood pressure 
regulation, hemostasis, and production of the inflammatory response. During 
inflammation, prostanoids are synthesized in greater quantities than what is normally 
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 
present during basal conditions within the body. When a tissue is exposed to a 
noxious stimulus, such as a sprain or strain, arachidonic acid is released from cell 
membrane phospholipids by activation of the enzyme phospholipase A2 (Smith, 
1992), due to an increased calcium ion influx. Arachidonic acid is converted to 
prostaglandin H
2
 (PGH
2
) by prostaglandin G/H synthase 1 or 2 and PGH
2
 is then 
converted into the active, paracrine prostanoid chemicals. There is growing evidence 
that show that prostaglandin G/H synthase 1 or 2 -derived prostaglandins are critical 
in many cellular processes including cell proliferation, differentiation, and apoptosis, 
which are all inflammation-associated responses. In addition, prostaglandins are 
associated with the pathogenesis of cancer, cardiovascular disorders, and 
inflammation (Zhang et al., 2010). Prostaglandin E
2
 (PGE
2
), an active prostanoid, is 
critical to the pain associated with inflammation by increasing the responsiveness of 
peripheral nociceptors in inflamed tissues, and is formed via the conversion of PGH
2 
by PGE synthase. 
Once PGE
2
 is formed, it is transported through the membrane of the cell where 
it is produced by the ATP-dependent multidrug resistance protein-4 in order to act on 
the cell it was secreted from (autocrine action) or near (paracrine action) its site of 
secretion (Park et. al, 2006). PGE
2 
acts via four subtypes of metabotropic receptors 
named E-prostanoid receptors, or EP
1
-EP
4
. The binding to EP
1
 receptors on the 
primary afferent neuron plays a major role in hyperalgesia, a classic symptom of 
inflammation that will be subsequently discussed, by acting on peripheral sensory 
neurons innervating structures at the site of inflammation (Moriyama et. al, 2005; 
Dubois et. al, 1998). Studies have shown that EP
2
 receptors located in the dorsal horn 
of the spinal cord, and EP
4
 receptors located on the primary afferent neuron, are 
significantly upregulated in induced paw edema in rats (Yuhki et. al, 2004), showing 
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 
the importance of PGE
2
 in mediating inflammation. Activation by PGE
2
 of EP
1
 –EP
4
 
receptors on pain sensing neurons results in a lowered action potential threshold, 
allowing for a painful response to be elicited under innocuous situations or 
exaggerated. Behaviorally, this effect is observed as hyperalgesia and allodynia. 
Sensitization describes a change in neuronal function following repeated 
stimulation. When a pain signal activates an afferent neuron, there is an increase in 
neuronal excitability and a lowering of pain thresholds, allowing for an increased 
response to stimuli (Woolf, 1983; Woolf and Thompson, 1991; Dubner and Ruda, 
1992; Coderre et al., 1993), resulting in hyperalgesic and allodynic responses. These 
altered pain-related responses develop in animal and human subjects following a 
cutaneous injury (Cervero and Laird, 1996). Tissue damage often leads to an increase 
in the response to subsequent noxious stimuli, a phenomenon called hypersensitivity 
or hyperalgesia. Allodynia, also a result of sensitization, is a painful response to a 
stimulus that would not normally produce pain, or an innocuous stimulus. 
Hyperexcitability of neurons related to the sensation of pain is produced by local 
inflammation. 
Acute, localized inflammation normally has a beginning and an end. The 
temporal aspects of the inflammatory response are mediated by the synthesis and 
release of chemicals that are pro-inflammatory and anti-inflammatory, respectively.  
Alterations to these temporal processes can lead to long-term, maladaptive 
inflammation. Sub-chronic inflammation is defined as inflammation lasting longer 
than acute inflammation, but shorter than chronic inflammation, usually around 6-12 
weeks. Chronic inflammation occurs when an inflammatory response is not 
terminated, so there is a continued production of pro-inflammatory, chemical 
mediators. This type of inflammation lasts longer than 12 weeks. Symptoms of 
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 
chronic inflammation include systemic effects such as body aches, sinus congestion, 
shortness of breath, swelling, stiffness, and weight gain (Reasoner, 2010). Normally 
the biochemical reactions of inflammation cease when homeostasis is restored, but in 
the case of pathological chronic inflammation, the communication is disrupted and the 
reactions do not “turn off” (Pick, 2011). Eventually, through temporal release, the 
inflammatory response does cease in chronic inflammation, going into remission. 
However, relapse is common. 
2. Experimental Models of Inflammation 
      In order to gain a better understanding of the signs and symptoms of 
inflammation, experimental animal models using a single injection of a pro-
inflammatory chemical into the hind paw or joint have been developed (Ji, 2004).  
The development of reproducible animal models of human ailments has been crucial 
to gaining a better understanding of underlying pathophysiologic factors and has 
contributed to the development of more effective treatments. One of the most 
commonly used agents in preclinical investigations has been Complete Freund’s 
Adjuvant (CFA; Stein, 1985). CFA is an injectable mixture of heat-killed bacteria 
suspended in sterile mineral oils that induces a sub-chronic inflammatory response 
(Freund, 1947). Another experimental model used to induce acute inflammation is an 
injection of carrageenan, a method originally developed as an effective way to 
standardize testing of anti-inflammatory drugs (Winters, 1962). Carrageenan and CFA 
are well-established experimental models of inflammation with a long history of 
effective use in preclinical studies, producing the same signs and symptoms 
associated with localized inflammation as mentioned previously. 
Actual and experimentally-induced inflammation results from tissue damage 
and elicits the sensation of pain. The ascending pain pathway is a three neuron 
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 
journey to the cerebral cortex with collateral branches transmitting noxious input to 
accessory areas of the brain as well as activating endogenous analgesia. 
B. Anterolateral System (ALS) 
The circuit anatomy of nociception in the human body consists of neurons and 
pathways leading to the cerebral cortex. The ALS (Fig. 1) is a group of sensory 
pathways ascending the spinal cord in the anterolateral portion of the white matter 
that conveys information about temperature and noxious stimuli to the brain. The 
ALS contains three pathways, including the spinothalamic (neospinothalamic) 
pathway, the spinoreticular (paleospinothalamic) pathway and the 
spinomesencephalic pathway. The cutaneous nociceptive pathways begin at primary 
afferent nociceptors, neurons with cell bodies located in the dorsal root ganglion. The 
central axonal process of this neuron synapses onto the second order neuron in the 
dorsal horn of the spinal cord, which sends its axons rostrally as the spinothalamic 
tract. The neurons of the spinothalamic tract eventually synapse onto a third order 
neuron located in the thalamus. The third order neuron terminates on neurons of the 
primary somatosensory cortex in the parietal lobe of the cerebral cortex. Together 
these pathways allow the site of noxious stimulation to be localized, to be consciously 
perceived, and allow for the activation of the descending inhibitory pathway. 
1. Nociceptor 
The first step in the perception of noxious stimuli occurs at the level of the 
sensory neuron. The behavioral response to a noxious stimulus is a combination of 
PNS-related nociception and CNS-related pain perception. Nociception is elicited by 
noxious thermal stimulation, pressures and mechanical stimuli, or chemical 
stimulation. Nociceptors are a class of peripheral sensory neurons that exhibit high 
activation thresholds and preferentially respond to noxious stimuli (Basbaum, 1984). 
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It should be mentioned that “nociceptor” is not only a term for describing the sensory 
receptor located in the cell membrane of the afferent neuron, but is also the entire 
afferent neuron that carries the initial action potential as well. 
a. Sensory Signal Transduction 
The first process in nociception is the transduction of a noxious stimulus into 
an action potential. Neurons are excitable cells that have a resting membrane potential 
(RMP) between -60mV and  -90mV established and maintained by ionic 
concentration gradients across the neuronal plasma membrane. Two important ions 
associated with membrane potentials are sodium (Na
+
) and potassium (K
+
). At the 
RMP Na
+
 tends to leak into and K
+
 tends to leak out of the neuron, while cells have 
protein pumps that compensate for the passive leakage by pumping K
+
 into and Na
+
 
out of the neuron. The Na+/K+ ATPase pump, a primary active transport protein, uses 
the hydrolysis of adenosine triphosphate (ATP) to catalyze the movement of three Na
+
 
ions out of the cell for every two K
+
 ions moving inward. The pump does not directly 
participate in the generation of action potentials, but serves to maintain the 
concentration gradients for Na
+
 and K
+
 across the cell membrane. In order for an 
action potential to be initiated in the peripheral axonal terminal of the nociceptor, the 
neuron must depolarize to a threshold potential (Hodgkin and Huxley, 1952). 
Depolarizing currents are produced by the movement of ions through a variety of 
plasma membrane-associated protein channels. 
 Transient receptor potential (TRP) channels are ion channels involved in the 
production of action potentials in nociceptive neurons. TRP channels are located 
within the plasma membrane of afferent neuron terminals and are non-selective cation 
channels, which when open permit the influx of Na
+
 and calcium (Ca
+2
). The TRPs 
resemble voltage-gated channels with the central pore closed at the RMP. Although 
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 
TRP channels may be weakly voltage-dependent, they lack the voltage sensor of 
voltage-gated Na
+
 or K
+
 channels. Instead, TRP channels act as sensors of osmotic 
pressure, temperature, and extracellular chemical changes. When homeostasis is 
disrupted, due to a noxious stimulus, TRP channel opening leads to a Na
+
 and Ca
+2  
influx into the afferent neuron terminal, producing a depolarizing current. TRP 
channels are able to produce the initial depolarizing current because they are located 
on nociceptors themselves.  
Ligand-gated ion channels open in response to the binding of an extracellular 
chemical stimulus and are referred to ionotropic receptors. These channels are formed 
by the non-covalent binding of several subunits facing one another to form an 
aqueous pore region. This pore can be highly selective for either Na
+
, K
+
, Ca
2+
, or 
chloride (Cl
-
).  The depolarization produced from the TRP channels, mentioned 
abover, initiates the opening of voltage-gated Na
+
 and Ca
+2  
 ion channels, causing a 
further depolarization due to the additional influx of Na
+
 and Ca
+2
. If the 
depolarization is sufficient to reach the threshold voltage, an action potential is 
initiated.  
Action potentials that carry nociceptive pain information are initiated in 
nociceptive neurons by noxious temperature, acidic chemicals such as hydrochloride 
(HCl), or noxious mechanical stimuli. At high temperatures, greater than 43º C, the 
rate of ionic diffusion increases, allowing for an increased influx of Na
+ 
 via 
temperature-sensitive TRP channels. This channel opening leads to the depolarization 
and the production of an action potential in the nociceptor. When HCl is introduced to 
the interstitial fluid, there is an influx of H
+
 through voltage-gated ion channels into 
the neuron. This positive influx results in nociceptor depolarization. Lastly, intense 
mechanical stimulation, such as hitting your thumb with a hammer, activates 
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 
mechanically-gated Na
+
 channels resulting in the production of a depolarizing 
potential with sufficient magnitude to initiate an action potential in the nociceptive 
neuron.  
b. Nociceptor- Primary Afferent Neuron 
Action potential signals, no matter the sub-modalities of noxious stimuli, are 
carried into the CNS by primary afferent nociceptor neurons known as A-delta and C 
fiber nociceptors. C- fibers, or C-mechano-heat receptors, are polymodal neurons that 
are sensitive to noxious thermal, mechanical, and chemical stimuli (Table 1). The 
axons of these neurons are unmyelinated, giving them a relatively slow action 
potential conducting time (0.5 – 2 m/sec). This produces a “burning” sensation when 
activated (Shepherd, 1994). C-fiber central axon terminals are widely distributed 
(Dubin, 2010), but mostly terminate in the superficial layers of the spinal cord dorsal 
horn, such as the substantia gelatinosa.  
A-delta fibers are thinly myelinated afferent nociceptors that can carry action 
potentials 5 to 30 meters per second, are generally responsible for noxious mechano-
sensation and heat, and are responsible for the fast on-set of pain sensation (Table 1). 
A-delta fibers also synapse onto second order neurons within the substantia gelatinosa 
of the dorsal horn. 
In 1965, Melzack and Wall introduced the Gate Control Theory of Pain 
(Melzack and Wall, 1965). This theory explains that nociceptive impulses are 
transmitted from peripheral nociceptor nerve endings within the skin to the substantia 
gelatinosa of the spinal cord dorsal horn through myelinated A-delta fibers and 
unmyelinated C fibers. The central axonal terminations and interneurons within this 
layer of the dorsal horn act as a “gate” which regulates the transmission of nociceptive 
impulses to the brain (Huether, 2004; Fig. 2). If an action potential is transmitted 
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 
through C fibers or A-delta fibers, the gate is “open,” allowing the pain information to 
be transmitted to the brain. If an action potential is also simultaneously conveyed by 
large, A-beta fibers, which carry information about touch, the gate is “closed,” 
blocking nociception and no signal is transmitted to the brain (Freudenrich, 2007).  
There are two excitatory neurotransmitters, glutamate and substance P, that 
mediate the chemical neurotransmission between the primary afferent neurons and the 
second order neuron within the spinal cord. Glutamate is the primary excitatory 
neurotransmitter in the CNS (Meldrum, 2000). It is an amino acid-based 
neurotransmitter synthesized from glutamine by glutamine synthetase. Glutamate is 
released at the central axonal terminals of each nociceptor and it binds to receptors 
located on the second order neurons. There is approximately 100 mM of glutamate 
within each pre-synaptic neurotransmitter vesicle and the release of a single vesicle 
produces a small, depolarizing excitatory postsynaptic potential (EPSP) in the 
postsynaptic neuron. The simultaneous release of multiple synaptic vesicles and 
activation of multiple postsynaptic glutamate receptors produces an EPSP that is 
sufficient to depolarize the second order neuron to threshold. The release of glutamate 
from the pre-synaptic neuron can be modulated by a variety of pre-synaptic receptors 
such as cholinergic, adenosine, or neuropeptide receptors, demonstrating the complex 
regulation and modulation of synaptic connections.  
Substance P (SP) is the second major excitatory neurotransmitter released 
from the central axonal terminal at the first synapse. SP is a short peptide that is a 
member of the tachykinin family of peptide neurotransmitters, and is derived from 
the pre-protachykinin-A (PPT-A)-gene (Hokfelt et al., 1975). This gene also codes 
for other tachykinins such as neurokinin A and neuropeptide K. Substance P is 
released by C-fiber nociceptor activation and binds to the specific tachykinin 
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 
receptors, called neurokinin-1 (NK
1
) receptors. NK
1 
receptors, along with substance 
P, are distributed in the brain and are specifically found in the hypothalamus, 
amygdala, and periaqueductal gray, regions that serve to regulate emotions, the 
autonomic nervous and endocrine systems, as well as nociception. In addition to its 
release from the central axonal terminals, substance P and other tachykinins are also 
released from the peripheral endings of nociceptors where they mediate effects called 
'neurogenic inflammation' (Geppetti, 1996). By binding to the NK
1
 receptor on 
vascular smooth muscle cells, substance P contributes to vasodilation and edema, a 
sign of localized inflammation (Laycock and Meeran, 2013).   
 
 
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 
                                         
 
Table 1: Nociceptors 
A-delta fiber nociceptors are thinly myelinated and are responsible for 
mechanical and thermal stimuli, carrying them quickly resulting in 
“fast pain.” C-fiber nociceptors are unmyelinated and are responsible 
for the slow conduction of action potentials from mechanical, thermal, 
and chemical stimuli. (Table taken from Bear, 2007). 
 
 
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 
 
Figure 1: Anterolateral System (ALS) 
The ALS is a group of sensory pathways ascending the spinal cord in the ante
rolateral portion of the white matter that conveying information about tempera
ture and noxious stimuli to the brain. The collateral terminations include the r
eticular formation, thalamus, and somatosensory cortex.(Figure from the Inter
active Neuroscience v3.0 Supplements). 
 
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 
 
 
                       
 
Figure 2: Gate Control Theory of Pain 
C-fiber and A- delta nociceptors are represented by small nerve fibers above. 
A-beta fibers are represented by large nerve fibers above A) If an action 
potential is transmitted through the small fibers, the gate is “open,” allowing 
the signal to travel to the brain.. B) If an action potential is transmitted 
through the large fibers, the gate is “closed,” blocking nociception and no 
signal is sent to the brain. (Figure taken from Linton et al., 1999). 
 
 
 
 
  
 
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 
c. Dorsal Horn-Second Order Neuron 
The first synapse in the transmission of pain information occurs within the 
dorsal horn of the spinal cord gray, between the nociceptive dorsal root ganglion 
neuron and a second order neuron. This region of the dorsal horn is called Rexed’s 
lamina. Laminae I to IV are primarily associated with exteroceptive sensation, making 
these regions highly involved in the modulation and transmission of nociceptive 
stimuli within the CNS. Laminae I, called the marginal zone, is located at the 
superficial tip of the dorsal horn and receive information from thermal stimuli and 
non-noxious thermal stimuli. Lamina II, the substantia gelatinosa, is comprised of 
interneurons that receive sensory information. Lamina III contains neurons that 
mainly receive information about mechanical stimuli and lamina IV contains cells that 
receive information about light mechanical stimuli. Lamina V plays an important role 
in transmitting nociceptive sensations from visceral organs. The axon of the second 
order nociceptive neuron then decussates to the opposite side of the spinal cord, where 
it enters the anterolateral quadrant and ascends via the spinothalamic tract. This tract 
passes superiorly through the brain stem and terminates in the thalamus.  
2. Thalamus- Third Order Neuron  
The spinothalamic tract transmits information pertaining to noxious chemical, 
mechanical, and thermal stimuli to the thalamus. The thalamus is a subcortical area of 
the CNS known as the brain's relay station, and plays an important role in the relay of 
a variety of ascending sensory information, including nociceptive information, to the 
higher sensory processing centers of the cerebral cortex. Studies conducted on rats 
following hindpaw inflammation show that thalamic neurons have lowered thresholds 
to noxious mechanical and thermal stimuli due to sensitization brought about by the 
constant transmission of pain information, producing hyperalgesic responses 
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 
(Guilbaud et al., 1986; Ahmad, 2006). The nociceptive neurons of the spinothalamic 
tract terminate in the ventroposterolateral nucleus (VPL; Fig. 1) of the thalamus 
where they synapse onto third order thalamic neurons. These thalamic neurons then 
send their axons to the primary somatosensory cortex.  
3. Primary Somatosensory Cortex 
Nociceptive sensory input from the VPL of the thalamus terminates in the 
primary somatosensory cortex, located in the postcentral gyrus of the parietal lobe, 
which determines the location and intensity of a noxious stimulus (Bushnell et al., 
1997; Kanda et al., 2000; Fig. 1).   
 The spinothalamic tract is not the only pathway responsible for the sensation 
of pain. There are several collateral pathways that branch from the spinothalamic tract 
that are responsible for the transmission of noxious input to accessory areas of the 
brain. These pathways have collectively been called the “pain matrix.” 
4.   Pain Matrix 
The term pain matrix was coined by Tracey and Manyth in 2007 as a means to 
describe the complexity of pain sensation. Rather than a simple, linear pathway from 
receptor to cortex, the idea of a “matrix” brings to mind a net of neuronal connections. 
The pain matrix is not a simple regulatory pathway, but is a collection of 
interconnected brain regions involved in cognition, emotion, motivation, and the 
sensation of pain (Tracey and Mantyh, 2007; Tracey and Johns, 2010). The pain 
matrix can be considered analogous to a train station, with the 3 neuron, ascending 
afferent pathway serving as the grand central station, or core, of the pain matrix. The 
areas that serve as this pain matrix “routing station” include the primary 
somatosensory cortex, insula, amygdala, and brain stem (Tracey and Mantyh, 2007).  
 The brain stem serves as an area of afferent as well as efferent nociceptive-
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 
related signals in the pain matrix (Fig. 1). The spinoreticular tract brings afferent 
nociceptive information to the reticular formation of the brainstem and allows it to be 
processed without synapsing in the thalamus. Collateral afferent pathways of the pain 
matrix also target the rostral ventromedial medulla (RVM) nuclei of the medulla 
oblongata, the periaqueductal gray (PAG) in the midbrain, the locus coeruleus (LC) in 
the midbrain, and the nucleus raphe magnus (NRM) in the medulla oblongata. These 
areas contribute to the descending, efferent pain suppression pathway, inhibiting or 
modulating incoming nociceptive information at the spinal cord level (Ossipov, 
2010).  
Activation of the ascending nociceptive pathway elicits a simultaneous 
response from the descending nociceptive pathway. This pathway is an endogenous 
system that modulates the transmission of pain. The brain receives sensory input, as 
well as interprets these signals and makes constant adjustments in lieu of the 
changing conditions. The descending nociceptive pathway begins in the 
periaqueductal gray (PAG) located within the midbrain, an area with many opioid 
receptors and rich in endogenous opioid peptides, and projects to the medulla 
oblongata. The neurons that begin within the PAG produce an excitatory influence on 
neurons located within the RVM. The RVM contains the nucleus raphe magnus 
(NRM) which is abundant in serotonin. These serotonergic neurons act to inhibit 
action potentials carrying nociceptive information in order to reduce the transmission 
of pain. They do so by  descending within the spinal cord and terminating in the 
dorsal horn where they activate enkephalinergic interneurons, causing the release of 
endogenous opioids. These endogenous opioids produce presynaptic inhibition of the 
primary afferent neuron by reducing their release of neurotransmitters and 
postsynaptic inhibition of the second order neuron, causing in a K
+
 efflux and 
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 
inhibitory postsynaptic potential (IPSP). This IPSP, in turn, causes the release of 
endogenous serotonin and norepinephrine from axons that originate in the thalamus, 
reticular formation, midbrain, and spinal cord which acts to “block” pain signals by 
decreasing the action potential frequency within the spinothalamic tract (Vanegas et 
al., 2004).  
While the descending pathways are activated to begin suppression of 
nociceptive sensations, afferent collateral pathways are also sending information to 
emotional areas within the CNS. Emotional pathways are those pathways responsible 
for taking pure nociceptive responses and storing them as the pain experience. It must 
be remembered that pain is a network phenomenon that is mediated, not by a single 
protein or brain region, but by multiple pathways at cortical, subcortical, spinal, and 
peripheral levels. These regions involve branches of neurons that lead to sites within 
the brain that store emotional memory and are involved in learning, including the 
amygdala and the secondary somatosensory cortex. These pathways are multisynaptic 
and pass through the reticular formation of the brainstem, allowing for the awareness 
of the stimulus to be identified. Once the nociceptive information is sent to the 
brainstem, it synapses in the central nucleus of the amygdala (“nociceptive 
amygdala”). The amygdala specializes in processing emotion and is essential for the 
expression of fear and anxiety, especially situations involving noxious stimuli 
(Phelps, 2004), and is active in the first stage of memory encoding. It can clearly be 
seen that the structures of the pain matrix play a vital function in the central 
processing of incoming signals and result in the conscious perception of a painful 
event.  
C. Hypothalamic-Pituitary-Adrenal Axis (HPA-axis) 
The pain matrix is activated not only during physical pain, but also in times of 
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stress, via the hypothalamic-pituitary-adrenal axis (HPA axis). The HPA axis is 
involved in the regulation of glucocorticoid stress hormones within the body. It is 
composed of the hypothalamus, the anterior pituitary gland, and the adrenal cortex of 
the adrenal glands. Stress can be both adaptive and maladaptive (Chapman, 2008), 
serving as a survival mechanism for an animal to escape a threat or actually hindering 
that animal in the time of threat. This axis consists of direct influences and feedback 
between the hypothalamus, pituitary gland, and adrenal gland, demonstrating an 
anatomical link as well as physiological link between the nervous and endocrine 
systems.  
Within the hypothalamus lies the paraventricular nucleus (PVN) which 
produces corticotropin releasing hormone (CRH) that initiates the stress response. 
Nociceptive signaling acts directly on the PVN in order to increase CRH levels 
within the body (Chapmann, 2008). CRH is released into the median eminence, and 
then carried by the hypothalamic-hypophyseal portal circulation to the anterior 
pituitary, where it stimulates the release of adrenocorticotropin releasing hormone 
(ACTH). ACTH is then released into systemic circulation where it travels to the 
adrenal glands and stimulates the release of cortisol.  
The main effects of cortisol generally last for a few hours and include 
increased blood pressure, increased glucose levels, suppression of the immune 
system, and decreased activity of the sympathetic nervous system which helps the 
body regain a state of 'normality' (Sapolsky, 1998). Cortisol binds to glucocorticoid 
receptors that are distributed in almost every cell of the body.  Metabolic effects of 
cortisol include elevating blood glucose levels by stimulating glycogenolysis and 
counteracting the effects of insulin. Cortisol also works to regulate levels of 
electrolytes such as Na
+
 and K
+
. If the body needs Na
+
 to be conserved, cortisol has 
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 
the intestines increases absorption of Na
+ 
by inducing vasodilation of afferent 
arterioles (Laycock, 2013). Normally, cortisol acts via negative feedback at the PVN 
level. Excess activation/stimulation of the PVN can cause a dysregulation, or 
abnormalitiy, of the HPA axis (Shepherd, 1994). If the HPA axis is no longer able to 
return to normal functioning following a stressful or painful situation, cortisol is 
continually produced and the hypothalamus and anterior pituitary become 
desensitized to potentially harmful situations.  
D. Environmental Enrichment  
Environmental enrichment (EE) is a broad area of research, with the core 
focus on understanding the effects of environmental variables on captive and research 
animals. The data collected from EE studies have wide-ranging implications, from 
improving animal welfare to applications in clinical biomedicine. Research into 
enriched environments has been conducted since the 1940’s with the positive effects 
of EE on animal learning and memory introduced by Hebb. Hebb allowed his 
laboratory rats to freely roam throughout his house and observed improved behaviors 
when compared to rats housed in a standard laboratory environment (Hebb, 1947).   
Since then, many studies have confirmed that rats housed in EE show 
improved learning and memory (Young, 1999). It has also been shown that being 
exposed to EE induces biochemical and structural changes in many brain regions, 
such as the hippocampus, which shows an increase in neuroplasticity (Diamond et al., 
1976; Kempermann et al., 1997; Nithianantharajah and Hannan, 2006 and van Praag 
et al., 2000).  
Recent studies concerning environmental enrichment have shown a decrease 
of stress in animals housed in an enriched environment when compared to those 
housed in a standard environment (Arco, 2007; Gabriel, 2010).  Also, a decrease in 
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the degree of thermal hyperalgesia in those rats exposed to EE has been seen, 
suggesting that EE has a significant effect on nociceptive responses (Tall, 2009).  
 Environmental enrichment affects a range of physiological and behavioral 
effects on animals (Young, 2003). Regulatory agencies in the United States have been 
trying to conduct a movement to include physical enrichments as standard protocol 
for housing environments, rather than an as optional supplement (Hutchinson, 2005).  
There are certain regulations that need to be met under Federal Law when housing 
research animals in order to insure the animals’ well-being. Currently, there are no 
mandates that EE must be provided in a laboratory by the Animal Welfare Act 
(AWA), however there have been many legal changes concerning EE as the primary 
housing condition for rodents, such as in Alternative Research and Development 
Foundation v. Veneman (Hutchinson, 2005). During this case,  the Alternative 
Research and Development Foundation filed a petition for rulemaking, requesting 
that the term “animal” as defined in the AWA include birds, mice, and rats bred for 
the use in research.  It was suggested that these animals be housed in a “natural” 
environment, taking into consideration the animals’ structural, social, and activity 
needs (NRC, 1996). 
Although the founding research in EE was conducted in rodents, the physical 
enrichment of animals such as horses and pigs has produced interesting findings. 
First, horses that were exposed to a pasture all day, every day (enriched group) 
showed the drive to exercise and graze more willingly than those horses that stayed in 
a pasture for a limited amount of time (control group; Houpt et al., 2001; Christensen 
et al., 2002; Chaya et al., 2006). The enriched horses also displayed a more varied 
rolling behavior, interpreted as a sign of comfort (Hansen et al., 2007). Pigs placed in 
an enriched environment displayed anticipation and “play” as measured through 
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 
observation of rooting around and head-butting cage mates. Also, providing visual 
barriers by placing boards or hay bales in the cages allowed the pigs to have a place 
to hide their heads (a target of aggression) in order to avoid aggressive pen-mates 
(Stolba and Wood-Gush, 1984; McGlone and Curtis, 1985; Fraser et al., 1986; 
Pedersen et al., 1993; Waran and Broom, 1993; Anderson et al., 1999).  
From the perspective of Federal Law, EE is a husbandry issue, but to a 
preclinical biomedical researcher, EE can become a distinct cohort in a study. EE can 
be divided into five categories based on the subtype of enrichment provided. The first 
category is social enrichment, consisting of animals being in contact directly or 
indirectly with animals of the same species or humans (Bloomsmith et al., 1991). The 
second category is known as occupational enrichment, including devices that provide 
animals with control or challenges and promotes exercise. The third category is 
physical enrichment, including alterations of home cage size and inclusion of objects. 
The fourth category is sensory enrichment that includes visual, olfactory, tactile, 
auditory, or taste stimuli given at testing times in the home cage. The last category is 
nutritional enrichment, including changes in the nutritional content provided to the 
animal (Bloomsmith et al., 1991). In the present study, EE served as a treatment, 
specifically physical EE and its effects on nociception. 
1. Preclinical Environmental Enrichment - Nociception 
      The use of animals in the studies of environment and nociception has played 
an important role in helping researchers to elucidate the relationships between the 
external environment and pain sensation. Many studies have shown that 
morphological and biochemical indices in the brain undergo alterations in response to 
the influence of the environment (Ickes et al., 2000). The brains of animals housed in 
EE show an increase in synapse number on pyramidal neurons, the main projecting or 
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 
output neurons from the cerebral cortex (Pham et al., 1999).  With an increase in the 
number of synapses present, there is an increase in synaptic activity as well as an 
increase in size and number of the glial cells used for support. The above findings 
could be directly related to how EE affects nociception. Once an action potential is 
produced, it will travel to every presynaptic axon ending in that neuron and if more 
synapses are produced as a result of EE, alternative pathways are now present for the 
transmission of pain information. Since sensitization is a result of repeated 
stimulation, the additional synapses  allow for a reduced level of activity in each 
individual synapse, resulting in a prevention of sensitization and reduced 
hyperalgesia and allodynia.  
  Along with producing anatomical changes within the CNS, EE also has 
anxiolytic effects. Stress can be either adaptive or maladaptive to an organism but 
some detrimental effects include behavioral/cognitive deficits and brain damage 
(atrophy).The majority of preclinical studies of EE and stress have shown that EE 
serves to temper animals' emotional reactivity to stressors (Chamove, 1989). The 
benefits of EE for combating stress can best be seen with hippocampal neurogenesis. 
Studies have supported that the brain’s ability to adapt and change in given situations, 
by creating new pathways, could be a direct effect of EE (Kempermann, 2006). The 
positive preclinical findings of EE have clinical applications. However there is, 
limited data demonstrating physical and psychological benefits of EE in humans. 
2. Clinical Environmental Enrichment - Pain  
Clinical studies have shown that pain is not only a result from nociception, but 
the sensation of pain is also strongly influenced by emotional and social factors. A 
study done by Ulrich (1984) supports the hypothesis that changes in an environment 
can affect a person's sensation of pain. This retrospective analysis compared patients 
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 
with a view through a postoperative hospital room window of a natural scene versus a 
brick wall. The patients who were in the rooms with the natural scenes required less 
pain medication and were discharged from the hospital more rapidly, suggesting that 
external environmental variables affect pain. With slight data showing EE has 
positively affected nociception, the question arises if the benefit of EE was due to 
pre-pain or post-pain environment. 
E. Cross-Over Research Studies 
Common in clinical investigations, cross-over studies include patients or 
human volunteers receiving a prescribed sequence of treatments, then at a set point in 
the investigation a proportion of the subjects “cross” to the other treatment. This type 
of study uses fewer patients than what would normally be required because the 
patients are able to serve as their own control. A cross-over study begins with two 
cohorts: a control group (AA) and an experimental group (BB). At a particular point 
in the study a cross-over will occur and four cohorts will proceed in the subsequent 
investigations; AA, AB, BB, BA. 
       There is a paucity of data on the effect of EE on nociception in animals and a 
lack of data examining cross-over of home cage environments. Based on previous 
work done in this lab (Tall, 2009) on enriched environments alone, it can be 
hypothesized  that EE rats should be less affected by nociceptive stimuli than rats in 
standard home cage environments. The current investigation expanded on previous 
work from this laboratory examining the effects of home cage environment on 
nociceptive behavior by looking at home cage environment prior to and following 
experimentally-induced inflammation. What would happen if a cross-over cohort 
study was conducted; would the same results as seen by tests previously conducted in 
this lab hold true if rats were raised in an enriched environment then switched to a 
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 
standard environment once experimental nociception was induced? What if rats 
raised in a standard environment were switched to an enriched environment after 
induced inflammation? It was hypothesized that subjects in enriched housing 
condition throughout the entire experiments would exhibit the highest thresholds to 
mechanical and thermal stimuli after inflammation, indicating an analgesic effect of 
enrichment. This group would be followed by the standard-enriched, showing the 
second highest, the enriched-standard showing the second lowest, and the standard-
standard showing the absolute lowest thresholds after inflammation was induced. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 
Chapter 2 
II. Methods 
A. Animals  
Studies were performed on male Sprague-Dawley rats 27-32 days old upon 
arrival, obtained from Charles River Laboratories Inc. (Wilmington, MA). Four 
groups of rats were used with n=6 per housing condition, for a total of 24 rats. Rats 
were maintained on a 12/12 light/dark cycle, with lights off at 10:00 a.m. and lights 
on at 10:00 p.m. The animal facility and behavioral testing laboratory was maintained 
at 21-22 degrees Celsius. Food (Lab Diet 5P00 Prolab RMH 3000 PMI Nutrition 
International Brentwood, MO) and water were provided ad libitum. Fresh bedding 
(aspen pine shavings and Bed-O-Cobb) was provided during the weekly cage 
change/cleaning by the animal facility staff. 
B. Housing Conditions 
Animals were randomly placed, by the animal facility staff, in polycarbonate 
cages measuring 20" long x 16" wide x 8 1/2" tall upon arrival (N=3 rats per cage). 
Food, water, and bedding were included in all home cage housing conditions. After 
one week of habituation, the rats were randomly assigned a housing environment by 
cage; meaning, 12 rats (4 cages) were assigned as standard, and the remaining 12 rats 
(4 cages) were assigned as enriched or supplemented (Fig. 4). The physically 
supplemented cages included nylabones (4.5" x 1.25"), rat tunnels (round and 
square), crawl balls, barbells, and chew toys that were changed during the cage 
changing each week. After a five week pre-treatment period, the cross-over occurred 
(rats were 62-67 days old); 6 of the 12 rats in the standard cages remained standard 
while the remaining 6 were crossed to enriched housing. Six of the original 12 
enriched remained enriched while the remaining 6 were crossed to standard housing. 
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 
A representation of each housing condition is shown in Figure 3. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 
A.) 
 
 
B.) 
 
 
Figure 3: Housing Conditions 
A.) A standard housing cage supplied with food, water, and bedding. B.) An 
enriched housing cage supplied with various toys, food, water, and bedding. 
 
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 
C. Inflammation Model  
Following the 5 week housing pre-treatments, while under brief isoflurane 
anesthesia (3%), an intraplantar injection of 100 microliters of 100% Complete 
Freund's Adjuvant (CFA) was administered into the left hind paw on the plantar 
surface, between the foot pads (Stein, 1988; Fig. 4). 
D. Edema Quantification 
Thickness measurements of the dorso-ventral hind paw were made using a 
caliper in order to assess the degree of edema in the CFA-injected hind paw. Baseline 
data were first collected before the injection of CFA and post-inflammation edema 
data were collected at multiple points after the injection (2h, 7d, 14d, 21d, 28d, and 
35d; Fig. 4).  
E. Behavioral Assessments 
Animals were habituated to the behavioral testing laboratory, testing 
equipment and investigators for 2 hours, 3 days a week for 1 week. Baseline 
measurements were collected subsequent to the habituation and animals’ weight was 
collected each week. Post inflammation behavioral data were collected at multiple 
times after injection (2h, 7d, 21d, 28d, and 35d). All testing was performed between 
10:00 a.m. and 3:00 p.m., during the dark phase. 
F. Paw Withdrawal Threshold (PWT) to a Mechanical Stimulus 
The withdrawal threshold to a mechanical stimuli was measured using a set of 
nine calibrated von Frey monofilaments (0.2-32.0 grams force) using the Up-Down 
Technique (Chaplan et al., 1994; Dixon, 1980). Rats were placed in plexiglass 
chambers on an elevated stainless steel mesh screen and allowed 5-10 minutes to 
habituate. The von Frey monofilaments were applied for 5 seconds with a small 
amount of pressure to cause a buckling of the monofilament against the plantar 
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 
surface of each hind paw. A positive withdrawal was considered as a raising or 
licking of the hind paw. 
G. Paw Withdrawal Latency (PWL) to a Heat Stimulus 
Paw withdrawal from a heat stimulus was tested using the Hargreaves method 
(Hargreaves et al., 1988). Rats were placed on a glass table in plexiglass chambers 
and were allowed to habituate for 5-10 minutes. In order to quantify paw withdrawal 
latency, a heat source was focused on each hind paw in the area between the pads. 
Each hind paw, CFA injected and non-injected, was tested four times with at least 5 
minutes between successive application of the radiant heat source in order to avoid 
sensitization. The PWL of each rat was calculated as a mean of trials 2-4. A positive 
withdrawal was considered as a raising or licking of the hind paw. 
H. Statistical Analysis 
The difference scores for paw thickness, PWL, and PWT measurements were 
calculated by subtracting the right paw measurement from the left paw measurement, 
allowing the right paw to serve for the control for each animal. The data were then 
expressed as the standard error of the mean (S.E.M.) and analyzed using a repeated 
measure analysis of variance (ANOVA) in IBM SPSS Statistics version 20. A p value 
of < 0.05 was considered statistically significant 
 
 
 
 
 
 
 
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 
 
 
 
 
 
 
 
 
 
 
 
Figure 4: Experimental Design 
The line graph above depicts the timeline each group of rats followed 
upon arrival to the final testing day. Following habituation, h=hours 
and d=days.  
 
 
 
 
 
 
 
 
 
 
5 week habituation 
Baseline, CFA 
injection, and 
Cross-over 
2h 7d 14d 21d 28d 35d 
Behavioral Data Collections 
Arrival to anim
al care facility 
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 
Chapter 3 
III. Results 
A. Paw Thickness 
Paw thickness was measured using a caliper in order to assess the relative 
degree of inflammation induced from the injection of CFA. It was demonstrated that 
at 2 hours post-CFA injection, standard-standard showed the thickest paws, followed 
by standard-enriched, enriched-enriched, and enriched-standard. At 7 days post, all 
four housing conditions showed an increase in thickness when compared to 2 hours. 
Thickness values for all four housing conditions were seen to continue to decrease 
each week until 35 days post-CFA injection (Fig. 5). 
A repeated measures ANOVA was conducted on paw thickness for all four 
treatment groups (standard-standard, standard-enriched, enriched-standard, enriched-
enriched) at baseline, 2 hours, 7 days, 14 days, 21 days, 28 days, and 35 days post-
inflammation. There was no significant difference among home cage treatment groups 
(p-value = 0.180; F
10.125,44
=1.403 ). There was, however, a significant difference 
across testing times (2h, 7d, 14d, etc.; p-value < 0.001; F
3.485,44
=1.403). 
B. Paw Withdrawal Threshold (PWT) 
Animals were tested for PWT to mechanical stimuli using a set of nine 
calibrated von Frey monofilaments (0.2-32.0 grams force) with the Up-Down 
Technique (Chaplan et al., 1994; Dixon, 1980). This method was used in order to 
determine if an enriched environment affects mechanical-related nociceptive 
behavior. At 2 hours post-CFA injection standard-standard, standard-enriched, and 
enriched-standard groups all showed a significant decrease in PWT values from 
baseline, while the enriched-enriched group showed a slight decrease. Values 
recorded at 7 days post injection showed an increase in threshold for the standard-
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 
standard group, a slight decrease for the standard-enriched group, a decrease for the 
enriched-standard group, and a significant decrease for the enriched-enriched group. 
The threshold values recorded at 14 days and 21 days post stayed relatively the same 
as the data recorded at 7 days for each group. The standard-standard and enriched-
standard groups at 28 days showed an increase in threshold from 21 days. Standard-
enriched and enriched-enriched groups showed a significant increase in threshold 
value. At 35 days post-CFA injection, standard-standard showed a significant increase 
in threshold value while the other three housing groups showed significant decreases 
(Fig. 6) 
The repeated measures ANOVA conducted on PWT revealed and no 
significant difference among the four housing groups (p-value = 0.311; F
3.801,44 
 =
 
2.520). There was a slight significant difference across behavioral testing times (p-
value = 0.046; F
11.402,44
=1.169),. 
C. Paw Withdrawal Latency (PWL)   
Animals were tested for PWL from a heat stimulus using the Hargreaves 
method (Hargreaves et al., 1988). This method was used in order to determine if an 
enriched environment affects thermal-related nociceptive behavior. PWL values 
recorded at 2 hours post-CFA injections were seen to be significantly lower than those 
collected at baseline. At 7 days, standard-standard, enriched-standard, and enriched-
enriched showed slight increases from 2 hours while standard-enriched showed a 
slight decrease. All four housing groups showed a significant increase in PWL at 14 
days. At 21 days, standard-standard and enriched-standard showed slight increases, 
standard-enriched showed a slight decrease, and enriched-enriched showed a 
significant increase. PWL values remained similar to those recorded at 21 days until 
35 days (Fig.7).  
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 
The repeated measures ANOVA conducted on PWL showed no significant 
difference  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 
 
 
 
 
 
 
 
 
 
 
 
Figure 5: Paw Thickness Measurements 
This figure shows a comparison of rats housed in standard/standard, 
standard/enriched, enriched/standard, and enriched/enriched cages. 
Thickness was found using calipers. Data is presented for treatment 
groups at post 2 hours, 7 days, 14 days, 21 days, 28 days and 35 days. 
Values are represented as the difference score of the S.E.M. Values 
were analyzed using a repeated measure ANOVA, which revealed 
F
10.125,44
=1.403 with  no significant difference among home cage 
treatment groups (p-value = 0.180). There was, however, a significant 
difference across testing times (2h, 7d, 14d, etc.; p-value < 0.001) and 
F
3.485,44
=1.403. 
. 
 
 
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 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 6: PWT Measurements 
This figure shows a comparison of rats housed in standard/standard, 
standard/enriched, enriched/standard, and enriched/enriched cages. 
PWT was found using von Frey monofilaments to perform the Up-
Down technique. Data is presented for treatment groups at baseline and 
post 2 hours, 7 days, 14 days, 21 days, 28 days and 35 days. Values are 
represented as the difference score of the S.E.M. Values were analyzed 
using a repeated measure ANOVA, revealing revealed F
3.801,44 
 =
 
2.520 
and no significant difference among the four housing groups (p-value 
= 0.311). There was a slight significant difference across behavioral 
testing times (p-value = 0.046), and F
11.402,44
=1.169. 
 
 
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 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7: PWL Measurements 
This figure shows a comparison of rats housed in standard/standard, 
standard/enriched, enriched/standard, and enriched/enriched cages. 
PWL was found Hargreaves method. Data is presented for treatment 
groups at baseline and post 2 hours, 7 days, 14 days, 21 days, 28 days 
and 35 days. Values are represented as the difference score of the 
S.E.M. Values were analyzed using a repeated measure ANOVA, 
which revealed no significant difference among home cage housing 
groups (p-value = 0.446) and F
11.244,44
=1.004.There was a significant 
difference across behavioral testing times (p-value < 0.001) and 
F
3.748,44
=22.405. 
 
 
 
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 
among home cage housing groups (p-value = 0.446) and F
11.244,44
=1.004.There was a 
significant difference across behavioral testing times (p-value < 0.001) and 
F
3.748,44
=22.405. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 
Chapter 4  
V. Discussion 
The present study examined the effects an environmental cross-over on 
inflammation-induced nociception. In this investigation, rats were reared in two 
distinct housing treatment groups prior to the establishment of a model of 
experimental inflammation. Following the onset of sub-chronic inflammatory 
response, half of the rats were crossed to the other environment, while the other half 
stayed in the same environment. Prior to collecting data on inflammation - induced 
nociception, Thickness, PWT and PWL data were collected and served as a baseline 
point of comparison. After five weeks of living in their initial home cage 
environments, subjects were injected with CFA into their left hind paw to induce 
inflammation. The subjects were then crossed-over into their post inflammation 
environments and remained there for 35 days,with post inflammation data being 
collected once a week. 
In support of previous work, paw thickness, a tool to quantify the degree of 
inflammation, showed a significant difference across behavioral testing times (Fig.5). 
This demonstrates that the injection of CFA produced a localized inflammation in the 
hind paw. A significant difference among housing treatment groups was not found, 
suggesting that the severity of inflammation produced from an intraplantar CFA 
injection is not affected by the physical home cage environment. This finding 
supports previous work done in this laboratory (Tall, 2009) and work done by K. 
Militzer (Militzer, 1975), along with almost all other studies done using CFA to 
produce localized inflammation within the hindpaw. 
According to the literature, PWT is not affected by environmental conditions 
(Tall, 2009). This was supported by the present study, as no significant difference 
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 
among housing groups was found. There was, however, a slight significant difference 
seen across behavioral collection times (Fig. 6). These data indicate that there is a 
difference in mechanical and thermals hyperalgesia, which was not unexpected 
(Meller, 1994 and Tall, 2009). 
PWL to a radiant thermal stimulus showed no significant difference across 
behavioral testing times, not in support of PWL being affected by an intraplantar 
CFA injection (Fig. 7). A significant reduction in withdrawal latency was shown 
across all four housing conditions, but there was no significant difference among the 
housing treatment group. This finding suggests that PWL is not affected by the 
physical home cage environment. This finding does not support many other studies 
done on PWL (Hargreaves, 1988; Dubner, 1983; Schneider, 2006). It does, though, 
refute previous work done in this lab (Tall, 2009). 
When looking at Fig. 6, a few trends can be seen. First, looking at the 
standard-standard housing environment, PWT values were increased greatly from 
baseline (0). The values slightly decreased and increased over the course of data 
collection, but by day 35 post, standard-standard revealed the lowest PWT difference 
values, meaning a high PWT. It could be speculated that being raised in a standard 
environment and staying in a standard environment after localized inflammation was 
introduced could allow for a faster recovery when exposed to mechanical stimuli 
because no additional stress was introduced by the changing of an environment. As 
mentioned previously, stress can be either positive or negative. When relating to the 
crossover to a new environment, stress would be considered to have  negative effect 
on the body, causing excess activation of the HPA-axis and/or nociceptive pathways 
(Chapman, 2008). When comparing the other three housing environments (standard-
enriched, enriched-standard, enriched-enriched) values were seen to be very sporadic 
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 
with no identifiable trend, as expected from previous studies.  
Difference values for PWL (Fig. 7) show an evident trend. Standard-standard 
was seen to have the highest PWL thresholds from 2 hours post to 7 days post 
injection. Animals in this housing condition were then seen to have a slowed 
recovery, resulting in standard-standard having the lowest thresholds at 35 days post. 
It could be speculated that the animals in standard-standard were subjects of 
sensitization of the nociceptive pathway, resulting in lower activation thresholds and 
increased activation of pain transmission, as mentioned previously. The standard-
enriched showed a similar pattern as the standard-standard housing condition, 
suggesting that being reared in a standard environment at a young age leads to 
excessive activation of synapses, resulting in sensitization, when placed in a 
stressful/painful situation (Larsson, 2002; Baroncelli, 2010; Fox, 2006; Benaroya-
Milshtein, 2004). The enriched-standard environment showed the lowest PWL 
threshold at 2 hours post CFA injection, but rapidly being to increase as the study 
progressed. By day 35 post, enriched-standard revealed the highest values. The 
enriched-enriched environment showed a rapid increase in PWL threshold until 21 
days post. At 28 days post, although values increased from the previous week, 
enriched-enriched still showed the highest threshold. The data collected from these 
two housing conditions once again suggest that the initial housing environment plays 
a bigger role than the post inflammation housing environment. Being reared in an 
enriched environment allows for more synapses to be developed, thus reducing 
sensitization in synapses within the nociceptive pathway (Larsson, 2002; Baroncelli, 
2010; Fox, 2006; Benaroya-Milshtein, 2004; Diamond et al., 1976; Kempermann et 
al., 1997; Nithianantharajah and Hannan, 2006). Pain information now has more 
pathways to travel through, lowering the activation frequency of individual synapses, 
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 
“exhausting” them slower.  
  In future studies, improvement and further examination could be made on 
some variables. Increasing the number of pre-treatment data recorded after the 5 
week habituation would allow researchers to have more data to compare post-
treatments to, rather than just a single baseline. This would also determine whether or 
not housing has an effect on non-injured rodents. Also, extending the amount of time 
data is collected post-inflammation could be beneficial, allowing more time for 
recovery. The enriched-enriched group for PWL at post 35 days shows the threshold 
still increasing. By increasing the amount of post-inflammation time points, it could 
be determined how long it takes for the thresholds to level out or stay steady for each 
housing condition. In previous studies performed in this lab, it was shown that home 
cage numbers (socially vs. isolated) has an effect of nociceptive thresholds. Socially 
housed animals would include three animals per cage, while housing isolated would 
be a single animal per cage. It could be beneficial to conduct a cross-over study using 
isolated versus socially housed subjects. These data would elucidate if social housing 
and cross-over study affects inflammation-induced nociception. A few of the 
limitations of this study include only using male rats and starting with young rats. 
Using various ages would determine if the same results would be shown with rodents 
that were adult throughout the whole experiment.  
 Although this study did not support that crossing over from one environment 
to another significantly affects inflammation induced nociception, it is important to 
remember that being reared in an enriched environment does show significant 
improvements in nociceptive thresholds when compared to being reared in a standard 
environment (Hutchinson, 2005). When an animal is housed in an environment that 
allows them to exhibit natural behaviors, “less” pain is felt when subjected to 
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research. With this being said, EE is becoming a key standard when housing 
laboratory animals because it improves animal welfare overall (Hutchinson, 2005). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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