The Assessment of Hanging Behavior as a Measure of Mouse Welfare

by

Ingita Patel

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Pharmaceutical Sciences

Leslie Dan Faculty of Pharmacy University of Toronto

© Copyright by Ingita Patel 2018

ii

The Assessment of Hanging Behavior as a Measure of Mouse

Welfare

Ingita Patel

Master of Science

Pharmaceutical Sciences University of Toronto

2018

Abstract

A decline in mice welfare can be indicative of underlying pathology. Often, these

changes in mouse behaviors are difficult to detect before the pathology has significantly

advanced. Our experiments have revealed that cage-lid hanging, a “luxury” behavior in

mice, can serve as an indicator of welfare in different models of pain. The hanging

behavior is identified as an event where a mouse climbs onto the metal lid of the

laboratory cage, suspending itself off the cage floor. The goal of this project was to

characterize and evaluate the hanging behavior of mice as a robust early indicator of a

decline in physiological welfare. I observed that both acute and chronic pain models

robustly reduced the frequency and duration of hanging behavior in mice. My results

demonstrated that hanging behavior is a marker of mouse physiological and

psychological welfare, that can be easily incorporated to facilitate the early detection of

disease.

iii

Acknowledgments OM SHR E E GA NE SHA Y NA MAH; HA R E KR ISHNA

I enthusiastically offer a heartfelt thank you to my supervisor, Dr. Robert Bonin, who’s

inspiring mentoring ability has shaped my academic career and provided me with a

plethora of skills both inside and outside of research. I am grateful beyond words to him

for taking me on as his graduate student and will always appreciate his generosity and

kindness. I am incredibly grateful for his patience, time, and mentorship throughout

this degree. He is a leader in the field, though he would never admit this, and I am thankful

to have had the opportunity to learn from one of the best. My time with Dr. Bonin will not

soon be forgotten. Without Dr. Bonin, this exhilarating journey would not have been

possible, and for this, I am forever grateful.

Many thanks to my program advisory committee members Drs. David Dubins and Hance

Clarke for their continued guidance and valuable insight. They are both devoted experts

in their fields and I can only hope to live up to their exemplary standards. Their honest

enthusiasm for science is both admirable and encouraging, and I cannot thank them

enough for the time they dedicated to my thesis.

Drs. Irene and Yufeng, thank you for making me feel both welcome and a part of the

team. Without your help, this experience would not have been the same. I cannot thank

you enough for consistently taking time out of your busy schedules to guide me through

the inner workings of animal research. Specifically, thank you Dr. Irene, for your constant

willingness to assist me whenever I needed technical instruction. Your welcoming nature

and encouragement made the preparatory phase of my experiments significantly less

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daunting, which in turn gave me the confidence to use these acquired skills

independently.

Thank you to my fellow colleagues Abigail D’Souza, and Virginia Yini for their infinite

guidance, and support. Abby, you have become a great friend and I am indebted to you

for mentoring me through Microsoft Office. You are one of the most generous, kind and

social people that I have met. You are an excellent surgeon and I wouldn’t think twice if

you had to cut open my wound! Virginia, thank you for your constant willingness to answer

my seemingly never-ending list of questions with such patience and enthusiasm. You are

a meticulous and a driven graduate student and I am honored to have worked with you.

Above all, thank you for always supporting me by asking “What is there to cry about?!”. I

will never forget that!

This degree would also not have been possible without the constant support from the

Graduate Office at Leslie Dan. For their unwavering support, words of encouragement, a

listening ear, and resources throughout my thesis for which I am eternally grateful.

I would also like to thank my wonderful family, Momo, and friends for their unconditional

love and support. Most especially to my father, momma-bear and my baby brother.

Without their patience, endurance and support during the long days and weeks and all

those working “holidays” and “vacations” this degree would not have been possible. They

cannot imagine the influence their love, intellect and insights have had on this project.

Thank you for always encouraging me to be positive and kind. Krish Ohri, my Dr. Momo,

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I cannot even begin to express my gratitude towards you, as your endless support has

played a pivotal role in my success as a graduate student. You are a wonderful and

compassionate man, but most importantly a very loving boyfriend, and my strength. I am

appreciative for all of the time you devoted to ensuring my degree went smoothly.

Knowing that my family, boyfriend and friends believed in me was enough to get me

through the stressful times. I hope that I have made them proud and will continue to strive

to do so in the future.

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Table of Contents

Acknowledgments ................................................................................................................ iii

List of Figures ....................................................................................................................... ix

Table of Abbreviations .......................................................................................................... xi

Chapter 1 .............................................................................................................................. 1

Thesis Summary ............................................................................................................. 1

1.1 Overview ...........................................................................................................................1

1.2 Preliminary Data and Thesis Rationale ................................................................................2

1.3 Hypothesis & Specific Aims ................................................................................................4

Chapter 2 .............................................................................................................................. 5

Introduction ................................................................................................................... 5

2.1 Pain ...................................................................................................................................5

2.2 Definitions of Pain .............................................................................................................5

2.3 Components of Pain ...........................................................................................................6

2.4 Risk Factors .......................................................................................................................6

2.4.1 Age and Sex .............................................................................................................................. 6

2.4.2 Individual Factors ..................................................................................................................... 7

2.5 Preclinical Pain Research ....................................................................................................8

2.5.1 Do Animals Experience Pain? ................................................................................................... 8

2.5.2 Methods of Assessing Pain in Animals ..................................................................................... 8

2.5.3 Common Models in Pain Research .......................................................................................... 9

2.5.4 Animal Models Used in Current Study ................................................................................... 10

2.5.5 Challenges in Current Animal Models of Pain ....................................................................... 14

2.5.6 Current Developments in Animal Models of Pain ................................................................. 16

2.5.7 Monitoring Animal Welfare to Study Pain and Drug Action .................................................. 18

Chapter 3 ............................................................................................................................ 20

Materials and Methods ................................................................................................ 20

3.1 Animals ........................................................................................................................... 20

Table of Contents .................................................................................................................. vi

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3.2 Drugs ............................................................................................................................... 21

3.3 Experimental Schedule ..................................................................................................... 21

3.3.1 Reverse Light/Dark Cycles ...................................................................................................... 21

3.3.2 Mice Sensitivity to Red Light .................................................................................................. 22

3.4 Experimental Equipment .................................................................................................. 22

3.4.1 Cage ........................................................................................................................................ 22

3.4.2 Behavioral Recordings ........................................................................................................... 23

3.4.3 Data Processing ...................................................................................................................... 23

3.5 Software used to Analyze Video Recordings...................................................................... 23

3.5.1 CleverSys HomeCageScan® .................................................................................................... 23

3.5.2 Ethovision® ............................................................................................................................. 25

3.6 Forced Swim Test – Behavioral Assay................................................................................ 26

3.7 Data Analysis ................................................................................................................... 26

Chapter 4 ............................................................................................................................ 28

Physiological parameters affecting hanging behavior .................................................... 28

4.1 Introduction .................................................................................................................... 28

4.2 Characterization of Mouse Activity over 24-hours ............................................................. 28

4.3 Effects of Age and Sex on Hanging Behavior ...................................................................... 30

Chapter 5 ............................................................................................................................ 39

Physiological parameters affecting hanging behavior .................................................... 39

5.1 Introduction .................................................................................................................... 39

5.2 Visceral Pain .................................................................................................................... 39

5.3 Cyclophosphamide effect reversal by NSAIDs ................................................................... 40

5.4 Illness Model of Pain ........................................................................................................ 41

5.5 Monoamine-depletion model of depression ..................................................................... 42

Chapter 6 ............................................................................................................................ 55

Discussion .................................................................................................................... 55

6.1 Comparison to Literature on Animal Welfare Testing ........................................................ 60

6.2 Study Limitations ............................................................................................................. 62

6.3 Future Directions ............................................................................................................. 63

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6.4 Conclusions ..................................................................................................................... 65

References .......................................................................................................................... 66

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List of Figures

Figure 1. Quantification of behavioral activities over a 24-hour period of 1-6 month-old

mice.

Figure 2. Off ground activity over a 15-hour period of 1-6 month-old mice.

Figure 3. Number and duration of hanging episodes over a 9-hour period of 2, 4, 6-

month-old mice.

Figure 4. Distance traveled over a 9-hour period of 2, 4, 6-month-old mice.

Figure 5. Hanging episodes of 2-month-old male mice in a visceral pain model.

Figure 6. Hanging duration of 2-month-old male mice in a visceral pain model.

Figure 7. Distance travelled by 2-month-old male mice in a visceral pain model.

Figure 8. Hanging episodes of 2-month-old male mice in an illness model of pain.

Figure 9. Hanging episodes of 2-month-old male mice in an illness model of pain.

Figure 10. Distance travelled by 2-month-old male mice in an illness model of pain.

Figure 11. Hanging episodes of 2-month-old male mice in a pharmacological model of

depression.

Figure 12. Hanging duration of 2-month-old male mice in a pharmacological model of

depression.

Figure 13. Distance travelled by 2-month-old male mice in a pharmacological model of

depression.

Figure 14. Forced Swim Test of 2-month-old male mice in a pharmacological model of

depression.

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Methods Figure (MF):

Figure M1. Hanging behavior, inside a mouse home-cage setting.

Figure M2. Experimental set-up.

Figure M3. Screen shot of a video analysis using Ethovision®.

Figure M4. Forced Swim Test.

Supplementary Figures (SF):

Figure S1. Reduced hanging behavior in traditional mouse models of pain. A) Acute

pain models (formalin and capsaicin). B) Long-term pain models – Complete Freund’s

Adjuvant (CFA) and Spared Nerve Injury (SNI). C) Animal model of cancer pain.

Figure S2. Reduced hanging behavior in mouse models of pain which do not target the

paw. A) Post-surgical pain model via craniotomy.

Table T3. Post-hoc experimental power analyses.

Table T4. Number of outliers identified and removed in each experimental condition

xi

Table of Abbreviations

CYP Cyclophosphamide

LPS Lipopolysaccharide

TCA Tricyclic Antidepressants

IASP International Association for the Study of Pain

NSAIDs Nonsteroidal Anti-inflammatory Drugs

TRP Channels Transient Receptor Potential Channels

CFA Complete Freund’s Adjuvant

SNI Spared Nerve Injury

CNS Central Nervous System

i.p. Intraperitoneal

CNS Central Nervous System

PNS Peripheral Nervous System

1

Chapter 1

Thesis Summary

1.1 Overview

Every year, millions of mice are used worldwide to evaluate the efficacy and safety profile

of pharmaceutical drugs. The cost to develop and approve a new drug has been

increasing at an exponential rate; from $179 million in 1970s to a whopping $2,558 million

in 2013 (Dimasi, et al., 2016). The high cost of an approved drug arises in part from the

increasing rate at which drugs are removed from the development and testing pipeline

(Cook, et al., 2014). Additionally, the current behavioral toxicity assays cause

controversially high levels of pain, distress and mortality in mice (Richardson, 2015). One

strategy to mitigate these shortcomings is to improve measurement of mouse welfare

during preclinical testing. Improved measurement of welfare will allow for earlier and more

sensitive detection of drug-related toxicity or pathology, and ultimately ensure more

humane end-points and reduced animal usage.

I have observed that cage-lid hanging behavior is a ‘luxury’ behavior of mice that declines

with animal welfare. The behavior is defined as the event (and duration) of a mouse

climbing onto the metal lid of its laboratory cage, and suspending itself off the floor (Figure

M1). Preliminary data from our lab, using 2-month old male mice have determined that

cage-lid hanging behavior serves as a measure and test of mouse welfare. Using an

automated video tracking system, I observed that various pain models with different

temporal profiles reduced hanging behavior over a time course that paralleled the typical

expression of pain in these models. I have also explored the potential of hanging behavior

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to serve as an indicator of psychological welfare by studying the effects of reserpine-

induced depression on hanging behavior.

1.2 Preliminary Data and Thesis Rationale

The costs of drug development are extremely high; these could be reduced by improved

approval rates and reduced preclinical testing costs. A major driver of project closure in

clinical testing is the discovery of safety concerns, such as liver dysfunction, or unwanted

side effects of drugs such as psychosis, dizziness, or migraine (Bars et al., 2001). These

are particularly evident in Phase I testing, and can account for closure of more than 60%

of trials at this early stage (Cook et al., 2014). Existing behavioral testing paradigms are

often inadequate at detecting subtle changes in animal behavior, which could partially

explain why preclinical animal testing often fails to identify drug side effects. A potentially

effective strategy to improve safety screening may be to increase monitoring frequency

and/or sensitivity of animal welfare during preclinical testing thereby allowing for earlier

detection of drug-related toxicities or pathologies. I have observed that the frequency and

duration of a rodent’s interaction with the metal lid of a standard rodent home cage are

affected by the rodent’s welfare, and are reduced by pain and illness.

Preliminary data from our lab shows that this cage-lid hanging behavior declines with

animal welfare. Hanging behavior was monitored using video tracking technology

(CleverSys HomeCageScan® & Noldus Ethovision®) with minimal animal-experimenter

interaction. First, we studied the effect of traditional murine pain models on hanging

behavior (Bars et al., 2001). Short-term nociceptive pain was induced using an injection

of capsaicin (0.5% w/v, 5 µL) or dilute formalin (1% v/v, 5 µL) into the plantar hind paw of

mice. We observed a robust but transient decrease in hanging behavior following the

3

injection of the irritant or noxious chemicals, which recovered within 24 hours. (Figure

S1A). Injection of control solution had no effect on hanging behavior (Figure S1A).

Next, in collaboration with Dr. Jeff Mogil, we studied the effects of long-term pain on

hanging behavior. Inflammatory pain was induced using intraplantar injection of complete

Freund’s adjuvant (CFA), into the paw (10 µL). Neuropathic pain was induced by

permanent injury of the portions of the sciatic nerve that innervates the leg and

hindquarter using the spared nerve injury model (SNI) (Costigan, et al., 2009). We

observed that CFA significantly decreased hanging behavior on the first five days

following the injection with significant recovery in the second week post injection,

consistent with the development and resolution of hyperalgesia in this pain model(Figure

S1B). Similarly, SNI caused immediate decrease in hanging behavior but the effect was

far more pronounced and showed no signs of recovery a month following the surgery,

which is also consistent with the irrerversible hyperalgesia observed in this model (Figure

S1B).

While traditional pain models drastically decrease hanging behavior, they are insufficient

to show a correlation between hanging behavior and mouse welfare. Another confound

is that these standard pain models inflict pain by targeting the mouse paw, and thus may

impede the ability of the mouse to hang. In order to determine whether hanging behavior

can be reduced by painful stimuli that do not target the paw, in collaboration with Dr.

Chereen Collymore, we first looked at a model of post-surgical pain caused by surgical

drilling of the skull, or a craniotomy. Craniotomies are commonly performed for a variety

of research applications including intracranial injections, cannula implantation, and

creation of models of stroke or epilepsy (Gray et al., 2005). Like other surgeries,

4

craniotomies give rise to systemic inflammation, eliciting pain and distress in mice (Gray

et al., 2005). It was found that hanging behavior was significantly reduced at 1 hour

following craniotomy but recovered to baseline levels 8-hours post-surgery (Figure S2).

Notably, no analgesics were administered post-surgery in these experiments to observe

whether craniotomy impairs behaviour post-surgery. Overall these data demonstrate that

hanging behavior may provide a measure of ongoing pain and illness and consequently

can be used to test the efficacy of drug treatment when the disease-state model involves

pain. We further speculated that hanging behaviour may reflect overall animal well-being

and be impaired non-specifically in states of reduced well-being such as depression.

1.3 Hypothesis & Specific Aims

The goal of this project was to fully characterize hanging behavior as an early indicator of

declining mouse welfare and validate its measurement in the study of drug efficacy.

Based on our preliminary data I hypothesized that hanging behavior of mice will be

reduced in models of pain, illness and depression in mice, and that this reduction

will be reversed by pharmacological treatment of these conditions.

This project had two major aims:

Aim 1: To characterize the effects of age and sex on hanging behavior in mice.

Aim 2: To determine whether hanging behavior is impaired in mouse models of pain and

illness, and depression.

5

Chapter 2

Introduction

2.1 Pain

Pain can have serious and debilitating consequences for patients, their families, and

cause considerable economic burden. Pain acts as an early alarm system; its biological

purpose is to signal potential harm, and initiate withdrawal from harmful threats (IASP;

www.iasp-pain.org; Deuis, Dvorakova, & Vetter, 2017; Boyd et al., 2011). Although pain

is associated with injury and/or disease, pain is a subjective experience that cannot be

objectively measured (Deuis, Dvorakova, & Vetter, 2017). This subjectivity of pain

presents a great challenge in pre-clinical pain research, as we cannot directly assess the

subjective experience of animal, thus necessitating the use of direct sensory or

behavioural measures in animals.

2.2 Definitions of Pain

Although pain is subject to numerous definitions, one of the most widely cited definitions

of pain is by The International Association for the Study of Pain (IASP; www.iasp-

pain.org). They define pain as “[an] unpleasant sensory and emotional experience

associated with actual or potential tissue damage, or described in terms of such damage”

(IASP). While this definition focuses on damage done to the body, it also accounts for the

individual’s emotional experience in response to the pain (Mersky & Bogduk, 1996). This

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duality of physiological and psychological distress illustrates that a physiological event

within the body cannot exist without the psychological experience (Lumley et al., 2011).

2.3 Components of Pain

The experience of pain is multidimensional and involves sensory, affective, motivational,

environmental and cognitive components (Lumley et al., 2011). Nociception involves the

detection and encoding of a harmful stimulus (e.g. thermal, mechanical or chemical) that

causes tissue damage (Tyrer, 1992). These signals are transmitted from the sensory

environment to the CNS mostly through A delta and C fibers (Cassell, 1982). Notably, in

some pathologies the perception of pain can also be felt in the absence of a noxious

stimuli (Loeser, & Melzack, 1999). Pain perception is also greatly influenced by other

affective disorders, such as depression or anxiety (Tyrer, 1992).

2.4 Risk Factors

The risk of developing chronic pain varies between individuals. The main risk factors are

highlighted below. In particular, age, sex, social group factors, and specific individual

factors will be discussed in depth.

2.4.1 Age and Sex

Age and sex are important variables in the perception and incidence of pathological pain.

In children and adolescents, girls typically report more pain episodes than boys (Swain et

al., 2014). This trend becomes more pronounced in adulthood as women report increased

pain frequency and severity and overall longer duration of pain when compared to men

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of similar age (Apley et al., 1958). While, there is still much debate in the literature on the

physiological mechanisms or genetics that contribute to pain and how psychological and

social factors mediate this difference.

The literature on age and pain indicates that pain, especially low back pain, increases

from childhood through adolescence to adulthood (Swain et al., 2014). Recent studies

have shown that the prevalence of pain in any form increases with age, and this increase

is dependent on a host of environmental factors (Thomas et al., 2011). For example, the

prevalence of chronic pain in older adults with age 64 or above who lived in a retirement

community ranged from 25.0% to 76.0%. While the prevalence of chronic pain in older

adults living in residential care was much higher and ranged from 83.0% to 93.0%

(Abdulla, 2013). Interestingly however, in older adults, there seems to be a decrease in

pain sensitivity as indicated by an increase of pain thresholds, especially for heat stimuli

(Abdulla, 2013). Thus, the pain threshold increases with age, but the pain tolerance

decreases (Abdulla, 2013). This poses a clinical challenge, as external threats may be

detected later and older adults may run higher risks of injuries on an everyday basis.

2.4.2 Individual Factors

There are a wide range of personal factors that have been linked to the occurrence of

pain conditions. The most common job-related risk factors for the development of any or

combinations of pain conditions are: high level of physical job demands, job insecurity,

sedentary work position, job dissatisfaction, and low levels of social support in the

workplace (Taylor et al., 2014). Additionally, the most common personality-related risk

factors include: stress, anxiety, depression, low self-esteem, and the presence of chronic

8

health problems (Hoy, 2010). Lastly, lifestyle-related risk factors that have been noted to

lead to a variety of pain conditions are: smoking, obesity, and poor health status (King,

2011). Despite all these risk factors, a previous episode of pain is the best predictor and

the most consistent risk factor for development of any type of pain condition in the future

(Henschke et al., 2015).

2.5 Preclinical Pain Research

Much of our understanding of pain physiology and mechanisms comes from preclinical

studies in animal models of pain.

2.5.1 Do Animals Experience Pain?

In the literature, there seems to be two extremes regarding pain perception in vertebrates

other than humans (Dickety, 1992). One extreme claims that all vertebrates can

experience and perceive pain (Bateson, 1991). The other extreme believes that pain can

only be perceived by adult humans (Bateson, 1991). In middle of these extremes lies a

range of schools of thought, that accept a broader definition and assessment of pain.

Here the focus will be on pain in vertebrates, specifically rodents.

2.5.2 Methods of Assessing Pain in Animals

One way to determine whether animals experience pain is to expose animals to noxious

stimuli, then observe and compare their behaviors to animals that have not been exposed

to pain. However, studying pain in live animals raises ethical, philosophical and technical

problems. Philosophically, we cannot directly assess the ‘experience’ of pain in animals,

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but rather rely on observational research of animal pain behaviors. Nevertheless, to study

the perception of pain, animals are subjected to a variety of noxious stimuli like thermal,

mechanical or chemical. These stimuli are manipulated in combined in ways to model

acute pain, chronic pain, arthritis pain, inflammatory pain and visceral pain (Shriver,

2006). After exposure, animals are kept under observation to record behaviors from

simple spinal reflexes such as withdrawal of the paw from a heat source, to complex

behaviors like writhing behaviors after i.p. injection of chemical irritants (Sidhu et al.,

2004).

2.5.3 Common Models in Pain Research

Animal models of pain or nociception have two main components: the method of insult

and the measurement of an end-point. Pain models can be acute and involve reflexive

responses, such as hotplate and tail flick assays, or chronic and involve physiological

changes and adaptation, such as nerve injury models. Many early studies investigating

pain in rodents used the acute application of noxious stimuli such as heat from a hot-

plate, or a focused beam of intense light on their tails. These noxious stimuli would

typically result in behavioral responses, including paw withdrawal or tail flicking in

response to the noxious stimulus. This response would be used as an indicator of pain

(Cervero, 1996). In fact, a review by Le Bars and colleagues (2001) noted that the majority

of studies conducted between 1970 to 1999 used either the hot-plate test or the tail-flick

test to study nociception in mice. One advantage is that the source of noxious stimuli can

be applied from a distance, such as a laser beam, thus not relaying on any physical

10

contact between the experimenter and the mouse, while the mouse is being tested for

pain behaviors (Le Bars et al., 2001).

Another method to study pain in rodents is to chemically induce pain or discomfort to

study mechanisms of tissue injury. Rodent models of inflammatory hyperalgesia can

involve the administration of a variety of inflammatory agents (Lewis, 1939). For example,

subcutaneous injection of chemical irritants including formalin or acetic-acid use different

mechanisms to induce nociception. An advantage of this method is that the inflammatory

agents can be injected into an entire structure, like joints and muscle tissues to model the

persistent pain encountered in humans (Le Bars et al., 2001). In addition, nerve injury

models, such as the Spared Nerve Injury (Pertin et al., 2012), can cause long-lasting or

permanent damage with subsequent changes in nociceptive networks. Nerve injury

models are associated with changes in mechanical and thermal sensitivity that model

human neuropathic conditions.

Overall, these experimental designs are considered “behavioral studies” due to their need

for conscious animals to study nociception. The animal’s behaviors are used to study

responses to noxious stimuli; like an input-output system. Thus, in tests of this nature, it

is imperative to define and describe the stimulus (the input) and the behavioral response

of the animal (the output).

2.5.4 Animal Models Used in Current Study

I have used cyclophosphamide (CYP) as a noninvasive rodent model of acute bladder

pain (Boucher et al. 2000; Leventhal & Strassle 2008; Auge et al. 2013). It has been well-

documented that a single intraperitoneal injection of CYP results in bladder inflammation

11

(Stillwell & Benson 1988). CYP injection results in pronounced edema, massive

inflammatory cell infiltration, tissue hemorrhages, and mucosal ulcerations. In the kidney

and liver, CYP is converted to acrolein, which accumulates in the bladder. Prolonged

contact of acrolein with the bladder wall during urine retention generates painful cystitis.

This acute pain is accompanied by pelvic pain, frequent urination, and inflammation due

to edema, infiltration of inflammatory cells followed by tissue hemorrhages, and mucosal

ulcerations (Smaldone et al., 2009; Juszczak et al., 2010; Auge et al., 2013). The CYP

pain model has a rapid onset. Specifically, full bladder inflammation is reached within four

hours, and this inflammation can be easily studied with behavioral and histological

observations (Lanteri-Minet et al., 1995). Together, these data demonstrate that chronic

CYP‐induced bladder inflammation is a suitable mouse model of acute pain (Auge et al.,

2013; Juszczak et al., 2010; Smaldone et al., 2009).

Next, we aimed to reverse the effects of CYP by using ketoprofen. Ketoprofen is used

frequently as an analgesic non-steroidal anti-inflammatory drug (NSAID) in rodents.

Ketoprofen inhibits the cyclooxygenase-catalyzed metabolism of arachidonic acid to

prostaglandin precursors thereby inhibiting the synthesis of prostaglandin production in

tissue (Fornai et al., 2005; Humes et al., 1981; Kido et al., 1998; Legen et al., 2002).

Ketoprofen has been shown to reduce pain induced by CYP in rodents (Takagi-

Matsumoto, Tsukimi & Tajimi, 2004). In vivo, ketoprofen greatly (>95%) binds to plasma

albumin and is primarily confined to the plasma compartment (Royer et al., 1986).

Typically, cell membrane damage triggers the release of membrane-bound arachidonic

acid and its cyclooxygenase-catalyzed metabolism to short lived endoperoxides

(Johnston & Fox, 1997). By inhibiting cyclooxygenase-catalyzed formation of

12

prostaglandins, NSAIDs provide pain relief by blocking prostaglandin-mediated release

of inflammatory mediators associated with hyperalgesia like bradykinin and prostaglandin

E2, (Armstrong et al., 1999). NSAIDs also reduce the hyperalgesia that occurs in

inflammation. The half-life of ketoprofen in mice is approximately 1.1hr. (Sanoh et al.,

2011). Overall, ketoprofen can potentially reduce the bladder inflammation and pain

caused by CYP.

Second, an overall systemic inflammation was induced by lipopolysaccharide (LPS). LPS

is found in the outer membrane of Gram-negative bacteria and has been shown to

activate Toll-like receptor (TLR)-4 (Burkovskiy, Zhou, & Lehmann, 2013). Activation of

TLR-4 has previously been linked with initiating an overall inflammatory response in

animals and humans, leading to a systemic activation of the innate immune system (Fink,

2013). LPS also has been shown to indirectly stimulate the production of inflammatory

cytokines, such as tumor necrosis factor-α, interleukins, and interferons (Gouel-Chéron,

& Montravers, 2013). As for the pharmacokinetics of LPS, LPS binds to a LPS-binding

protein (LBP) in blood, and this interaction triggers the monocytic secretion of several pro-

inflammatory cytokines (Hao et al., 2013; Shapiro, L., & Gelfand, 1993). All tissues,

organs and systems are affected by LPS (Liu et al., 2017). However, the dynamics of

responses are different; some cell signaling systems (e.g., TLRs) respond within few

minutes, while others may require hours. The inflammatory response of the primary

lymphoid tissues and vascular and cardiopulmonary systems were observed to be the

fastest (Liu et al., 2017). LPS disappears rapidly from the circulation, with a half-life of 2–

4 min in mice (Yao et al., 2017). Overall, these data demonstrate that LPS is a suitable

model to investigate systemic inflammation.

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Lastly, in the present study, reserpine was used to induce depression-like symptoms

(Brodie et al., 1955). Reserpine is known to impair the vesicular transporters in the pre-

synaptic neuron of serotonin and norepinephrine. Specifically, reserpine was shown to

decrease adrenergic effects through a deficiency of norepinephrine in the

parasympathetic nervous system (Shore et al., 1957). Thus, the sedation experienced by

the animal is due to the decrease of catecholamine concentrations in the brain and CNS.

These observations led to the theory that alterations of mood are mediated by

modifications of the levels of serotonin, noradrenaline, among other catecholamines

(Brodie and Shore, 1957). Reserpine is an irreversible inhibitor of VMAT2 (Schuldiner,

Lui, & Edwards, 1993). Additionally, reserpine, drastically decreases the sequestering of

synthesized monoamines into synaptic vesicles. Thus, preventing monoamines from

being released into the extracellular space (Naudon et al., 1995). Reserpine is rapidly

metabolized, with 30-40% of reserpine appearing in urine as trimethoxybenzoic acid in

the first four hours and another 35% as un-metabolized reserpine in fecal excretion in this

time frame (Numerof, Gordon, & Kelly, 1955). Reserpine reduces synaptic release of

monoamines by blocking vesicular monoamine transporter (VMAT) and preventing

vesicular transport and storage of monoamines (Naudon et al., 1995). Since reserpine

irreversibly blocks VMAT, the rate of restoration of a vesicle monoamine concentration

depends upon the rate of monoamine synthesis (Ponzio et al., 1984). The recovery to

normal monoamine levels after reserpine is slow: specifically, even three weeks after

reserpine injection, the monoamine levels were still below normal (Häggendal, &

Dahlström, 1971). The reduced vesicular storage of monoamines therefore increases the

rate of monoamine metabolism and causes a reduction in monoamine concentrations for

14

up to several weeks (Ponzio et al., 1984). In this study, I investigated if mouse hanging

behavior is affected by depressive-like symptoms induced by reserpine treatment.

I aimed to reverse the effects of reserpine by desipramine. Desipramine is a tricyclic

antidepressant (TCA) which is used in the treatment of depression. It acts as a relatively

selective norepinephrine reuptake inhibitor (Kim et al., 2010; Lucki et al., 2001).

Desipramine is extensively metabolized in the liver by CYP2D6 (major) and CYP1A2

(minor) to 2-hydroxydesipramine, an active metabolite (Weinshenker et al., 2002). The

antidepressant effects of TCAs are thought to be due to an overall increase in

serotonergic neurotransmission; thereby exerting a positive effect on mood (Cooper,

Leonard, & Schnieden, 1972; Chen, 2002). Desipramine has a long half-life of elimination,

ranging from 7 to 60 hours in various species (Lemke et al., 2012). Overall, desipramine

can reverse the effects of reserpine.

2.5.5 Challenges in Current Animal Models of Pain

Although there might be parallels between humans and rodent nociception and

perception, it is important to note that none of the existing models in pain research can

replicate all the symptoms of human pain. There are many reasons for this deficit. One

problem with the above-mentioned methods is that they heavily rely on the rodent’s

reflexes. The use of reflexes as a readout of pain perception is limiting in many ways.

Firstly, the mechanisms involved in generating pain-related reflexes do not involve the

cerebral cortex. Through fMRI studies, the cerebral cortex has been shown to be

activated in both, acute and chronic pain condition in humans and in rodents (Borsook &

Becerra, 2006). Tests such as tail-flick and paw-withdrawal measure rodent’s reflexes,

15

and do not directly involve the cerebral cortex. Secondly, these models problematically

depend on interaction with the experimenter, confounding the results due to many

extraneous factors such as sex of the experimenter (Sorge et al., 2014).

A further challenge in animal models of pain is fundamentally in the clinical definition that

pain is an emotional experience as well as a sensory one, wherein the emotional

experience is harder to quantify. Additionally, pain is often co-morbid with depression and

anxiety that are difficult to measure in animal models. Clinically, pain is also measured

through subjective measures collected through self-reporting or scoring. Such subjectivity

in assessing pain in animals is unfortunately not a possibility, as we lack the ability to

understand the animal’s self-assessment of painful feelings through words or scores (Le

Bars et al., 2001).

While these challenges are methodology-based, there are several other variables that

complicate pain research in animals. Various studies have identified that pain and pain-

related behaviors are extremely sensitive and dependent on wide range of variables such

as: circadian rhythmicity, ambient temperature, humidity in the laboratory and animal

facilities, bedding texture, and cage density. Moreover, genetically modified knock-out

mice differ in their pain behaviors when compared to their other similar knock-outs

companions (Minett et al., 2014; Mogil, 2009). There is a substantial body of research

examining sex differences in rodent pain behaviors. For example, female rodents are

more sensitive and respond more quickly to lower drug doses than male rodents (Mogil,

2009). A more surprising variable that affects pain behaviors in rodents is the sex of the

experimenter. One study demonstrated that exposure to male experimenters caused an

inhibition in pain behaviors of rodents, but resulted in a strong physiological stress

16

response that caused stress-induced analgesia (Sorge et al., 2014). Such observations

were not found in rodents exposed to a female experimenter. Thus, what seems to be

minor factor, might have a profound influence on the rodents and subsequently their pain-

related behaviors.

2.5.6 Current Developments in Animal Models of Pain

Over the past few decades, many animal models of pain have been devised and

employed. However, due to the above noted issues and the complexities of human

behaviour and experience that cannot be fully captured in animal studies, there remains

a translational gap between research and clinic. Recently, there has been a considerable

interest in developing animal pain models that go beyond the traditional reflexive

responses. Specifically, researchers have been investigating and developing measures

that asses “suppressed” pain-related behaviors to more closely resemble pain

experienced by humans (Stevenson et al., 2006). Pain suppressed behaviors include

behaviors that are otherwise considered ordinary, for example, feeding, grooming, and

locomotion.

The rationale behind studying pain-suppressed behaviors is that in most models, pain is

delivered to the rodent and the subsequent reflexive output behaviors, that are otherwise

not present such as a tail-flick is observed. These pain-related behaviors are used to test

the effects of potential analgesics. While there many advantages, one major

disadvantage is that the potential drug’s effects and the drug’s unwanted side effects

cannot be separated in such models. For example, many non-analgesic drugs such as

cholinergic agents, antihistamines, and dopamine antagonists not only decrease pain-

17

related behaviors (e.g. increased paw withdrawal latency from a heat source) but they

also induce motor sedation, thereby creating false-positives. In addition, pain is commonly

associated with the suppression of many adaptive behaviors like feeding. Thus, it would

be crucial for anti-nociceptive drugs to restore such behaviors. These two disadvantages

could be a reason for drug failure in human clinical trials, as the drug may only provide

sedative effects without actually relieving pain. To overcome these challenges,

researchers have also started examining motor effects of potential drugs, to distinguish

between the sedative and anti-nociceptive effects of the drug (Stevenson et al., 2006).

There are two main reasons to include pain-suppressed behaviors in studies of pain and

anti-nociceptive drugs. Firstly, an ideal anti-nociceptive drug should be able to reverse

the pain-suppressed behaviors to baseline levels, thus, restoring feeding, grooming, and

locomotion. In contrast, drugs that have a sedative effect would not be able to restore

these behaviors, and in fact could worsen locomotion. Secondly, pain suppressed

behaviors are used as a method of assessing pain in humans. Thus, it would be

reasonable to include suppressed behaviors in the study of animal pain and in the

evaluation of potential analgesics (Stevenson et al., 2006).

So far, there have been few studies examining the effects of pain-suppressed behaviors

specifically on wheel-running and burrowing (Deacon, 2006). Researchers have

illustrated an overall decrease in these behaviors as a result of post-surgical and

inflammatory pain. Interestingly, the burrowing behavior can be restored by the

administration of gabapentin, naproxen, ibuprofen, morphine or pregabalin (Deacon,

2006). Advantages to investigating burrowing behavior is that it measures a non-reflexive

behavior, is responsive to analgesics and holds the potential to analyze pain in rodents.

18

2.5.7 Monitoring Animal Welfare to Study Pain and Drug Action

Behavioral changes that occur as mice become sick have been characterized in a number

of ways, which include a reduction in ‘luxury behaviors’ such as playing, grooming and

socialization (Ohl & Staay, 2012). Pain-suppressed behaviors, also called ‘sickness

behaviors’, are behavioral changes following exposure to infectious agents. These

behaviors have been particularly well described: animals are typically less active, sleep

more, exhibit postural changes and consume less food/water (Mogil, 2009). Disease is

frequently induced in laboratory mice to model pathophysiological processes and

investigate potential therapies, but despite what is known about behavioral changes as

animals become sick, behavioral phenotyping of mice involved in disease studies is

relatively rare (Mogil, 2009; Richardson, 2015).

Animal welfare can be determined from animal behaviors such as locomotion, activity,

exploration, sleep and feeding (Ohl & Staay, 2012). Unfortunately, assessing these

multiple parameters typically requires manual observation, repetitive animal testing and

handling by trained personnel. This approach to animal monitoring has several major

drawbacks: 1) it is costly and time-consuming, 2) it does not provide continuous

monitoring of animal behavior, 3) it requires interaction with the animals that may

confound behavior measurement. Therefore, there is a need for an animal monitoring

assay that is robust and sensitive to mouse welfare that can be easily incorporated to

facilitate the early detection of disease (Richardson, 2015).

Preliminary data from our lab indicate that cage-lid hanging behavior is a luxury behavior

in mice that could be used to detect pain and illness. Since mice are by far the most

commonly used experimental animal (Ohl et al., 2012), the ability to detect early

19

pathological behavior may generate overall reductions in laboratory animal suffering. It is

anticipated these results will reveal hanging behavior as a readout of mouse welfare that

can be easily incorporated to facilitate the early detection of disease.

20

Chapter 3

Materials and Methods

3.1 Animals

Eight-week old C57BL/6J mice were obtained from the Charles River Laboratories, Saint

Constant, Quebec, Canada. In many experiments of this study, both male and female

mice were used and compared for Aim 1. Thereafter, male mice were used. The mice

colony was maintained in the University of Toronto’s Animal Care Facility. Mice were

housed in same-sex cages containing 3-4 animals in each cage. Each plastic cage

included a wire top and contained corncob bedding and one plastic dome. Food and water

were provided ad libitum. Cages were maintained in a temperature-controlled (23 ± 4 °C)

environment with 15/9h light/dark cycle. C57BL/6 mice were used exclusively. Animals

were 9 weeks old at the time of testing, except in Aim 1 where the effect of age was

assessed. Mice were housed for 1 week prior to testing, to allow for acclimatization. For

Aim 2, specific methods for detailed experimental protocols are discussed below. For all

tests in Aims 1 and 2, animals were randomly assigned across treatments. Each animal

was subjected to experimental manipulations only once. The experimenter was blinded

to animals’ treatment groups. Testing was done during the night cycle. All experimental

procedures adhered to federal and institutional guidelines, and were approved by the

University of Toronto Animal Care Committee. All efforts were made to minimize animal

suffering.

21

3.2 Drugs

The following drugs were used: CYP, ketoprofen, LPS, reserpine, and desipramine. All

drugs were obtained from Sigma Chemical Co. (Toronto, Canada), except for reserpine,

which was obtained from https://racehorsemeds.com. Drugs were dissolved in

physiological saline, while, appropriate vehicle treated groups were assessed

accordingly. Specifically, CYP, ketoprofen, LPS and desipramine were dissolved in

physiological saline. The control for these drugs was physiological saline. Reserpine was

pH-balanced to the physiological pH of 7.4, using 0.05 M NaOH. Here, physiological

saline was determined to be the appropriate control. All drugs were administered

intraperitoneally (i.p.) in a constant volume of 10 mL/kg body weight.

3.3 Experimental Schedule

3.3.1 Reverse Light/Dark Cycles

Over the 24-hour cycle, the circadian rhythms dictate many changes in the physiology

and behavior of mice. One of the main reasons researchers study mice is to understand

human physiology and disease. However, mice are a nocturnal species, and are typically

more active during the night cycle. A study by McLennan and Taylor-Jeffs (2004)

suggests that studying mouse behavior during the night phase more closely parallels

human behavior than during the light/dark phase. Thus, to better capture mouse activity

and behavior, all the experiments in this study were conducted during the dark cycle. For

convenience, from the day the mice arrived to the day of the experiment, mice were kept

in a reverse light/dark cycle room. This reverse light/dark cycle room enabled researchers

22

to study nocturnal mice behavior during the day, in a dark room. Red lights were used to

illuminate the room, allowing the experimenters to see.

3.3.2 Mice Sensitivity to Red Light

Light plays a crucial role in regulating physiological and behavioral characteristics, both

in mouse and humans. However, mice differ from humans in that they are less sensitive

to longer wavelength light, such as the red spectrum. Red light, which mice cannot see,

has been recommended for observations during night-phase observations (Jennings et

al., 1998). Thus, in this experiment, I used four red LED bulbs to illuminate the room.

Bulbs were strategically placed to allow for maximum light while recording.

3.4 Experimental Equipment

3.4.1 Cage

Mouse behavior was recorded in a standard clear plastic 30 x 12 x 13 cm cage with metal

wire grating for a lid. The metal wire lid contained a food hopper and a water bottle holder.

Corncob bedding was provided inside the cage. Plastic dome/mouse huts and cotton

nest-building materials were removed from the cage to provide for less impeded views of

the mice during recordings. This clear visibility at all times decreased the ability of mice

hide, and improved data quality. Mice were individually placed inside the cage for the

duration of the experiment.

23

3.4.2 Behavioral Recordings

To record mouse activity, a SONY HDR-CX405 camcorder was placed in front of the

cage. Camera zoom and focus were adjusted such that the entire mouse cage was

recorded while minimizing white space in the background/surroundings, and to maximize

the visible cage area in order to track mouse movements closely. In order to keep track

of each mouse, their tails were marked using blue Sharpie pen. Their cages and cameras

were numbered accordingly. Immediately after injections, mice were placed in their

respective cages, and recording was started. Use of the experiment room door was

minimized until the end of the experiment cycle.

3.4.3 Data Processing

The videotapes were digitized in MTS format, and transferred to a computer. A video

format converter, AnyVideoConvertor was used to convert MTS files to MPEG2 and mp4

formats. The converted files were then analyzed using CleverSys HomeCageScan® and

Ethovision®, respectively.

3.5 Software used to Analyze Video Recordings

3.5.1 CleverSys HomeCageScan®

For Aim 1, the CleverSys HomeCageScan® (CleverSys Inc, Reston, VA, USA) software

was used to analyze the 24h video recordings. This software uses MPEG2 files to detect

a moving mouse from a stable and unchanging cage background. The software

determines the position, posture, and movement of the mouse to detect 34 pre-

24

determined behaviors (see Table 1). The behaviors where then reclassified into five

combined categories for further analyses, as previously done (Roughan et al., 2009).

Table 1. The behaviors used and their reclassification for analysis with HomeCageScan.

Re-Classified Behavior HCS Behaviors

Immobile • Urinate

• Pause

• Sleep

• Remain Low

• Stationary

• Groom

Locomotion • Walk slowly

• Walk right

• Walk left

• Run Right

• Run Left

• Turn

• Come down (from hanging)

• Stretch Body

• Circle

Feeding • Chew

• Eat Zone 1,2,3

• Drink

Exploration • Rear up

• Sniff

• Dig

• Forge

• Come down from partially reared

• Remain Rear Up

• Rear up partially

• Remain partially reared

• Come down to partially reared

25

Off-ground activity • Hang Vertically

• Hang Cuddled

• Remain Hang Cuddled

• Hang Vertically From Rear Up

• Remain Hang Vertically

• Hang Vertically from Hang Cuddled

3.5.2 Ethovision®

To specifically explore the effects of age and sex on hanging behavior (Aim 1), the

EthoVision behavioral tracking system EthoVision® (EthoVision XT, version 10.1, Noldus

Information Technology b.v., Wageningen, The Netherlands) software was used. The

software identifies the spatial location of the center point of the mouse, which can be used

to determine hanging behavior of mice based on their proximity to the cage lid. Hanging

behavior was identified by mice climbing onto the metal lid of the cage and hanging,

suspended off the cage floor. Hanging zones were drawn as shown in Figure M3. Hang

Zone arena (depicted in green in figure M3) was established and validated by first

manually scoring for hanging behavior in few sample videos; then adjusting the hang zone

arena as needed to get similar results as manual scoring. Ethovision was used for all

other experiments in Aims 2 and 3 as it provided rapid analysis of hanging behavior. The

software also calculates distance travelled by each mouse over the course of the

experiment.

26

3.6 Forced Swim Test – Behavioral Assay

The Forced Swim Test (FST) assay was conducted according to the previously described

protocols (Can et al., 2011). Briefly, in FST a mouse is introduced to a beaker of water

and forced to swim in an environment from which there is no escape. After an initial period

of vigorous activity, it will eventually cease to move altogether, making only minimal

movements to keep its head above water. The amount of time spent immobile or not

swimming is measured as a proxy of depression. In our FST experiments, a mouse was

placed in a glass cylinder (height: 30 cm; diameter: 10 cm; containing 15 cm of water at

24 ± 1°C) for 6 min (test; figure M4). A camera was mounted beside the forced swimming

cylinder, and all the test events were recorded. An experienced observer independently

scored the behavior blindly. The duration of immobility was analyzed during the last 4

minutes of the 6-minute testing period.

3.7 Data Analysis

Statistical analyses were performed using Graphpad Prism® Version 7.0. Results were

expressed as the mean ± standard error of the mean (SEM). First, the data were tested

for normality under the Shapiro-Wilk, D’Agostino-Pearson and Kolmogorov-Smirnov test.

Next, an outlier was defined as an observation that lies an abnormal distance from other

values in a random sample from a population. Thus, before outliers can be singled out, it

was necessary to characterize normal observations. First, I examined the overall shape

of the graphed data for important features, including symmetry and departures from

assumptions. Grubbs' test (Grubbs 1969) is used to detect a single outlier in a univariate

data set that follows an approximately normal distribution. Specifically, the Grubbs' test

27

statistic is the largest absolute deviation from the sample mean in units of the sample

standard deviation. Since the data were normally distributed, I used Grubbs’ test as an

analytical tool to detect outliers in my data. Lastly, data were analyzed for significance

and interactions between variables using a one-way or two-way analysis of variance

(ANOVA) as appropriate. One-Way ANOVA was followed by a post-hoc Dunnet’s test to

compare each treatment condition to the control saline/control group, while two-way

ANOVA were followed by Bonferroni post-hoc tests. Differences with P < 0.05 were

considered statistically significant.

28

Chapter 4

Physiological parameters affecting hanging behavior

4.1 Introduction

The objective of Aim 1 was to characterize the hanging behavior of mice across various

physiological parameters. First, I characterized the effect of sex and age on behavioral

activity in male and female mice over the course of 24 hours. Next, I specifically explored

the effects of age on hanging behavior by observing 2, 4, and 6-month-old male and

female mice for over the dark cycle, where activity is increased, using Ethovision.

4.2 Characterization of Mouse Activity over 24-hours

The primary aim for this protocol was to assess mouse activity over a 24-hour period.

Previous findings from our group suggest that the incidence of hanging will be higher

during the mouse’s active (dark) cycle. In this experiment, I therefore characterized

mouse off ground activity over the course of 24 hours, via grouping all off ground activity

into a broader category called off ground activity (see below). In addition, I assessed the

effects of sex and light/dark cycle on hanging behavior. First, individual male and female

mice were weighed and then placed in separate cages containing bedding, food and

water. As described above, each cage was placed in front of a camera which continuously

recorded the movement of the mice over a 24-hour period (Figure M2). I started the

experiment using 1-month-old male and female mice, with 8 mice of each sex. Mice were

then re-tested for 24 hours monthly until the age of 6 months.

First, videos were analyzed using HomeCageScan. Given the large number of behavioral

measures, I grouped behaviors identified by the software into five broader categories:

immobility (I), locomotion (L), feeding (F), exploration (E) and off ground activity (O). This

29

categorical breakdown was based on a published study (Roughan et al., 2008). After the

categorical breakdown, data were normalized within each category, for each month, with

0 being the lowest and 1.0 being the highest. Data were imported into a python script, to

create heat-maps based on the normalized activity measures during each hour of the 24-

hr recording session (Figure 1). The heat-maps depicts that an increase in off ground

activity during the night-cycle. There were no sex differences in this behaviour.

Because off-ground activity was highest during the dark phase, we further compared how

much time male and female mice spent engaging in this category of behaviour specifically

during the dark phase to assess sex differences in this activity. The combined male and

female data are shown in figure 2a, demonstrating a similar tendency for off-ground

activity to peak around 2 months of age in both sexes. However, a two-way ANOVA of

age and sex did not reveals significant differences (F (5, 168) = 1.059, p=0.3850). We

further analyzed off-ground behaviour of each sex by month using one way ANOVAs.

Figure 2b depicts a significant change in off ground activity in female mice as they age (1

month female vs 2 month female ** p<0.01, 1 month female vs 4 month female ** p<0.01,

2 month female vs 5 month female **** p<0.0001, 2 month female vs 6 month female ****

p<0.0001, 3 month female vs 5 month female ** p<0.01, 3 month female vs 6 month

female *** p<0.001, 4 month female vs 5 month female **** p<0.0001, 4 month female vs

6 month female **** p<0.0001). Similarly, this trend was also evident in male mice figure

2c (1 month male vs 2 month male * p<0.05, 1 month male vs 4 month male ** p<0.01, 2

month male vs 5 month male *** p<0.001, 2 month male vs 6 month male **** p<0.0001,

3 month male vs 5 month male ** p<0.01, 3 month male vs 6 month male **** p<0.0001,

4 month male vs 5 month male *** p<0.001, 4 month male vs 6 month male ****

30

p<0.0001). Thus, male and female mice decreased their off ground activity as they aged

(Figure 2). Given that we observed differences in off ground activity, we sought to specify

whether this applies to hanging behaviour.

4.3 Effects of Age and Sex on Hanging Behavior

In this experiment, I aimed to test the effect of age on hanging behavior. I used 2, 4, and

6-month-old male and female mice with 10 mice in each group for a total 60 mice. As

described above, mice were placed in separate cages and their movements were

recorded over a 9-hour dark cycle period. The 9-hour period was chosen, as the prior

experiment showed an increased off ground during the dark phase.

In these experiments we specifically examined hanging behaviour of the mice. Hanging

behavior is when a mouse climbs onto the metal lid of the cage, and completely suspends

itself off the cage floor. The first variable of interest was hanging episode frequency, which

was operationally defined as the total frequency a mouse hung from the cage lid, over the

duration of the experiment. The second measure was total hanging duration. Hanging

duration was calculated as the sum of the total time a mouse spent hanging from the cage

lid, over the duration of the experiment. The third variable was locomotion. Locomotion

was calculated as the total distance travelled by a mouse inside its cage over the duration

of the experiment as a control measurement.

I first measured the number of hanging episodes in 2, 4 and 6-month-old mice. Figure 3

depicts a trend towards a decrease in hanging episode of male mice as they age (F (2,

52) = 1.454, p=0.2430). However, this change in behavior was not evident in female mice.

31

Interestingly, the hanging episode in male and female mice was similar in 2-month-old

mice (see Figure 3).

I then measured total hanging duration in 2, 4 and 6-month-old mice. As seen in the

previous analysis, there was a decrease in hanging duration in female mice as they age.

However, this change in duration was not evident in males. Male mice reduced both

hanging behavior frequency and duration as they aged (Figure 3). A two-way ANOVA

shows that females, at the age of 2, 4, 6 months, hang more than their male counterparts,

although this difference did not reach significance (age x sex: F (2, 52)=2.93, p=0.0623).

Lastly, I examined locomotion in 2, 4 and 6 month-old mice. The distance travelled in

female mice decreased significantly with mouse age (Figure 4). This change in behavior

was not observed in male mice. These results are in agreement with trends in hanging

episode frequency and hanging duration, as males experienced an overall decrease in

locomotion (including hanging frequency and duration) with age, in comparison to their

female counterparts (Figure 3). Two-way ANOVA shows that females, at the age of 2, 4,

6 months, travel significantly more than their male counterparts (age x sex: F(2, 52) =

6.825 p= 0.0023).

32

Figure 1. Graphical representation of behavioral activity over a 24-hour period of 1-6 (months numbered on the far left of the figure) month-old female and male mice. (n = 8 females, n = 8 males).

D: Day time

N: Night time

I: Immobile. This category represents behavior such as “stationary”, “sleep” and “remain low” over a 24h period.

L: Locomotion. This category represents behavior such as “walk”, “turn” and “circle” over a 24h period.

33

F: Feeding. This category represents behavior such as “chew”, “eat” and “drink” over a 24h period.

E: Exploration. This category represents behavior such as “sniff”, “dig” and “forge” over a 24h period.

G: Off Ground Activity. This category represents behavior such as “hang verically”, “hang cuddled” and “remain hanging” over a 24h period.

34

Figure 2a. Two-way ANOVA of off ground activity over a 15-hour period of 1 to 6

month-old male and female mice. In both males and females, there is a significant

decrease in off ground activity in older mice (5 and 6 months) compared to younger

mice, with a maximum amount hanging seen between 2 and 4 months of age (n = 8

females, n = 8 males for each month).

35

Figure 2b. One-way ANOVA of off ground activity over a 15-hour period of 1 to 6

month-old female mice. We see an increase in off ground activity from 2-4 months in the

female mice, which eventually decreases significantly as the mice age.

36

Figure 2c. One-way ANOVA of off ground activity over a 15-hour period of 1 to 6

month-old male mice. We see an increase in off ground activity from 2-4 months in the

female mice, which eventually decreases significantly as the mice age.

37

Figure 3. Hanging episodes and duration over a 9-hour period of 2, 4, 6-month-old female and male

mice. These figures depict the male mice in blue and female mice in red bars. There is a significant

decrease in hanging episodes and hanging duration by 4 and 6-month female mice when compared to

the 2-month female mice. Interestingly, this trend was not observed in male mice for either hanging

episodes or hanging duration. Two-way ANOVA shows that hanging episodes for females are

significantly more than their male counterpart. (n = 10 females, n = 10 males). Two-way ANOVA shows

that females; hanging duration is significantly more than their male counterpart. (n = 10 females, n = 10

males) ** p<0.01, *** p<0.001, **** p<0.0001.

2 4 60

500

1000

1500

2000

2500

Month

Hangin

g E

pis

odes

Hanging Episodes

****

Male

Female

2 4 60

2000

4000

6000

8000

10000

Month

Hangin

g D

ura

tion (

s)

Hanging Duration

*******

Female

Male

38

Figure 4. Distance traveled over a 9-hour period of 2, 4, 6-month-old female and male

mice. This figure depicts the male mice in blue and female mice in red bars. There is a

significant decrease in distance travelled by 4 and 6-month female mice when

compared to 2-month female mice. Interestingly, this trend was not observed in male

mice. Additionally, two-way ANOVA shows that females, at the age of 2, 4, 6 months,

travel significantly more than their male counterparts. (n = 10) and male (n = 10) mice.

** p<0.01, *** p<0.001, **** p<0.0001.

2 4 60

10000

20000

30000

Month

Dis

tance T

ravelled (

cm

)

Distance Travelled

Male

Female

********

Chapter 5

Physiological parameters affecting hanging behavior

5.1 Introduction

The objective of Aim 2 was to determine whether hanging behavior was impaired in mouse

models of pain and disease. Preliminary data from our group indicate that pain models

involving plantar injections or nerve injuries affecting the leg reduce hanging. However,

these approaches can lead to confounding results as mice need all their paws to suspend

themselves from the metal lid cage (to hang). I therefore used a pain model that does not

involve limbs. Pain was elicited in mice using CYP, which can cause a painful bladder

cystitis. We further investigated whether hanging behaviour may be impaired by decreases

in well-being that are not explicitly caused by pain, such as illness. To investigate whether

illness can similarly impact hanging, mice were injected with LPS i.p. to model septic illness.

5.2 Visceral Pain

I studied the effects of long-term pain on hanging behavior. The traditional pain models

investigated in our lab’s preliminary studies drastically decrease hanging behavior; but

since standard pain models inflict pain by targeting the mouse paw, they may have impeded

the ability of the mouse to hang. Thus, in order to determine whether hanging behavior can

be reduced by painful stimuli that do not affect the paw, I investigated the effect of painful

bladder cystitis on hanging in mice. I.P injection of CYP (30, 100, 300 mg/kg) was used to

cause a painful cystitis and allodynia in the lower abdomen of mice. CYP injection results

in pronounced edema, massive inflammatory cell infiltration, tissue hemorrhages, and

mucosal ulcerations. Induction of bladder cystitis does not affect the paw directly, and gives

rise to an inflammatory response which results in pain and distress in mice. This dose range

was selected based on prior work, where a single dose of 300 mg/kg CYP was found to

produce acute visceral pain (Olivar et al., 1999). We also tested the 30 mg/kg and 100

mg/kg doses to identify a threshold for CYP impairment of hanging behaviour.

The results for CYP’s effect on hanging frequency, duration, and locomotion are presented

in Figures 5, 6, and 7, respectively. At the lowest dose tested, 30 mg/kg, CYP did not cause

impairments of hanging or locomotor behavior. However, at 100 mg/kg, I observed

significant reduction in distance travelled during the experiment (Fig 7, p < 0.05), but not in

hanging episodes (F (2, 43) = 0.5191 p=0.5988), hanging duration (F (2, 42) = 2.627

p=0.0841), or distance travelled (F (2,46) = 1.76 p= 0.1835). At the highest dose of 300

mg/kg, CYP significantly reduced hanging frequency (Fig 5, p < 0.05), hanging duration

(Fig 6, p < 0.01) and distance travelled (Fig 7, p < 0.01).

5.3 Cyclophosphamide effect reversal by NSAIDs

I next investigated whether administration of the analgesic, ketoprofen attenuated the

reduction in hanging and locomotor behavior induced by CYP. Ketoprofen is used

frequently as an analgesic non-steroidal anti-inflammatory drug (NSAID) in rodents.

Ketoprofen inhibits the cyclooxygenase catalysis of arachidonic acid and prostaglandin

precursors thereby inhibiting the synthesis of prostaglandin production in tissue (Fornai et

al., 2005; Humes et al., 1981; Kido et al., 1998; Legen et al., 2002). NSAIDs, specifically 5

mg/kg of ketoprofen, have been shown to reduce pain induced by 100 and 300 mg/kg CYP

in rodents (Takagi-Matsumoto, Tsukimi & Tajimi, 2004). A dose of 5 mg/kg of ketoprofen

was used here, as higher dose have been reported to cause gastrointestinal lesions that

may confound possible analgesic actions of the drug (Lamon et al., 2008).

The administration of ketoprofen alone (5 mg/kg) did not significantly alter hanging or

locomotor behavior of mice (Figs 5-7), with no statistical significance observed between the

control and the treatment groups (hanging episode F (2, 43) = 0.5191 p=0.5988; hanging

duration F (2, 42) = 2.627 p=0.0841; distance travelled F (2,46) = 1.76 p= 0.1835). Thus,

co-administration of ketoprofen did not significantly restore hanging to control levels.

To check whether the non-significant results were due to a lack of statistical power, I

conducted post hoc power analyses (Rosner, 2016) with power (1 - β) set at 0.80 and α =

05, two-tailed. Based on this analysis using the observed variability, the sample sizes would

have to be increased substantially to observe a statistically significant analgesic effect. This

suggests that the lack of differences observed here do not solely arise from insufficient

experimental power and likely reflect a lack of ketoprofen effect in this assay. The full list of

sample sizes needed to reach significance of 0.05 for each experiment based on observed

variability is provided in supplementary table 3 (T3).

5.4 Illness Model of Pain

I further investigated whether hanging behavior is reduced by inflammation. To induce

systemic inflammation, mice were injected with LPS (5, 15, 50, 150, 450 µg/kg, i.p.). Studies

have demonstrated that LPS administration of 50, 100 or 200 µg/kg significantly decreased

locomotor activity (Bazovkina et al., 2012). Thus, in this study I tested a wide range of LPS

doses to test their effects on hanging behavior and locomotion.

I observed a dose-dependent effect of LPS on mouse behavior, with LPS causing a

decrease in both hanging behavior and locomotor activity. Notably, I observed a decrease

in hanging frequency at all doses of LPS tested, including a significant decrease in hanging

episode (saline vs 5 µg/kg *p<0.05, saline vs 15 µg/kg *p<0.05, saline vs 50 µg/kg ****

p<0.0001, saline vs 150 µg/kg **** p<0.0001, saline vs 450 µg/kg **** p<0.0001) and

hanging duration (saline vs 50 µg/kg **p<0.01, saline vs 150 µg/kg **p<0.01, saline vs 450

µg/kg **p<0.01) after administration. At an LPS dose of ≥50 µg/kg, I observed a reduction

in locomotor activity (Fig 10; saline vs 50 µg/kg **p<0.01, saline vs 150 µg/kg **** p<0.0001,

saline vs 450 µg/kg **** p<0.0001). Thus, as the dose of LPS increases, the hanging

behavior, particularly the frequency and hanging episodes decrease significantly. This

suggests that hanging and locomotor activity may both serve as sensitive measures of

inflammation and systemic illness in mice.

5.5 Monoamine-depletion model of depression

I next investigated if mouse hanging behavior can be affected by modulation of a CNS

function. Specifically, I explored depressive-like symptoms in mice and its effect on hanging

behavior. Given that presynaptic catecholamine depletion is cited as a cause of depression

in humans (Brodie et al., 1965), I used the reserpine model of monoamine depletion to

pharmacologically induce neurotransmitter reduction associated with depression.

Reserpine inhibits the uptake of neurotransmitter into storage vesicles resulting in depletion

of catecholamines and serotonin from central and peripheral axon terminals (Kim et al.,

2010; Lucki et al., 2001). Thus, the effects of reserpine are characterized by slow onset of

action and sustained effects (Brodie et al., 1965). To test the effect of reserpine on hanging

behavior, 2-month-old male mice received either reserpine (1 or 2 mg/kg) or vehicle (saline)

on Day 1 (Figs 11-13). In the present study, I chose to test mice on Day 3 to allow for

reserpine to act on neurons within the CNS. Hence, after injection, mice were placed back

into their home cages with their littermates until Day 3. On Day 3, each mouse was placed

in a separate cage, and were recorded over a 9-hour dark period. On Day 4, I verified

depressive behavior in the mice induced by reserpine using the Forced Swim Test (FST;

Figure 14). Notably, the doses chosen for the experiment do not impair motor activity in

mice (Brodie et al., 1965). I further tested whether any possible change in mouse activity

induced by reserpine could be reversed by the antidepressant, desipramine. Desipramine

is a tricyclic antidepressant (TCA) which is used in the treatment of depression. It acts as

a relatively selective norepinephrine reuptake inhibitor (Kim et al., 2010; Lucki et al., 2001).

A desipramine dose of 10 mg/kg i.p. was chosen, because a recent study for desipramine

effects in the mouse FST revealed that single i.p injection with a 10 mg/kg dose before the

test was sufficient to reduce the immobility time in otherwise healthy mice (Sugimoto et al.,

2008). Immediately following reserpine injection, mice were injected with desipramine (10

mg/kg i.p.) or vehicle (saline).

No significant differences in hanging episodes were detected after treatment with either

reserpine, with or without desipramine (Fig 11; F (2, 75) = 0.8273, p=0.4412). Although a

two-way ANOVA (reserpine dose vs desipramine) did not reveal significant interaction

between reserpine dose or desipramine treatment, post-hoc tests indicated that hanging

duration was reduced by reserpine at both doses tested. (Fig 12; F (2, 78) = 0.5, p= 0.6085;

post-hoc tests: saline vs 1 mg/kg *p<0.05, saline vs 2 mg/kg ***p<0.001). Additionally, there

is a significant difference between reserpine and reserpine + desipramine groups (Fig 12;

p < 0.0001), suggesting the reduction in hanging duration induced by reserpine was caused

by a reduction in neurotransmitter levels. Notably, I observed no change in locomotor

behavior of mice after either reserpine or reserpine and desipramine (Fig 13; (F (2, 78) =

0.5, p=0.6085). The lack of locomotor effect of reserpine suggests that the reduction in

hanging behavior seen after reserpine does not arise from generally reduced locomotion

and may reflect the selective modulation of hanging.

To confirm whether changes in hanging behavior were associated with depressive behavior

induced by reserpine, I tested in mice in the FST (Figure M4). Briefly, the characteristic

behavior of immobility in a FST is a commonly used proxy of depression. In our

experiments, I observed that immobility significantly increased after reserpine at 1 mg/kg

and 2 mg/kg was reversed in mice given desipramine (Fig 14; saline vs 1 mg/kg ****

p<0.0001; saline vs 2 mg/kg **** p<0.0001). Thus, suggesting that the reduction in mobility

induced by reserpine was caused by a reduction in neurotransmitter levels, modeling

clinical depression in mice post-reserpine injections.

Figure 5. Reduced hanging episodes of 2-month-old male mice in a visceral pain model.

Mice were injected with Saline (n = 15), CYP 30 mg/kg (n = 8), CYP 100 mg/kg, (n = 7) or

CYP 300 mg/kg, (n = 6) Saline + Ketoprofen (n = 7), CYP 100mg/kg (n = 8), and 300 CYP

+ Ketoprofen (n = 6). *p<0.05.

Sa

l i ne

30

mg

/ kg

10

0m

g/ k

g

30

0m

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g

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E

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C Y P H a n g i n g E p i s o d e s

*

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H a n g i n g E p i s o d e s

Ha

ng

ing

E

pis

od

es

C Y P

C Y P + K e t o p r o f e n

Figure 6. Reduced hanging duration of 2-month-old male mice in a visceral pain model.

CYP saline (n = 15), CYP 30 mg/kg (n = 8), CYP 100 mg/kg, (n = 7) and CYP 300 mg/kg,

(n = 6) Saline + Ketoprofen (n = 7), CYP 100mg/kg (n = 7), 300 CYP + Ketoprofen (n = 6)

**p<0.01.

Sa

l i ne

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mg

/ kg

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C Y P H a n g i n g D u r a t i o n

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D

ur

at

io

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(s

ec

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Ha

ng

ing

D

ur

atio

n (

s)

C Y P

C Y P + K e t o p r o f e n

Figure 7. Reduced distance travelled by 2-month-old male mice in a visceral pain model.

CYP saline (n = 16), CYP 30 mg/kg (n = 8), CYP 100 mg/kg, (n = 8) and CYP 300 mg/kg,

(n = 7) Saline + 5 mg/kg Ketoprofen (n = 7), CYP 100mg/kg (n = 8), 300 CYP + 5mg/kg

Ketoprofen (n = 6) *p<0.05, **p<0.01.

Sa

l i ne

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mg

/ kg

10

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D i s t a n c e T r a v e l l e d

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ra

ve

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(c

m)

***

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Dis

ta

nc

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Tr

av

ell

ed

(

cm

) C Y P

C Y P + K e t o p r o f e n

Figure 8. Reduced hanging episodes of 2-month-old male mice in an illness model of pain.

Saline (n = 12), LPS 5 µg/kg (n = 6), LPS 15 µg/kg (n = 4), LPS 50 µg/kg (n = 6), LPS 150

µg/kg (n = 8), and 450 µg/kg (n = 8) *p<0.05, **** p<0.0001.

Saline

5µg/kg

15µg/kg

50µg/kg

150µg/kg

450µg/kg

0

1000

2000

3000

Hangin

g E

pis

odes

LPS Hanging Episodes

**

********

****

Figure 9. Reduced hanging episodes of 2-month-old male mice in an illness model of pain.

Saline (n = 11), LPS 5 µg/kg (n = 6), LPS 15 µg/kg (n = 4), LPS 50 µg/kg (n = 5), LPS 150

µg/kg (n = 8), and 450 µg/kg (n = 7). **p<0.01. At the lower dose of 5 µg/kg and 15 µg/kg

of LPS, no statistically significant differences were detected when compared with saline.

However, hanging frequency was reduced at higher doses of LPS.

Saline

5µg/kg

15µg/kg

50µg/kg

150µg/kg

450µg/kg

0

2000

4000

6000

LPS Hanging Duration

Hangin

g D

ura

tion (

sec)

******

Figure 10. Reduced distance travelled by 2-month-old male mice in an illness model of

pain. Saline (n = 12), LPS 5 µg/kg (n = 6), LPS 15 µg/kg (n = 4), LPS 50 µg/kg (n = 6), LPS

150 µg/kg (n = 8), and 450 µg/kg (n = 7). **p<0.01, *** p<0.001, **** p<0.0001. No

statistically significant differences were detected in locomotion at 5 µg/kg and 15 µg/kg of

LPS.

Saline

5µg/kg

15µg/kg

50µg/kg

150µg/kg

450µg/kg

0

20000

40000

60000

80000

LPS Distance Travelled

Dis

tance T

ravelle

d (

cm

)

**

****

****

Figure 11. Hanging episodes of 2-month-old male mice in a pharmacological model of

depression. Saline (n = 16), Reserpine 1 mg/kg (n = 13), Reserpine 2 mg/kg (n = 11), Saline

+ Desipramine 10 mg/kg (n = 18), Reserpine 1 mg/kg + Desipramine 10 mg/kg (n = 13),

Reserpine 2 mg/kg + Desipramine 10 mg/kg (n = 12).

Sa

l in

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R e s e r p i n e

R e s e r p i n e + D e s i p r a m i n e

R e s e r p i n e H a n g i n g E p i s o d e s

Figure 12. Hanging duration of 2-month-old male mice in a pharmacological model of

depression. Saline (n = 16), Reserpine 1 mg/kg (n = 14), Reserpine 2 mg/kg (n = 11), Saline

+ Desipramine 10 mg/kg (n = 18), Reserpine 1 mg/kg + Desipramine 10 mg/kg (n = 14),

Reserpine 2 mg/kg + Desipramine 10 mg/kg (n = 12). *p<0.05, ***p<0.001. ****p<0.0001.

Sa

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R e s e r p i n e H a n g i n g D u r a t i o n

*

***

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R e s e r p i n e H a n g i n g D u r a t i o n

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g

Du

ra

tio

n

(s

ec

)

R e s e r p i n e

R e s e r p i n e + D e s i p r a m i n e

****

Figure 13. Distance travelled by 2-month-old male mice in a pharmacological model of

depression. Saline (n = 15), Reserpine 1 mg/kg (n = 14), Reserpine 2 mg/kg (n = 12), Saline

+ Desipramine 10 mg/kg (n = 18), Reserpine 1 mg/kg + Desipramine 10 mg/kg (n = 13),

Reserpine 2 mg/kg + Desipramine 10 mg/kg (n = 12).

Sa

l i ne

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R e s e r p i n e D i s t a n c e T r a v e l l e d

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(c

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R e s e r p i n e

R e s e r p i n e + D e s i p r a m i n e

R e s e r p i n e H a n g i n g D u r a t i o n

Figure 14. Forced Swim behaviour of mice in a pharmacological model of depression.

Reserpine treatment increased the amount of time mice spent immobile in in the forced

swim test and this increase was not observed in mice treated with reserpine and

desipramine. Saline (n = 16), Reserpine 1 mg/kg (n = 14), Reserpine 2 mg/kg (n = 12),

Saline + Desipramine 10 mg/kg (n = 18), Reserpine 1 mg/kg + Desipramine 10 mg/kg (n

= 14), Reserpine 2 mg/kg + Desipramine 10 mg/kg (n = 12). **** p<0.0001.

Saline

1mg/

kg

2mg/

kg

Saline

1mg/

kg

2mg/

kg

01020304050

200

300

400****

Forced Swim Test

Tim

e S

pent Im

mobile

(s)

Ketoprofen (10mg/kg)

****

Desipramine (10 mg/kg)

Chapter 6

Discussion

In the present study, I quantified hanging behavior to determine whether this behaviour

provides a measure of well-being in animal models of pain, illness, and depression, in mice.

I explored whether hanging behavior can be used as a readout of mouse welfare that can

be easily incorporated to facilitate the early detection of disease in mice. This work

provides, to the best of my knowledge, the first detailed description of hanging behaviour

in mice in terms of physiological determinant of hanging, and how hanging can be

modulated by pain, illness, or neurochemical manipulation. The findings of this study

indicate that hanging behaviour provides a valuable and sensitive behavioural measure for

the investigation of mouse well-being, and may be of utility for pharmacological studies.

I first investigated the physiological parameters of hanging behavior. I observed that on

average, younger male and female mice hung more than older mice. A similar pattern was

also observed with locomotion. Furthermore, female mice hung more on average than male

mice; a pattern that was consistent with locomotion. However, hanging duration and

distance travelled failed to show a similar pattern; wherein hanging duration and distance

travelled remained similar as the mice got older. This lack of change in male mice may

reflect their overall lower amount of hanging compared to female and the possible existence

of a “floor effect” or lower boundary of hanging behaviour in healthy mice. Nevertheless,

these findings strongly indicated that 2-month old mice are the ideal age for further

investigation of hanging behaviour. Although female mice exhibited more hanging, my

additional studies were conducted using male mice for two reasons: 1) studies of pain and

illness are historically conducted using only male mice and it was unclear whether the pain

and illness models would be as reliable in female mice due to the lack of published literature

in females; 2) I conducted the pain and illness studies in parallel with the age and sex

assessments, and it was not clear that females would hang substantially more than males.

Further work with female mouse models of pain, illness, and depression is warranted given

these novel findings.

I investigated mouse behavior under various traditional mouse models of pain and distress.

I was careful to eliminate all models of pain that targeted the paw, as this could very well

have confounded the findings. Thus, I started my study with injecting CYP i.p. to induce

visceral pain in mice. It has been well documented that after just one injection of CYP, there

is bladder inflammation that results in acute bladder pain (Boucher et al., 2000; Leventhal

and Strassle 2008; Auge et al., 2013). Cyclophosphamide can also be administered without

any anesthesia, freeing the study of potential confounding factors. I found that hanging

episode frequency was greatly affected only in the 300 mg/kg treatment group, with lower

doses not affecting hanging episode frequency. Interestingly however, hanging duration

(defined at the total time the mice spent hanging in the nine-hour experiment) was

significantly affected at the dose groups of 100 mg/kg and 300 mg/kg. Thus, hanging

duration showed a greater sensitivity to cyclophosphamide dose in comparison to hanging

episodes. A similar trend was observed with locomotion (defined as the total distance

travelled by the mice over the course of nine hours), where CYP reduced locomotion at 100

mg/kg and 300 mg/kg. Interestingly, a similar trend was observed by Bon and colleagues

(2003) when they injected mice with cyclophosphamide 100 mg/kg, 200 mg/kg and 300

mg/kg. They found that mechanically hypersensitivity, measured via von Frey test, was the

highest at 300 mg/kg dose and decreased as the dose of cyclophosphamide decreased.

They further analyzed locomotion, observing a trend comparable with the present study.

Locomotion in their study was greatly affected at higher dose of 300 mg/kg when compared

to 100 mg/kg. I further attempted to reverse the effects of cyclophosphamide with

ketoprofen, a non-steroidal anti-inflammatory drug, having analgesic and antipyretic

properties. It is also one of the most commonly used therapeutic drug in rodents.

Ketoprofen has been shown to have inhibitory effects on prostaglandin and leukotriene

synthesis, since it inhibits the cyclooxygenase catalysis of arachidonic acid and

prostaglandin precursors thereby inhibiting the synthesis of prostaglandin production in

tissue (Fornai et al., 2005; Humes et al., 1981; Kido et al., 1998; Legen et al., 2002). Thus,

I identified ketoprofen as having potential to reduce the pain caused by cyclophosphamide.

In the reversal treatment groups, 100 mg/kg and 300 mg/kg cyclophosphamide was mixed

with 5 mg/kg of ketoprofen and injected into mice. In the 100 mg/kg + 5 mg/kg ketoprofen

groups, hanging episodes, hanging duration and distance travelled were not significantly

different from the saline group. However, there is a trend towards analgesic effects of

ketoprofen in hanging episodes, hanging duration and distance travelled in the 300 mg/kg

+ 5 mg/kg ketoprofen groups, compared to their respective saline groups. Perhaps

ketoprofen is not a strong enough analgesic, since it did not completely reverse the effects

of 300 mg/kg of cyclophosphamide but there a trend towards analgesic effects of

ketoprofen in the 100 mg/kg of cyclophosphamide group.

Next, I assessed whether hanging behavior was reduced by septic illness, when animals

were injected with LPS. LPS is a chemical present in Gram negative bacterial cell walls,

and produces a systemic inflammatory response that mimics sepsis (Fink 2013). It has

been noted that this generalized inflammatory process is largely due to a

macrophage/monocyte-mediated event, which results in excessive production of pro-

inflammatory cytokines (including IL-1 and TNF-) in response to LPS injection (Matsuura,

2013). Even doses of LPS in the low µg/kg range can elicit strong immune responses in

rodents (Marra et al., 1994). Since the overarching goal of the study was to determine if

hanging behavior is a sensitive indicator of declining mouse welfare, I chose to inject mice

with a range of LPS from very low (5 µg/kg) to moderately high (450 µg/kg) doses. In the

present study, I found that even the lowest dose of 5 µg/kg had a significantly decreased

hanging episode frequency. This decrease in hanging episodes became more pronounced

at higher levels of significance as the drug dose increased. Thus, it was observed that with

a model of overall systematic inflammation, hanging frequency in mice decreased

significantly even at the lowest LPS dose. A similar trend was observed in hanging duration,

where 50 µg/kg resulted in a significant reduction in hanging time, and the same effect

carrying forward at higher LPS doses. In contrast, locomotion was significantly reduced in

the 50 µg/kg, 150 µg/kg, and 450 µg/kg groups and not affected at the lower LPS doses of

5 µg/kg and 15 µg/kg treatment groups. Overall, these data show that pain models not

targeting the mouse paw specifically, impeded the ability of the mouse to hang, still resulted

in a decrease of hanging behavior at higher doses.

Finally, I examined whether hanging behavior could serve as an indicator of psychological

welfare using the reserpine model of depression. Two doses were chosen based on

anticipated effect sizes observed in literature, where 1 mg/kg is close to a threshold dose

for reserpine effects but 2 mg/kg reliably produces changes in behaviour or physiological

parameters such as temperature. While there was no significant change detected in the

hanging episode frequency of mice, there was no statistically significant reduction detected

in the 1 mg/kg and 2 mg/kg reserpine groups (see Figure 13 & 14). However, in the hanging

duration group, there was a significant reduction detected in the 2 mg/kg reserpine group

when compared to saline treated mice. Locomotion failed to show any significant decrease

in distance travelled, contrasting with the ability of hanging behavior to detect minor

changes in animal welfare. It is notable that the effects of reserpine on hanging behavior

and Forced Swim Test were observed 2 and 3 days after a single dose of reserpine,

respectively. More strikingly, these changes were reversed by a single dose of desipramine

given at the same time as reserpine. Based on the pharmacokinetics of reserpine and

desipramine there would be minimal levels of these drugs present in the mouse at the times

of testing (Kim et al., 2010; Lucki et al., 2001). Specifically, given the relatively well-

described mechanism of action of reserpine, I can speculate on the possible

neurobiological mechanisms driving hanging behavior. Monoamines, particularly dopamine

and serotonin, are strongly implicated in the modulation of luxury behaviors, such as wheel

running (Waters et al., 2013), and play behavior (Swallow et al., 2016) that are sensitive to

animal wellbeing (Ohl & Staay, 2012). It is therefore likely that similar biological systems

modulating these behaviors also underlie the modulation of hanging behavior. Although

further research is necessary to definitively make this mechanistic connection, this possible

link further supports our hypothesis that hanging behavior provides a measure of animal

wellbeing.

6.1 Comparison to Literature on Animal Welfare Testing

As stated in the introduction, millions of mice are used worldwide annually to evaluate the

efficacy and safety profile of pharmaceutical drugs. However, record levels of drug failures

during clinical trials indicate that current preclinical screening poorly predicts drug toxicity

in humans. One strategy to mitigate these shortcomings is to improve measurement of

mouse welfare during preclinical testing. Currently, mouse welfare is primarily determined

by the absence of aversive physical or psychological sensations such as illness, distress,

hunger, anxiety, fear and pain (Ohl & Staay, 2012). Animal welfare can be inferred from

animal behaviors such as locomotion, activity, exploration, sleep and feeding (Mogil, 2009;

Richardson, 2015). Unfortunately, assessing these parameters typically requires manual

observation as well as repetitive animal testing and handling by trained personnel. This

approach to animal monitoring has several major drawbacks: firstly, traditional animal

welfare testing models require highly trained and skilled experimenters, have time

consuming protocols and are usually very expensive to carry out. Secondly, the traditional

animal welfare testing models also comprise of static measures, rather than measuring

more dynamic, on-going progressions of welfare over time. There is therefore a need to

measure on-going changes in animal behaviors to gain a better understanding of animal

welfare. Thirdly, current animal welfare models require interaction with the animal that may

confound the behaviour-based measurements. This handing and interaction may affect the

study measures, as it was discovered that rats and mice show increased stress levels and

stress hormones when exposed to male experimenters compared to female (Sorge et al.,

2014).

The concept that play behaviors can be used as potential measures of animal wellness is

not novel (Fagen 1981; Lawrence 1987). In fact, many studies in the past have explored

the potential of play behaviors as a measure of animal welfare, since it is exhibited by

almost all mammals, is extremely non-invasive and does not require as much technological

equipment or human interaction as compared to the traditional methods of assessing pain

(Fraser & Duncan 1998; Spinka et al., 2001; Barnard 2011). Play behaviors decrease in

the presence of threats or illness, and increase when the animal is satiated (Lawrence

1987; Fraser & Duncan 1998; Spinka et al., 2001; Dawkins 2006). For example, it was

illustrated that after a 24-hour period of food deprivation, rats decrease their play behavior

over threefold (Siviy & Panksepp, 1985). In contrast, access to unlimited food increased

the play behavior twofold (Sharpe et al., 2002). These findings suggest that play behaviors

may be a useful indicator of overall absence of stressors, and by extension, an indicator of

animal welfare. This led early scientists like Lawrence (1987) and Fraser & Duncan (1998)

to conclude that play behavior can be regarded as a luxury behaviour: one that is only

displayed when there are no stressors, and basic physiological needs have been met,

which can ultimately indicate animal welfare (Dawkins 1983).

In the present study, I used an automated video tracking system to observe hanging

behavior in the mouse. This greatly decreased the cost of experiments, reduced the need

for highly trained and skilled experimenters, and obviated direct human interaction with the

rodents.

6.2 Study Limitations

While care was taken in designing and executing the experiments and the overall study,

there are a few limitations with the present study. Firstly, in the cyclophosphamide,

lipopolysaccharide and reserpine experiments, only 2-month-old male mice were used, and

female mice were not included. There is considerable evidence that pain behavior is

modulated by sex (Bartley & Fillingim et al., 2016; Mogil, 2005). The parallels between sex-

dependent pain behavior and hanging behavior would be an intriguing avenue for further

research, and the inclusion of females should be considered in future experiments.

Secondly, baseline measurements were not done for each mouse used in the CYP, LPS

and reserpine experiments. However, the data reveal that there is considerable variability

in the hanging behavior of saline treated mice. Thus, it would have been beneficial to record

individual mice baseline hanging to gain a better understanding of how hanging behavior

changes post drug exposure. This high variability of hanging behavior in saline treated mice

may partially explain the difference in hanging behavior observed control mice across the

cyclophosphamide, lipopolysaccharide and reserpine experiments. Alternatively, the i.p.

injection itself may reduce hanging behavior, possibly through the stress induced by

injection. Although we did not directly assess the impact of stressors on hanging behavior,

this may be an area of interest for future studies. Additionally, the season when the

experiments were performed could contribute to the variability in the saline treated groups,

although our experiments were largely interleaved to contribute to possible temporal

confounders.

Additionally, I used the FST to measure depression-like symptoms in mice, induced by

reserpine. This assay provided a positive control that reserpine modulated mouse

behaviour in a manner consistent with depression, and which was reversible by

antidepressants. However, it would have increased the validity of my findings to quantify

pain behaviors in the cyclophosphamide and lipopolysaccharide via other behavioral

methods such as von Frey, Randall-Selitto and the Hargreaves tests (Bon et al., 2003;

Juszczal et al., 2010). However, the drug dose used in these experiments have been used

many times to induce pain and have been quantified extensively in the literature (Auge et

al., 2013; Bon et al., 2003; Boucher et al., 2000). Additionally, it would have been beneficial

to measure the LPS-induced cytokine response in mice post-injection of LPS to quantify

the effects of LPS observed. Similarly, measuring the change in monoamine levels, due to

reserpine injection, would have further added to the assessment of pharmacological-

induction of depression in mice.

Finally, the mice had to be taken out of their home cages and were individually placed into

new cages. This could have confounded our results as mice might have been stressed due

to the change in environment and isolation from their littermates.

6.3 Future Directions

In the present study, I aimed to cover a wide range of pain and emotional distress models

to explore hanging behaviour. I also analyzed various physiological parameters such as

age and sex to further characterize hanging behaviors. In future studies, it would be

worthwhile to further characterize hanging behaviors across different strains of mice. The

C57BL/6 strain was selected as it is one of the most commonly used inbred mouse lines.

However, it would be interesting to assess hanging behavior in several other commonly

used strains of mice to assess the generalizability of this measure, such as 129 mice

(inbred), BALB/c mice (inbred), CD-1 mice (outbred), Swiss Webster mice (outbred), and

immunocompromised SCID mice (inbred). These strains would be particularly valuable

since they are quite popularly used in animal studies of pain. It would also be interesting to

characterize hanging behaviors across different age groups for each strain of mice, as the

present study determined a significant decrease in hanging behavior as the age of mice

increased.

It would further be valuable to investigate if and why some mice have an increased

tendency to hang over others. Throughout this study, even after statistically removing

outliers, it was evident that there were some mice engaging in hanging behavior more than

others in their age group. A potential reason for these differences in hanging could be that

hanging behavior is like any other behaviour, where in some mice like to engage in it more

often than others.

Finally, it would be worthwhile to investigate effects of depression induced via chronic

unpredictable stress or learned helplessness models to understand how hanging behavior

is modulated under such emotional conditions (Gambarana et al., 2001; Monterio et al.,

2015) Additionally, since chronic stress can produce anxiety-like symptoms in mice, it would

be interesting to explore the effects of anxiogenic agents such as

metachlorophenylpiperazine, or the effects of behavioral anxiety models on hanging

behavior. This would help elucidate whether anxiety or depression symptoms, which are

observed in animal models of chronic pain, could account the decrease in hanging behavior

in these pain models.

Lastly, in all of the above-mentioned experiments it would be valuable to add females as

well as male mice to better understand the sex-dependence of hanging behavior.

Unfortunately, literature has largely focused on male, ignoring female populations. Notably,

one meta-study looked at scientific research as a whole and found that out of 1,200 papers

surveyed, only 42 disclosed the sex of their lab animals (Clayton and Collins, 2014). There

are many papers in the literature discussing sex differences on the pathways and rates of

drug metabolism. In some instances, females have been reported to have serious side

effects from the same drug where males do not; however, only males are used for testing

potential therapeutic drugs (Clayton and Collins, 2014). Thus, the reliance on male-only

subjects could potentially be devastating and costly from a scientific perspective.

6.4 Conclusions

While the potential of luxury behaviors have been explored in the literature, cage-lid

interaction has never been previously used for behavior measurement of animal welfare.

Hanging behavior is identified as a mouse climbing onto the metal lid of the cage, and

suspending itself off the cage floor. Overall, our data suggest that cage-lid hanging behavior

is a luxury behavior metric (in frequency and duration) in mice that can detect pain, illness

and emotional distress. The long-term and non-invasive measurement of hanging behavior

will facilitate the study of sensory disorders (including persistent pain), mood disorders

(such as depression and anxiety) and sickness behavior (induced by infectious agents).

Given the ease with which hanging behavior can be assessed, and the non-invasive and

ethological validity of this measure, I anticipate that results of this work will be valued by

veterinarians, animal care technicians, basic scientists, and pharmaceutical researchers.

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Methods Figures

Capsaicin, Formalin, CYP, LPS &

Reserpine

Overnight Recording – 9hr

Figure M3. Screen shot of a video recording being analyzed using Ethovision®, one of the automated video tacking systems used in this study.

Figure M1. Hanging behavior identified by mice climbing onto the metal lid of the cage and suspending themselves from the lid.

Figure M2. For each protocol described in the paper, each mouse was separately placed in front of a camera which continuously recorded the movement of the mice over 9hr during the dark cycle.

Figure M4. A mouse shows active behavior in an inescapable cylinder during the FST procedure. The tests were performed for 6min. All swimming sessions were recorded form the front view.

Figure S1. Reduced hanging behavior in traditional mouse models of pain. A) Acute pain models (formalin and capsaicin). B) Long-term pain models - complete Freund’s Adjuvant (CFA) and Spared Nerve Injury (SNI). C) Animal model of cancer pain. * P < 0.05, **P < 0.01, *** P < 0.001.

Figure S2. Reduced hanging behavior in mouse models of pain which do not target the paw. A) Post-surgical pain model – craniotomy. * P < 0.05, *** P < 0.001.

TumorSham

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Supplementary Figures

Figure T3. Table depicts the power analysis results for each experiment.

Experiment Comparison Current Sample Size

(with outliers removed) Power

Sample Size needed to reach

Power of 0.8

RES - Episodes Saline vs Desi 34 0.04 4991

1mg/kg RES vs 1mg/kg RES + Desi 26 0.82 N/A

2mg/kg RES vs 2mg/kg RES + Desi 23 0.48 50

RES - Duration Saline vs Desi 34 0.23 178

1mg/kg RES vs 1mg/kg RES + Desi 28 0.98 N/A

2mg/kg RES vs 2mg/kg RES + Desi 23 0.99 N/A

RES - Distance Saline vs Desi 33 0.59 55

1mg/kg RES vs 1mg/kg RES + Desi 27 0.34 89

2mg/kg RES vs 2mg/kg RES + Desi 24 0.97 N/A

CYP - Episodes Saline + Keto 22 0.04 6409

100mg/kg CYP vs 100mg/kg CYP + Keto 15 0.42 38

300mg/kg CYP vs 300mg/kg CYP + Keto 12 0.76 14

CYP - Duration Saline + Keto 22 0.77 24

100mg/kg CYP vs 100mg/kg CYP + Keto 14 0.42 36

300mg/kg CYP vs 300mg/kg CYP + Keto 12 0.57 21

CYP - Distance Saline + Keto 23 0.27 99

100mg/kg CYP vs 100mg/kg CYP + Keto 16 0.73 20

300mg/kg CYP vs 300mg/kg CYP + Keto 13 0.25 61

LPS - Episodes 43 0.999 N/A

LPS - Duration 41 0.981 N/A

LPS - Distance 43 0.998 N/A

Experiment Number of Mice Removed; Variable type

CYP – Saline Treatment 1; Hanging Episodes

CYP – 100mg/kg Treatment 1; Hanging Episodes

CYP – 300mg/kg Treatment 1; Hanging Episodes

CYP – Saline Treatment 1; Hanging Duration

CYP – 100mg/kg Treatment 1; Hanging Duration

CYP – 300mg/kg Treatment 1; Hanging Duration

CYP - 100mg/kg + 5 mg/kg Ketoprofen Treatment

1; Hanging Duration

LPS - 450µg/kg Treatment 1; Hanging Episodes

LPS – Saline Treatment 1; Hanging Duration

LPS - 50µg/kg Treatment 1; Hanging Duration

LPS - 450µg/kg Treatment 1; Hanging Duration

LPS - 450µg/kg Treatment 1; Distance Travelled

RES - 1mg/kg Treatment 1; Hanging Episodes

RES - 2mg/kg Treatment 1; Hanging Episodes

RES - 1mg/kg Reserpine + Desipramine 10 mg/kg Treatment

1; Hanging Episodes

RES - 2mg/kg Reserpine + Desipramine 10 mg/kg Treatment

1; Hanging Duration

RES – Saline Treatment 1; Distance Travelled

RES - 1mg/kg Reserpine + Desipramine 10 mg/kg Treatment

1; Distance Travelled

Figure T4. Table depicts the outliers removed via Grubbs’ test.