Prior voluntary wheel running prevents memory deficits and enhanced neuropathic pain presenting after morphine dosing ceases

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1 University of Colorado, Boulder CU Scholar Undergraduate Honors Theses Honors Program Spring 2018 Prior voluntary wheel running prevents memory deficits and enhanced neuropathic pain presenting after morphine dosing ceases Nathan Plattner Follow this and additional works at: Part of the Animal Experimentation and Research Commons, Behavioral Neurobiology Commons, Medicinal Chemistry and Pharmaceutics Commons, Molecular and Cellular Neuroscience Commons, Nervous System Diseases Commons, Other Immunology and Infectious Disease Commons, Pathological Conditions, Signs and Symptoms Commons, and the Pharmacology Commons Recommended Citation Plattner, Nathan, "Prior voluntary wheel running prevents memory deficits and enhanced neuropathic pain presenting after morphine dosing ceases" (2018). Undergraduate Honors Theses This Thesis is brought to you for free and open access by Honors Program at CU Scholar. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of CU Scholar. For more information, please contact

2 Prior voluntary wheel running prevents memory deficits and enhanced neuropathic pain presenting after morphine dosing ceases Nathan Plattner Department of Molecular, Cellular, and Developmental Biology University of Colorado Boulder Defended April 9th, 2018 Examining Committee: Dr. Linda Watkins Department of Psychology and Neuroscience Honors Advisor Dr. Kevin Jones Department of Molecular, Cellular, and Developmental Biology Honors Council Representative and Committee Member Dr. Monika Fleshner Department of Integrative Physiology Honors Committee Member

3 Abstract Clinical research studies show that chronic pain is a prevalent, difficult affliction to manage. The most common management for chronic pain is opioids, which research is showing to paradoxically induce persistent, worsened pain states as well as potentially having negative effects on memory. The necessity for understanding chronic pain and drug management options is apparent, and research is aiming to do just that. Exercise is one potential preventative measure or post hoc treatment possibility for chronic pain, as abundant research has shown. Knowledge of the interactions between exercise, morphine dosing, and peripheral nerve injury is nonexistent. As such, we desired to investigate whether prior to injury voluntary exercise could prevent paradoxical hyperalgesia experienced after morphine regimen cessation. We also desired to investigate these same parameters with relation to memory. Keywords: Neuroimmunopharmacology, Pain, Memory, Opioids, Exercise 2

4 Introduction Chronic pain is an extremely prevalent affliction in modern society with numerous causes; cancer, traumatic injury, neuropathy, headache, and arthritis are a few. Management for chronic pain is usually extreme due to the severity; opioids such as Morphine, Fentanyl, and Oxycodone are all commonly prescribed to treat severe pain. It is now known that opioid treatment has persistent paradoxical effects post-dosing that are contradictory to the analgesia experienced while dosing, deemed opioid induced hyperalgesia (OIH) 1, 2, 3. The threads tying pain experienced in these incidents/diseases together are specific, chronically overexcited neural and immune pathways. Current research is focusing on better understanding these pathways in order to find new treatments for chronic pain and preventative measures and treatments for the paradoxical effects of morphine 4, 5. Voluntary exercise has just recently been recognized for its protective capabilities prior to onset of pain such that recovery duration and severity are both significantly lessened 8. It should be of no surprise, then, that research is showing voluntary exercise to be mechanistically immunological 6, 7. Preconditioning is currently a popular research theory describing how basal immune system activity is reprogrammed during and after voluntary exercise. This preconditions, or trains, the immune system to have a lower response in the presence of pain, as compared to no exercise prior to pain 8, 9. Exercise is now known to be protective against 8, as well as a treatment for 7, peripheral pain. Exercise also has beneficial effects on learning and memory, stress, and anxiety in both naïve rats and rats with peripheral nerve injury 9, 10, 43. This introduction will discuss inflammatory systems, peripheral nerve injury as a chronic pain model, the role of opioids, and voluntary exercise in relation to researching and understanding chronic pain and memory. 3

5 Inflammation The immune system has historically been seen as an exclusive defense system interacting only with foreign invaders 11. It wasn t until recently, in the late 1970 s, that research began to show interaction between the immune system and nervous system, which has led to our current knowledge about the importance of neuroimmune function 12. Research is showing that pain is heavily based in neuroimmune signaling, and memory may be somewhat regulated by this as well. Inflammation is a unique process involving specific cell types and signaling cascades. Generally speaking, immunocompetent cells will constantly search for molecules deemed dangerous to the host with specific cell surface receptors and activate when they find said molecules. Once activated, they will begin secreting signaling molecules that allow cells in the surrounding tissue to know there is danger. This process repeats, causing signal amplification. We can think about this in three categories: cells, cell surface receptors, and signaling molecules and danger signals 5, 12. Some of the most important cells pertinent to pain and memory are neurons and immunocompetent cells such as macrophages, astrocytes, and microglia. For our purposes, we can say that macrophages and microglia are the first responders to danger molecules. These cells always have cell surface receptors expressed that are probing for danger molecules and are ready to initiate inflammatory cascades. Macrophages are circulating phagocytic lymphocytes associated with amplification and regulation of inflammation, among other functions not as pertinent to our discussion. Microglia are resident macrophages of the CNS that display heterogeniety to defend against a variety of dangers through initiation of inflammatory cascades 4

6 5, 12, 13. Astrocytes serve numerous purposes in the nervous system such as maintenance of neurons and regulation of synaptic signaling 5, 12. In relation to immune competency, astrocytes are capable of reacting to pro-inflammatory cytokines and modulating strength of nociceptive signaling at synapses in the dorsal horn 14. Neurons are not usually thought of as cells capable of immune signal reception, but a growing body of research shows that this is indeed true. Neural cell surface receptors are able to receive information from cytokines, such as interleukin-1β (IL- 1β), and alter signaling intensities from sensory stimulation 12. In the context of the neuroimmune interface, cell surface receptors reprogram cellular function based on the current environment and thus modulate pain and memory consolidation. Important cell surface receptors are toll-like receptor 4 (TLR-4), interleukin-1 receptor (IL-1R), cluster of differentiation molecule 11B (CD11B), and interleukin-10 receptor (IL-10R) 5, 12. TLR-4 is expressed on all cell types mentioned earlier but is only known to up-regulate proinflammatory cytokine release and inflammation by macrophages, microglia, and astrocytes 15, 16. IL-1R is also present on all cells mentioned earlier; IL-1β is the endogenous agonist associated with this receptor 17. CD11B is found on macrophages and microglia and is associated with activated state and chemotaxis. Glial fibrillary acidic protein (GFAP) is a protein associated with the cytoskeleton of astrocytes and is indicative of activation and pro-inflammatory states 12. IL-10R is present on all cell types mentioned and functions to moderate pro-inflammatory states 6. TLR-4, IL-1R, and CD11B are all associated with increasing inflammatory states, whereas IL-10R is associated with regulating inflammatory states. TLR-4 has a non-specific binding capacity and is activated by lipopolysaccharide, opioids, and many other molecules deemed to be harmful to the system. When TLR-4 is activated, it will begin signaling through 5

7 Myd88 and/or TRIF pathways which will lead to expression and synthesis of pro-inflammatory cytokines 4. An IL-1R agonist is IL-1β and when bound will potentiate inflammatory actions and sensitize central signaling for pain. IL-1β is only released by macrophages and microglia, but will act on these, neurons, and astrocytes 4. Lastly, IL-10 has anti-inflammatory properties through acting on IL-10R. These include lowered release of pro-inflammatory cytokines and a transition to M2 anti-inflammatory phenotypes of macrophages and microglia 18. Peripheral Nerve Injury Research has provided quality rat models of varying pain to account for heterogeneous neuropathy experienced by patients 19, 20. Pain models such as chronic constriction injury () of the sciatic nerve show that communication between neurons and immune cells increases as allodynia increases, and systemic inflammation increases 19, 20. Microglia and astrocyte activation and signaling in the ipsilateral lumbar dorsal horn are positively correlated with allodynia in this model, 19, 20 and the increased inflammation provides a good model to study memory performance. As previously discussed, pain is known to involve neurons, glia, and astrocytes, and spans through multiple locales of the nervous system. Beginning in the periphery, nociceptive sensory neurons travel along the spinothalamic pain pathway to transmit information to the brain for higher order processing. The somata of peripheral afferent nerves reside in dorsal root ganglia just lateral of the spinal column. Their axons will both receive sensory input and deliver information to where they synapse in the substantia gelatinosa of the spinal cord to second order neurons. These axons will then immediately decussate and travel to the thalamus 12. Conditional neuroimmune signaling pathways rely on cytokines, immunocompetent cells, and 6

8 neurotransmitters such as glutamate and substance P. Activation of immunocompetent cells through TLR-4 will begin inflammatory cascades preparing for the release of IL-1β 21. IL-1β mediates neuroimmune signaling and subsequently sensitizes for or intensifies pain signals; increased expression of AMPA, Kainate, and NMDA on post synaptic neurons, decrease in glut- 1 reuptake transporters on astrocytes, and increased calcium flow into presynaptic terminals are all results of increased IL-1β presence at lumbar synapses 5, 12, 22, 23. This means that neural synapses contain more glutamate, contain less GABA, which is an inhibitory neurotransmitter lessening pain signaling, and are more receptive to glutamate presence which increases pain signaling. Investigation of the neurobiology of memory and what alters memory capability is a hot topic in modern neuroscience research, and recently has been shown to depend upon immune state. Chronic constriction injury models pain and inflammation, and as such may have an effect on memory 7, 9, 10, 25. Recognition and contextual memory are subcategories of memory that specifically pertain to the ability to recall or associate previous experiences based on current perceptions 25. The limbic system is the primary set of brain regions and pathways that allows for recall of perceived stimuli such as environment, objects, smell, temperature, and painful stimuli 26. It is well known that the hippocampus and insular cortex are integral parts of the limbic system, and thus in memory formation, consolidation and recall 24, 26. Put simply, a stimulus will be perceived by the associated sensory neuron, a signal will be sent to either the spine and then to the brain or straight to a brain region, and then be processed 26. As heightened states of inflammation are associated with macrophages and microglia expressing more CD11B and astrocytes expressing more GFAP, seeing these upregulated may be associated with lower 7

9 memory performance in these regions as it has been shown that peripheral nerve injury has detrimental effects on memory 28. Opioids As discussed earlier, treatment for neuropathic pain commonly comes in the form of opioid dosing. Classical opioid receptors are present throughout the central and peripheral nervous systems 5. µ, κ, and δ receptors are associated with analgesic properties, and are all found on peripheral sensory neurons, neurons of different brain regions, and microglia and astrocytes. Endogenous neurotransmitters are associated with each of these receptors and they are distinct in function; the most pertinent receptor to opioid treatment, however, is the µ 1 -recetor 4, 5, 16. Classical opioid receptors belong to the G-protein coupled receptor (GPCR) class of cell surface signaling receptors. Binding of opioids to these classical receptors causes prevention of calcium flow into pre-synaptic terminals and thus prevention of glutamate and substance P release 44. The µ 1 -receptor is associated with microglia activation through the cell surface receptor TLR-4. TLR-4 functions as a non-specific receptor for endogenous danger signals such as cell debris from apoptosis or trauma, pathogenic components such as lipopolysaccharides, or lowdensity lipoprotein. When TLR-4 is bound, it initiates the MyD88 and/or TRIF signaling cascades that will eventually lead to activation of transcription factors such as NFκ-β or IRF3 (respectively) and expression of pro-inflammatory cytokines, NLRP3, or interferon. The main effect from all of this is that pro-il-1β and intracellular inflammasomes are primed for activation when opioids bind TLR-4. Endorphins do not bind to TLR-4; however, exogenous opioids are 8

10 shown to bind TLR-4. Interestingly, only (-)-opioids will bind the µ 1 -receptor, but will nonstereoselectively bind TLR-4 4, 5, 16. Another cell surface receptor, called P2X7R, activates immune signaling in tandem with TLR-4 4, 5, 16. P2X7R belongs to a class of receptors that bind ATP and other programmed cell death (apoptosis) associated markers present with neuropathic pain. It has been shown that the main functions of P2X7R are to activate primed inflammasomes, thus beginning the conversion of pro-il-1β to IL-1β, and initiate secretion of IL-1β 16. This, paired with TLR-4 activation by opioids, is what recent research has found to mediate the paradoxical effects of morphine 16. Grace et. all (2016) displayed how an opioid regimen soon after peripheral nerve injury primes immune cells through activation of TLR-4 and P2X7R signaling pathways. Upon morphine dosing cessation, this causes persistent sensitized immune signaling within the ipsilateral dorsal horn, which is indicative of chronic neuropathy. During morphine dosing, µ 1 receptors are relevant in that (-)-morphine will bind and cause analgesia. However, when morphine dosing stops what is left are the sensitized immune cells which will potentiate neuropathy without an analgesic present. These findings provide a mechanism for how opioid regimen post injury causes enhanced neuropathy for extended periods of time following cessation of morphine dosing. Voluntary Exercise Physical activity and exercise are proven to benefit overall health in multiple ways, including modulating the immune system. Applicable research procedures fall into three categories: prior to injury/dosing, following injury, or prior to euthanasia if the only variable is exercise. Many studies show that exercise, begun after injury, will aid in recovery of chronic 9

11 pain from injury. Decreases in sciatic nerve IL1-b and increases in regenerative markers, as well as decreased astrocyte and microglia expression in associated lumbar sections are characteristic of post injury running 7, 29, 30. Also, increased levels of muscle IL-10 are associated with decreased levels of hyperalgesia 6. Profoundly, prior exercise to injury has recently been shown to be protective against the onset of pain in a rat model of injury by Grace et. all (2016). This study displayed prior running decreased injury site macrophage presence, increased serum level of anti-inflammatory cytokine IL-10 and decreased inflammatory cytokines, and desensitized states and lowered levels of inflammation in the ipsilateral dorsal horn and dorsal root ganglia 16. This has far reaching implications, as the large majority of literature focuses on the benefits of post injury exercise on pain management and neuroexcitation. This indicates a new clinically relevant avenue for pain managment; instead of managing pain post injury, it may be possible to protect against the development of allodynia. Paradoxically, a modern study shows that brain neuroinflammation in female mice post wheel running is at a higher level than at sedentary; monocyte chemoattractant protein-1 (MCP- 1), pro and anti-inflammatory cytokines, and astrocyte activation are all at higher levels than when sedentary 9. Wheel running also improves cognitive function in the frontal cortex and hippocampus. This is associated with better memory performance and cognition 25, 31. Thus, central and peripheral immune systems are reprogrammed through exercise, which benefits neuropathic pain and potentially memory. This reprogramming is called exercise induced pre-conditioning and has several important points. Peripheral inflammation is decreased through deactivation of inflammatory cells and a down-regulation of membrane receptors. IL-10 is increased in muscle associated with exercise and through the periphery, which aids in controlling the level of inflammation. In 10

12 the central nervous system pro- and anti-inflammatory cytokines are up-regulated, MCP-1 is upregulated, and astrocytes are activated 9, 25, 31. Aims Due to their efficacy, opiates are the go-to pain management drugs for chronic neuropathic pain. Only recently have clinical studies shown the persistent debilitating consequences that morphine has long after treatment has ended. Though there is quite a knowledge base from research about the effects of morphine while dosing, little is know about consequences once dosing has ceased. Thus, the goal is to understand mechanisms of action better, find better treatment for pain, and understand how to prevent/treat enhanced neuropathy experienced after cessation of morphine dosing. The aims of this project were to see whether voluntary wheel running can be used as a preventative measure for persistent allodynia developed after morphine dosing post peripheral nerve injury, and to better understand the effects of voluntary wheel running, morphine, and peripheral nerve injury on memory. We expect to observe voluntary wheel running will act as a preventative measure for allodynia caused by morphine, post dosing. Our hypotheses are: I) Exercise will reduce persistent mechanical allodynia post and morphine dosing, II) exercise will decrease IL-1β in the ipsilateral lumbar dorsal horn, dorsal root ganglia, and injury site sciatic, III) IL-10 will be increased in these tissues, IV) prior exercise will improve stunted memory consolidation post injury and post morphine dosing, and V) GFAP and CD11B will be upregulated in the insular cortex, hippocampus, and hind limb sensory cortex. 11

13 Methods Animals Adult male Sprague Dawley outbred rats (pathogen free; n=6 for each group, 48 total; weeks old upon arrival from ENVIGO; Denver, CO) were used for all experiments. Habituation proceeded 1 week after arrival through three rounds of five minute long handlings to mitigate stress and anxiety from handling during behavioral assessments. Von Frey habituation was conducted in three one-hour sessions on separate days. Rats were either single housed in cages with a running wheel for the exercise groups, or single housed in standard cages without a running wheel for the sedentary groups. All rats were housed in the same light controlled (12 hour light dark cycle; light at 0600 hr, dark at 1800 hr) and temperature controlled (23 ± 3 C) room; consistent unrestricted access to standard rat chow and water was provided. All cages contained the same standard bedding; cage changes occurred weekly. Behavioral assessments were conducted with respect to cage changes to avoid potential cofounding stress and/or anxiety. All experiments and procedures were conducted during the light cycle. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado Boulder. Voluntary Wheel Running 24 animals were allowed voluntary access to running wheels, whereas 24 were sedentary controls. Running data were recorded by computer 24 hours per day the first 6 week duration of running (Vital View, STARR Life Sciences; Oakmont, PA). Distance was calculated by multiplying revolutions per week by the circumference of the wheel. No significant baseline differences in running were observed between groups. Running occurred for six weeks prior to 12

14 surgery, ending on the day of surgery. This running model was chosen based on previous studies shown to decrease inflammation states 8, 32, 33. Chronic Constriction Injury Peripheral neuropathic pain was modeled through chronic constriction injury () of the left sciatic nerve at mid-thigh level. This produces a predictable intensity of neuropathic pain over a known time course, as has been shown previously 19, 20. The hind left thigh was shaved, 70% EtOH was applied to sterilize, and the rat was anesthetized throughout the surgery using 3% isoflurane. A scalpel blade was used to open the skin, where the muscle was bluntly separated parallel to the fibers. The sciatic nerve was isolated using glass hooks, whereupon one sterile chromic gut suture (cuticular 4-0 Chronic Gut, FS-2; Ethicon, Somerville, NJ) was loosely tied around the sciatic proximal to bifurcations approximately half way up the thigh. To close, the sciatic was gently set back in, the muscle was sutured back together with silk suture (PERMA- HAND, FS-2; Ethicon, Somerville, NJ), and the skin was stapled closed. Rats were then weighed and monitored while recovering until fully ambulatory before being transferred back to their home cage. Sham surgeries would follow the same protocol, including isolation of the sciatic, except no suture was tied around the nerve. Staples were removed at day 10 post-. All procedures were preformed aseptically. Drugs Morphine (5 mg/kg, subcutaneous) was administered twice a day beginning day 10 post- and lasted through day 14. To prevent overdose, injections were performed at least seven hours apart and occurred between 0800 and 1000 hr and 1600 and 1800 hr. Morphine groups 13

15 received (-)-morphine sulfate dissolved in isotonic endotoxin-free saline. Controls received vehicle saline injections at the same volume as morphine doses. Injection volume was determined based on weight of animals at day 9 post-. 1-mL syringes were prepared with 26-guage needles and after injection were disposed of according to Environmental Health and Safety documentation of the University of Colorado Boulder. Von Frey Assessment of Mechanical Allodynia Von Frey testing was conducted the day before voluntary wheel running began (baseline), the day before surgery (day-1), the day before morphine dosing (day 9), the day following morphine completion (day 15), and the two following weeks (days 22 and 29). The Von Frey procedure proceeded as previously described 34, 35, 36. Assessors were blinded to which groups had received surgery and morphine. Rats were placed underneath NALGENE containers on top of wire-bar shelving (1/2 inch spacing between bars) approximately 6 feet off of the floor. The plantar surface of both hind paws were stimulated with calibrated Semmes-Weinstein monofilaments (from Stoelting) of varying force according to a logarithmic scale. The filaments were applied perpendicular to the paw and held for eight seconds at constant bend and pressure. This determines the threshold stimulus intensity required to elicit a paw withdrawal response. The log 10 scale ( ) is based upon grams of pressure ranging from g. The log values are 10 times the gram values in milligrams (ex:.407 grams = log(4070)). When plotted, this scale provides a logarithmically graded slope when inputting stimulus threshold intensity values 35. A Gaussian integral psychometric function was used to achieve this through calculating the absolute threshold (50% probability of response) from stimulus intensity threshold values, which can be parametrically analyzed from 14

16 normalization 16, 37, 38. This style allows for appropriate analyses that cannot otherwise be achieved 36, 39. Object Placement Recognition Assessment of Learning and Memory As previously described, exercise, morphine dosing, and inflammatory pain are all thought to effect learning and memory through regulation of inflammatory states the mechanisms of which are currently not well understood 9, 10, 25, 28, 31. To further research on these topics through testing recognition memory in rats, the object place recognition (OPR) experiment was conducted in collaboration with the Stress Physiology Lab under Dr. Monika Fleshner one week after morphine dosing completed. Because a new person would handle rats for this paradigm, handling by the experimenter for one minute each once per day for the two days preceding object placement recognition took place. The protocol is three days long, with two time points each day spaced two hours apart. The first two days consist of one ten-minute open chamber round, followed by one five-minute open chamber round. Three different clear-glass containers with different color and material of filling are introduced on the third day; the first round introduces the objects and locations, and the second round tests memory of the locations through moving the location of one object. The object moved, and the new location was randomized. Four apparatuses were set up in the room, so that four rats could run per cycle. The room was dimly lit and quiet with red light and a small amount of white light, had constant white noise in the background, and had cameras mounted above the boxes. ANY-maze video tracking software (Stoelting co., Wood Dale, IL) was used to track all movements. Boxes were made from black non-reflective Plexiglas (60cm x 60cm x 50cm; L x W x H), with distinctive patterns 15

17 on each internal wall for context cues. Boxes and objects were cleaned with EtOH between each cycle to reduce odor cues between subjects. Fear Conditioning Assessment of Recognition Memory Complementing OPR, fear conditioning was conducted to glean a broader scope of how exercise, morphine and regulate memory consolidation. This paradigm was conducted week 2 post morphine, and after OPR to avoid cofounding stress from foot shock. Fear conditioning protocol was similar to previous paradigms 27, 40, 41. The protocol is three days long with testing being conducted on days one and three, 48 hours apart. Day one consists of a two-minute contextual memory formation trial and day three consists of a six-minute contextual memory recall trial. The first day, rats are transported in a black bucket two at a time to the trail room where they are each placed in a separate chamber. At the end of six minutes, a tone would sound for 15 seconds prior to administration of one 2-second long, 1.5 ma shock. Freezing was recorded through the trial, which is characterized by no movement besides breathing. Rats were then removed and the chambers were cleaned with water. The second day rats are transported two at a time and put into the same chambers for six minutes, this time absent of tone; freezing is once again recorded during this. Chambers consisted of a clear Plexiglas box (26cm x 21cm x 24cm; L x W x H) contained within an Igloo ice cooler with a white interior (54cm x 30cm x 27cm; L x W x H). A 24 V DC light bulb and speaker were mounted inside the cooler. The floors of the Plexiglas boxes were made from stainless steel rods (1.5 mm diameter; 1.2 apart from center), which were connected to a shock generator and scrambler (Colbourn Instruments, Allentown, PA). 16

18 Tissue Acquisition and Preparation Day 35 post rats were deeply anaesthetized with Fatal Plus (sodium pentobarbital) via intraperitoneal injection. Blood was then collected via cardiac puncture for later serum collection. Rats were then transcardially perfused with ml of ice cold 0.9% saline solution. Once perfused, ipsilateral lumbar (left) dorsal horn (LDH; L4-L5) segments, dorsal root ganglia (DRG) from L4-L5, sciatic nerve injury site sections, gastrocnemius, and spleen samples were collected and flash frozen in liquid nitrogen. Blood serum and tissue samples (besides brains) were then stored at -80C for later RNA extraction, cdna synthesis, and PCR. Brains were extracted and post-fixed in 4% paraformaldehyde/0.1m phosphate buffer (ph 7.4) for 24 hr. Brains were then cryoprotected in three rounds of sucrose gradients (15%, 20%, 30%) with 0.1% sodium azide, snap frozen on dry ice, and then stored at -80C until slicing. Real-Time Polymerase Chain Reaction RNA Isolation and cdna Synthesis Total RNA from LDH, DRG, and sciatic samples was collected through Invitrogen protocol. Frozen tissue was homogenized in TRIzol reagent (Invitrogen), then kept at room temperature until the start of extraction. 200 µl chloroform was added per 1000 µl TRIzol used to homogenize; samples were then vortexed and centrifuged. The aqueous layer was transferred to tubes containing 500 µl isopropanol, briefly vortexed, 8 µl glycogen (Thermo Fischer Scientific; Waltham, MA) was added (10 µl for DRGs) and samples were vortexed again. Samples were then centrifuged, the supernatant decanted, and pellets carefully dried. The pellet was re-suspended in 10 µl RNase free H 2 O. RNA concentrations and purity were assessed 17

19 using a 1 µl nanodrop spectrophotometer (Thermo Fischer Scientific; Waltham, MA). RNA samples were stored at -80C until use. iscript tm cdna synthesis kit (Bio-Rad Laboratories; Hercules, CA) was used for first strand cdna synthesis. Approximately 4000 ng of total RNA (variable volume) was added to 1 µl of iscript Reverse Transcriptase (Bio-Rad) and 4 µl of 5x iscript Reaction Mix (Bio-Rad); H 2 O was added to make a final volume of 20 µl. The mixture was incubated at 25C for 5 minutes, 46C for 20 minutes, 95C for 1 minute, and then held at 4C until retrieved. Samples were stored at -20C. RT-PCR cdna amplification was conducted blinded using QuantiTect SYBR Green PCR Kit (Qiagen; Germantown, MD) in barcoded 96 well PCR plates (Bio-Rad) on a CFX96 Touch Real Time PCR Detection System (Bio-Rad). Primers were obtained from GenBank (National Center for Biotechnology Information; and are displayed in table. The 21/22 µl reaction mixture was composed of 13 µl SYBR (SYBR Green fluorescent dye I, MgCl 2, dntp mix, and Hot Start Taq polymerase), 1 µl of forward and reverse primers (company), 10 µl nuclease free H 2 O, and either 1 µl cdna for LDH/DRG samples or 2 µl cdna for sciatic samples; each sample was measured in duplicate. (insert cfx96 protocol). The threshold cycle (CT) was determined for each reaction from setting threshold for detection of PCR product at the log-linear phase of amplification. The comparative CT method (ΔCT) was used to quantify the level of target mrna relative to the glycolytic housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH levels did not differ significantly between samples. 18

20 DAB Immunohistochemistry Brains were mounted in OCT and sliced coronally at a thickness of 30 µm in a -20 C cryostat. 3,3'-diaminobenzidine (DAB) immunohistochemistry was conducted on 30 µm slices of CA1 hippocampus and medial (MDm) thalamus (Bregma -3.7 mm), Cg1 prefrontal cortex (Bregma 3.7 mm), and S1HL hind limb sensory cortex and GI granular insular cortex (Bregma -.92 mm) (Bregma regions were obtained from The Rat Brain in Stereotaxic Coordinates; Paxinos and Watson). Sections were mounted on charged slides and stored at -20 C. 3,3 - diaminobenzidine (DAB) oxidation by hydrogen peroxidase was used to visualize the presence of GFAP and CD11b, as is previously described 16. To prepare for blocking on day 1, slides were washed 3x five minutes with fresh.1 M PBS in coplin jars, then washed for fifteen minutes in 100 ml PBS + 1 ml of 30% H 2 O ml of 10% sodium azide solution. Afterwards, slides were again washed 3x five minutes in.1 M PBS. Slides were then blocked for one hour with 1:9 ratio solution of 10% natural goat serum to.3% Triton-x in.01 M PBS. Primary antibody solutions were prepared with the following ratios to blocking buffer: 1:200 rabbit α-gfap (Agilent; Santa Clara, CA) and 1:500 mouse α-cd11b (company). Solutions were then added to slides and incubated overnight at 4 C. At the beginning of day 2, slides were washed 3x five minutes with.01 M PBS. Either 1:200 goat α- rabbit (Jackson ImmunoResearch; West Grove, PA) to bl ocking buffer or 1:200 goat α-mouse (Jackson ImmunoResearch) to blocking buffer was added to slides, which were then incubated covered for 2 hours at room temperature. Slides were again washed 3x five minutes in.01 M PBS. ABC (avidin-biotin-horseradish peroxidase complex) solution, made up of 100 ml PBS-T, 4 drops A, 4 drops B (Vector Laboratories; Burlingame, CA), was prepared during these washes (30 minutes prior to incubation). Slides were then incubated for 2 hours in ABC solution. DAB 19

21 solution (50 ml.1 M PB, 640 µl 1% nickelous ammonium sulfate, 400 µl 1% cobalt chloride, 16 mg ammonium chloride, and 1 DAB tablet from Sigma) was made while samples went through 3x 10 minute washes in.01 M PBS. DAB was then split into 50 ml aliquots, where 40 µl of glucose oxidase was then added; slides were incubated for 10 minutes in the inactivated DAB solution. 2 ml of β-d glucose (concentration of 10 mg/ml) was then added to each coplin and incubated for 8 minutes. Slides were washed 3x 10 minutes in PBS to stop the reaction and dried overnight. Day three consisted of ethanol gradient dehydration (5 min 50%, 5 min 75%, 10 min 95%, 2x 15 min 100%) ending with one 30 minute histoclear bath, and cover slipping with DPX. Slides were dried overnight, cleaned with EtOH, and kept at room temperature until image acquisition. Image Analysis An Olympus BX-61 bright field microscope (Olympus of the Americas; Waltham, MA) equipped with Suite Cell Sens Dimension software (Olympus) was used to visualize and photograph brain regions. At least 6 images were taken for each animal at the same exposure settings; all images were taken at 20x resolution and analyzed through densitometry using ImageJ (NIH). One complete region was analyzed once images were converted to 32-bit resolution and corrected for threshold. Uniform neuronal darkness gradient was used to determine threshold consistently through all samples while blinded to groups. Data are expressed as total area positive for darkness threshold within the selected image areas. 20

22 Statistics Analysis of variance (ANOVA) was used to analyze fear conditioning, OPR, PCR, and IHC data; whereas von frey data was analyzed using a Gaussian integral psychometric function before ANOVA. Once run through ANOVA, a Fisher s post hoc repeated measures analysis was used to look at von frey interactions. Main effects and interaction were deemed statistically significant for P <.05; data are expressed in terms of mean ± SEM. Prism 7 (GraphPad Software; La Jolla, CA) was used to graph all data, PsychoFit (L.O. Harvey; was used to preliminarily interpret von frey data, and StatView (Scientific Computing; Cary, NC) was used to run repeated measures three way ANOVA. 21

23 Results Voluntary wheel running broadly reduced neuropathic pain and specifically reduced pain in morphine groups As described, it is well established that prior to injury voluntary wheel running prevents full development of neuropathic pain 8 and that morphine paradoxically amplifies neuropathic pain (allodynia) 16. The first paradigm sought to define whether in male rats subjected to surgery, voluntary wheel running prior to injury would suppress morphine initiated enhanced neuropathic pain seen after cessation of dosing. Data are shown separated by sedentary control or voluntary exercise, and then combined (Figure 1A-C respectively). Baseline values were normal and not significantly different for sedentary and exercise groups (Figs. 1A, B). Voluntary exercise groups showed similar, but more consistent, trends to sedentary groups (Figs. 1A, 1B). Fisher s post hoc analysis of the repeated measures three-way ANOVA revealed our paradigm replicated previous results 8, 16. Reductions in pain were seen from prior exercise at day 9 post, and day 1 and week 2 post morphine (P=<0.0001, and respectively). Significant increases in pain were seen after cessation of morphine dosing at day 1 and week 2 post morphine (P=0.037 and 0.014, respectively). We also observed a significant effect of exercise on pain in the sham group at day 9 (P=0.03). At week 2 post morphine we observed an increase in pain between HCC+ and HCC+sham from injury (P<0.0001). We also observed an increase in pain between VWR+ and VWR+sham at day 1, week 1, and week 2 post morphine from injury (P<0.0001, P=0.0018, P< respectively). We observed an increase in pain between +saline and sham+saline at all time points after baseline from injury (P< for all), and a reduction in pain from 22

24 exercise at day 9 post sham (P<0.001). Lastly, there was an increase from injury between +morphine and sham+morphine at all time points after baseline (P= day 9, P< for the rest), and a decrease in pain from exercise at day 9 post sham and day 1 post morphine (P< and P=0.0027) (Fig 1C). Object placement recognition memory was impaired by morphine in exercise groups and improved by exercise in saline groups The second behavioral paradigm served to explore whether negative impacts on recognition memory by heightened inflammation and morphine could be attenuated by prior exercise. We observed a significant main effect of morphine (p=0.016) and significant 2-way interaction between exercise and morphine (P=0.037) when running a three way analysis of variance (Fig. 2A). There was no significant interaction between exercise and surgery or surgery and morphine; no significance was found in the main effects of exercise or surgery either. Within saline groups, we found a significant main difference between +VWR and +HCC groups (P=0.03) (Fig. 2B) from running a 2-way ANOVA. Interaction between exercise and surgery, as well as the other main effect of surgery, were not significant from this analysis. Contextual fear conditioning was improved by exercise in saline groups and impaired by exercise in sham morphine rats We further investigated the effects of exercise,, and morphine on memory for paradigm 3. Previously we ran OPR to test learning and memory, whereas this paradigm tested consolidation and retrieval of memories associating environment and tone with stressful shock. All groups of rats displayed significant association of tone with administered shock, as displayed 23

25 by increased levels of freezing (Fig 3A). Two-way ANOVA displayed exercise significantly increased levels of freezing, indicative of stronger memory consolidation. saline groups showed prior to injury exercise significantly aided in establishing association of environment with shock, revealed through running Tukey s post hoc analysis (Fig. 3B). Interactions were observed between exercise and surgery as well as exercise and morphine in a three-way ANOVA (P=0.02, P= respectively). Tukey s post hoc analysis revealed exercise decreased memory consolidation of sham morphine groups, and morphine decreased levels of memory consolidation in sham groups that were exercised prior to surgery (Fig. 3C). Percent area analysis of brain sections displayed no significant differences in groups stained for GFAP and CD11b Our results from fear conditioning indicated exercise might have an effect on levels of inflammation in associated brain regions (Fig. 4B). We ran densitometry analysis on photographed brain regions, quantifying percent darkness of staining to represent presence of CD111B or GFAP (Fig. 5). As literature shows exercise improves memory consolidation, which involves the hippocampus and insular regions as well as the hind limb sensory cortex for pain, we preformed IHC for GFAP and CD11B on these regions 24, 25, 26, 31. We report that IHC and area analyses revealed no significant differences in staining for GFAP or CD11b for GI granular insular cortex, CA1 hippocampus, or S1HL hind limb sensory cortex. 24

26 Exercise promoted anti-inflammatory state gene expression in the CNS, and proinflammatory state gene expression peripherally Results from Von Frey indicated IL-1βb and IL-10 levels might have been altered within associated tissues. Location of pain development and processing is well known for our injury paradigm, and exercise and morphine introduction are important factors in this. Ipsilateral LDH, DRG, and injury site sciatic are fundamental tissues involved in pain processing 8, 16. As such, we analyzed these tissues through PCR in order to obtain mrna expression of IL-1β and IL-10. Exercise was found to prevent pro-inflammatory gene expression of IL-1b in lumbar spine and associated dorsal root ganglia, protect from morphine induced increases in pro-inflammatory gene expression of IL-1b in lumbar spine, and increase levels of IL-10 mrna in the left dorsal horn. Three-way ANOVA displayed a significant main effect of exercise in L4/L5 dorsal root ganglia for expression of IL-1b mrna (P<0.0001) (Fig 6A). In lumbar dorsal horn L4/L5 sections, three-way ANOVA showed exercise significantly reduced IL-1b gene expression (P<0.0001); Tukey s post hoc analysis revealed significant differences between HCC++morphine and VWR++morphine groups, and HCC++morphine and VWR++saline groups (P= and P= respectively) (Fig. 6B). Three way ANOVA showed that in the left lumbar dorsal horn, IL-10 mrna levels were found to be significantly increased from exercise (P=0.006) (Fig. 7B). Peculiarly, injury site sciatic pro-inflammatory gene expression of IL-1b was observed to increase due to exercise, as well as increase +morphine IL-1b gene expression. Three way ANOVA revealed main effects of surgery and exercise (P<0.0001, P=0.01 respectively), interactions of exercise and surgery, and exercise and morphine (P=0.015, P=0.048 respectively), and an interaction of exercise, morphine, and surgery (P=0.041). Tukey s post hoc analysis 25

27 displayed IL-1b gene expression was significantly increased in VWR++morphine compared to VWR++saline, and significantly increased expression compared to HCC++morphine rats (P=0.0009, P=0.007 respectively) (Fig. 6C). From three way ANOVA, Il-10 mrna levels in injury site sciatic nerves displayed significant main effects of surgery and exercise (P< for both), and significant interaction between exercise and surgery (P=0.0002). Tukey s post hoc analysis revealed increased IL-10 gene expression in HCC++morphine rats compared to VWR++saline (P=0.0007), increased IL-10 mrna in HCC++saline compared to VWR++saline (P=0.0002), and increased IL-10 mrna in HCC++saline compared to VWR++morphine (P=0.01) (Fig. 7C). Also perplexing is how a three-way ANOVA revealed a significant increase in HCC+ IL-10 mrna levels compared to VWR+ in dorsal root ganglia (P=0.025), as well as an interaction effect between exercise and surgery (P=0.028) (Fig. 7A). 26

28 Discussion This project sought to investigate the interactions of exercise on persistent enhanced neuropathy and lowered memory performance presenting after cessation of morphine dosing. Previous research has observed four important groups of information: I) morphine use for pain management paradoxically causes enhanced nociceptive sensitization and duration of pain after complete cessation of dosing 16, II) exercise prior to peripheral nerve injury elicits a protective effect on developing mechanical allodynia, accompanied by normalization of pro-inflammatory cytokine levels and attenuated macrophage activation 8, III) memory impairment is attenuated by exercise in female rats receiving morphine, and increased central immune states of activation are seen in mice allowed exercise 9, 10, and IV) peripheral nerve injury (spared nerve injury model) affects synaptic plasticity in the hippocampus 28. With this information, we posed the question can prior to injury voluntary exercise protect from detriments to memory and enhanced neuropathic pain caused after cessation of a short morphine regimen? We found that prior exercise attenuated expected hyperalgesia initiated by ceased morphine dosing (Fig. 1C). This finding was only significant at week 2 post morphine; however, considering how we replicated results displaying enhanced neuropathy after cessation of morphine, and prior exercise lowered neuropathic pain development from, this connection is a profound finding. This implies immunophysiological preconditioning from exercise prevents morphine dosing from sensitizing central neuroimmune signaling pathways. We hypothesized this would be consistent for memory consolidation. Both object placement recognition and fear conditioning revealed exercise improved memory in +saline groups, as we predicted (Figs 2B, 3B). However, we observed that VWR+morphine had worse memory performance than HCC+morphine in both object placement 27

29 recognition and fear conditioning, contrary to our predictions. OPR results display morphine sensitization may have been present in exercise groups but not in sedentary groups, regardless of surgery (Fig. 2A). Fear conditioning results display exercise significantly impaired memory for sham+morphine groups, and morphine significantly impaired memory in VWR+sham groups (Fig 3C). These findings indicate that exercise may further detriment memory consolidation and recollection for groups that had received a short regimen of morphine weeks before, which suggests that there is a complex paradoxical mechanism underling the pairing of exercise and morphine in the context of peripheral nerve injury. To explore this further, we employed immunohistochemistry and staining analysis of brain regions; PCR was used to analyze tissues more pertinent to allodynia. No significant results were obtained from our IHC analysis, which indicates that GFAP and CD11b may not play a role in modifying memory consolidation at this time point (Fig. 4). However, interesting results were observed from our PCR analysis. We found exercise to prevent development of ipsilateral dorsal horn sensitized states associated with neruopathy in rats that received morphine weeks prior; IL-1β expression was significantly decreased in VWR++morphine compared to HCC++morphine (Fig. 6B), and IL-10 expression was seen up-regulated in VWR rats compared to HCC (Fig. 7B). Exercise was found to prevent proinflammatory gene expression of IL-1β in dorsal root ganglia (Fig. 6A), indicating there is less allodynic neuroimmune signaling in DRG s from exercise groups. Interestingly, there was a significant three way interaction showing increased gene expression of IL-1β in VWR++morphine groups compared to VWR++saline and HCC++morphine groups in sciatics (Fig. 6C). IL-10 was seen at higher expression in both DRG and sciatic tissue of HCC+ compared to VWR+ groups (Figs. 7A, 7C). This peculiarity could mean that 28

30 exercise prevents morphine immunosuppression of IL-1β expression at sciatic injury site but is unable to prevent suppression of IL-10 expression, or that an interaction between exercise and morphine increases inflammation at the injury site. However, our behavioral data is not indicative of this, indicating that this may be just an anomaly. We report two novel findings: I) that exercise may have beneficial protective effects on morphine initiated increases in neuropathy and associated CNS pro-inflammatory states seen after dosing cessation and II) that exercise benefits memory consolidation for +saline rats but not for +morphine rats, and may even detriment memory in rats that received morphine weeks earlier. Serious consideration is needed when approaching these findings, however. Though IL-1β and IL-10 mrna expression in LDH support the Von Frey results, we only observed a separation between HCC++morphine and VWR++morphine groups at weak 2. Also, there was no significant three-way interaction between exercise,, morphine in expression of either gene. IL-1β expression in DRG s shows that there is only an exercise effect, so this aids only slightly to our finding as there was no three way interaction either. Also, DRG IL-10 and sciatic data suggest there are inconsistencies when considering both central and peripheral systems. Consistency here would mean increased IL-10 expression in wheel running sciatics and DRG s, and opposite data for IL-1β in sciatics. These shortcomings mean the differences we observed may not be as strong as an interaction between all of our variables. In other words, our data is indicative but not conclusive of our hypothesis with regards to neuropathic pain. Similar considerations must be made when approaching our findings pertaining to memory. Our behavioral data are only indicative of there being an interaction between exercise, morphine, and as there is no three way interaction observed from OPR or fear conditioning. Our IHC revealed GFAP and CD11b amount don t differ between groups, 29

31 which may mean that either GFAP and CD11b aren t pertinent in this particular scenario or that our behavior may not be accurate. The final consideration with all of our data is human error and rat inconsistency. It is always possible that our IHC staining or densitometry wasn t an accurate representation of the results, was done improperly, behavior was off because of abnormal rat anxiety, or other similar errors. As such, future direction is necessary to consider. The necessity of continuing this research is well founded in clinical relevancy. Because our results are only indicative of interactions between exercise,, and morphine, the first step is to establish whether or not these statements are accurate. Repeating this project with new markers of inflammation would be important to consider. Examining markers such as CD11b, NFκb, TNF-α, or P2X7R for ipsilateral dorsal lumbar spinal cord, MCP-1 or atf-3 for DRG, and/or CD11b or IFNγ for the injury site sciatic would provide a better understanding of underlying mechanisms. In terms of re-evaluating a new approach to brain region analysis, it may be a consideration to micro-punch regions and run PCR or SDS-PAGE/western blot to obtain mrna expression or total protein values. In terms of changing behavior analysis, it may be a consideration to change from Von Frey to an escape-avoidance experiment paradigm, which may allow for better analysis of pain processing 42. For learning and memory, it may be beneficial to run a dedicated project specifically for OPR and/or fear conditioning, as Von Frey may potentially add extra stress for rats. These measures may ensure better results, or at least different results that may contribute to understanding the future path. If there is indeed an interaction between exercise, morphine and, it is essential to extend the research to other branches. Exploring other clinically relevant opioid pain moderators such as fentanyl or oxycodone, different levels of voluntary exercise at different time points, and different injury paradigms are paramount to understanding this field better. This novel project 30

32 was potentially the first step in recognizing the important neuroimmune connections between voluntary exercise, morphine dosing sensitization after cessation, and peripheral nerve injury. The applications of these processes are far reaching and may lead to new pain management regimens, treatment for chronic neuropathy, or prevention of full pain development. 31

33 Figures A. B. Absolute threshold (g) C. BL 10 8 Von Frey Surgery + Drug for Home Cage n=6 per group D9 post day 1 week 1 time post Morphine week 2 Sham+saline Sham+morphine +saline +Morphine Absolute threshold (g) Von Frey Surgery + Drug + Exercise n=6 per group BL Von Frey Sham+saline Surgery + Drug for 6wk VWR n=6 per group Sham+morphine D9 post day 1 week 1 time post Morphine week 2 +saline +Morphine Sham+saline HCC Absolute threshold (g) *** * *** *** *** *** *** *** ** *** * ** *** *** *** *** *** *** * * Sham+morphine HCC +saline HCC +Morphine HCC Sham+saline VWR Sham+morphine VWR +saline VWR +Morphine VWR BL D9 post day 1 week 1 Time Post Morphine Figure 1. Wheel running attenuates allodynia caused by -injury as well as morphine treatment. Analysis of mechanical allodynia was conducted on male Sprague-Dawley rats with 1x 4-0 suture, morphine dosing, and or exercise. Von frey conducted post morphine dosing shows separation in levels of allodynia between sedentary sham and groups as well as sham saline and sham morphine groups (A). Both groups of sham runners show similar levels of allodynia, with sham morphine trending slightly lower; groups were consistently lower but began to recover to saline levels soon after dosing (B). Fisher s post hoc analysis of the repeated measures three-way ANOVA reveals various between group significances (C). week 2 32

34 Discrimination Index A Discrimination Index OPR Surgery + Drug + Exercise 5 minutes n=6 per group * * saline morphine 0.0 VWR sham VWR HCC sham HCC B. Discrimination Index Discrimination Index OPR + Exercise for Saline 5 minutes n=6 * p=.03 * Sham 0.0 HCC VWR Figure 2. Recognition memory performance is affected by voluntary exercise. OPR was performed at 1 week post morphine/saline on sedentary or active male Sprague-Dawley rats with 1x 4-0 suture or sham. The main effect of morphine dosing was found to be significant (P=.016); a significant interaction of exercise and morphine was also found (P=.037) (A). Significance was seen between VWR++saline and HCC++saline groups (P=.03) (B). 33

35 % Freezing A. B. Sed Cued Fear (Tone) Freezing + Morphine + Exercise n=6 per group Run Pre-Tone Sed Tone Run Sham/Saline Sedentary Sham/Morphine Sedentary /Saline Sedentary /Morphine Sedentary Sham/Saline Running Sham/Morphine Running /Saline Running /Morphine Running %Freezing Contextual Fear Freezing + Exercise for Saline n=6 per group HCC *P=.03 VWR sham C. % Freezing Contextual Fear Freezing + Morphine + Exercise n=6 *p=.04 *p=.02 Sham Saline Sham morphine saline 0 HCC VWR Morphine Figure 3. Contextual memory levels benefitted from exercise and suffered from morphine dosing. Fear conditioning paradigm was run week 2 post morphine/saline on sedentary or active male Sprague-Dawley rats with 1x 4-0 suture or sham. Cued tone freezing levels were all significantly different (P<.0001) (A). We found a significant main effect of exercise and significant difference between VWR++saline and HCC++saline groups (P=.03, P=.017 respectively) (B). Significant interactions were seen between exercise and morphine, and exercise and surgery (P=.0007, P=.02 respectively). Differences were also seen VWR+sham+morphine HCC+sham+morphine (P=.04), and VWR+sham+morphine and VWR+sham+saline groups (P=.02) (C). 34

36 % Area positive A. B Exercise for Saline CD11b insular cortex n=6 Sedentary VWR % Area positive Exercise for Saline CD11b Hippocampus n=6 Sedentary VWR % Area positive % Area positive 0.0 Sedentary VWR C. D Sedentary VWR E. F Exercise for Saline CD11b Hind Limb Sensory Cortex n=6 Exercise for Saline GFAP Hippocampus n=6 Sedentary VWR Sedentary VWR % Area positive 0 % Area positive Sedentary Sedentary VWR Exercise for Saline GFAP insular cortex n=6 VWR Exercise for Saline GFAP Hind Limb Sensory cortex n=6 Sedentary VWR Sedentary VWR 0 Sedentary VWR 0.0 Sedentary VWR Figure 4. No significant differences were found between glial activation in brain regions. Brains were pulled at week 2-post morphine. All ANOVA densitometry analyses ran on IHC photographs were non significant, indicating that the null hypothesis was true. 35

37 Insular Cortex CD11b Stain Figure 5. Photo of CD11b stain on an insular cortex section. Red arrows indicate examples of cell bodies, where the stain is darkest. 36