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Sex Differences In Cannabinoid And Opioid Mediated Analgesia


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Abstract: Orofacial musculoskeletal pain conditions, such as temporomandibular disorder (TMD), are debilitating and often difficult to treat. As with many chronic pain conditions, TMD occurs more frequently in women. Thus, understanding mechanisms underlying sex differences in pain and analgesia is essential for effective pain management in both sexes. This study introduces the potential therapeutic advantage of targeting cannabinoid 1 receptor (CB1) localized in primary afferent neurons under myositis conditions. Although sex differences in CB1 responses are recognized in the CNS, it is not known whether such sex differences exist in the periphery. Therefore, I investigated whether peripheral cannabinoid treatment leads to sex differences in anti-hyperalgesic effects, and whether the effects are mediated by sex differences in CB1 level in trigeminal ganglia (TG) under a rodent orofacial myositis model. Peripherally administered ACPA, a specific CB1 agonist, significantly attenuated inflammation-induced mechanical hypersensitivity in the masseter of male rats. In female rats, a 30-fold higher dose of ACPA was required to produce a reduction in mechanical hypersensitivity. CFA injected in masseter muscle significantly up-regulated CB1 mRNA expression in TG from males, but not from females, and CB1 mRNA levels in TG were positively correlated with the anti-hyperalgesic effect of ACPA. IL-1β and IL-6, elevated in the muscle tissue following inflammation, induced a significant up-regulation of CB1 mRNA expression in TG cultures from male rats. The up-regulation of CB1 was prevented in TG cultures from orchidectomized males, and was restored by testosterone treatment. The cytokines did not alter the CB1 mRNA level in TG from intact or ovariectomized female rats. Neither estradiol nor estrogen receptor blockade had any effect on CB1 expression. Similar results were obtained regarding cytokine-induced regulation of μ-opioid receptor (MOR) in TG, another important peripheral target for pain management. These data indicate that testosterone, but not estradiol, is required for the regulation of CB1 and MOR in TG under inflammatory conditions, which explains the sex differences in the anti-hyperalgesic effects of peripherally administered agonists. These data offer important new insights for the development of mechanism-based sex-specific pharmacological treatment alternatives that can be directed at the peripheral anti-nociceptive systems to ameliorate persistent pain.

 

 

Table of Contents

1. General Introduction ........................................................................................................1

1.1 Orofacial muscle pain and temporomandibular disorders (TMD) .............................1

1.2 Trigeminal nociceptive and anti-nociceptive processing ...........................................2

1.3 Cannabinoid and pain ................................................................................................6

1.4 Peripheral cannabinoid receptor-mediated analgesia .................................................8

1.5 Modulation of CBR and OR expression ....................................................................9

1.6 Sex differences in CB1- and MOR-mediated analgesia ..........................................11

2. Materials and Methods ...................................................................................................15

2.1 Animals ....................................................................................................................15

2.2 Experimental myositis (Masseter inflammation) .....................................................15

2.3 Assessment of local cytokine levels in masseter muscle .........................................16

2.3a Experimental groups .........................................................................................16

2.3b Tissue preparation .............................................................................................16

2.3c Luminex multi-analyte profiling (xMAP) technology ......................................17

2.3d Data analysis .....................................................................................................17

2.4 Behavioral studies ....................................................................................................17

2.4a Behavioral paradigm .........................................................................................17

2.4b Experimental and control groups – CB1 study .................................................18

2.4c Experimental and control groups – MOR study ...............................................19

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2.4d Experimental and control groups – cytokine antagonist study .........................20

2.4e Drug preparation and administration ................................................................20

2.4f Testosterone replacement for MOR experiments ..............................................20

2.4g Data analysis .....................................................................................................21

2.5 TG cultures ...............................................................................................................21

2.5a TG primary cultures for CB1 studies ................................................................21

2.5b TG tissue cultures for MOR studies ..................................................................22

2.6 Real-time RT-PCR ...................................................................................................23

2.6a Real-time RT-PCR for CB1 ..............................................................................23

2.6b Real-time RT-PCR for MOR ............................................................................24

3. Intramuscular cytokine levels under myositis condition in male and female rats .........26

3.1 Abstract ....................................................................................................................26

3.2 Introduction ..............................................................................................................27

3.3 Results ......................................................................................................................29

3.4 Discussion……........................................................................................................33

4. Sex differences in cannabinoid-mediated analgesia and modulation of CB1 receptor

expression ......................................................................................................................38

4.1 Abstract ....................................................................................................................38

4.2 Introduction ..............................................................................................................39

4.3 Results ......................................................................................................................41

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4.3a Sex differences in the effect of peripheral CB1 activation on masseter

hypersensitivity .................................................................................................41

4.3b Sex differences in inflammation-induced CB1 expression in TG ....................43

4.3c Inflammatory cytokines induce CB1 up-regulation in TG cultures in a sex-

dependent manner .............................................................................................45

4.3d Il-1β and IL-6 mediate CFA-induced CB1 up-regulation and ACPA

effects in vivo ....................................................................................................47

4.3e Testosterone, but not estrogen, modulates cytokine-induced CB1

up-regulation .....................................................................................................49

4.4 Discussion ................................................................................................................52

5. Sex differences in μ-opioid-mediated analgesia and modulation of MOR receptor

expression ......................................................................................................................57

5.1 Abstract ....................................................................................................................57

5.2 Introduction ..............................................................................................................58

5.3 Results ......................................................................................................................60

5.3a Sex differences in inflammation-induced MOR expression in TG ...................60

5.3b Effects of inflammatory cytokines on MOR expression ...................................61

5.3c Testosterone is required for cytokine-induced MOR mRNA up-regulation .....63

5.3d Sex differences in the effect of peripheral MOR activation on masseter

hypersensitivity .................................................................................................64

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5.4 Discussion ...............................................................................................................66

6. General discussion .........................................................................................................69

6.1 Summary of main findings......................................................................................69

6.2 Peripheral CB1 and MOR in chronic pain conditions ............................................70

6.3 Cytokines and their roles in regulation of CB1 and MOR expression in TG .........73

6.4 The role of testosterone in cytokine-induced regulation of CB1 and MOR in TG .76

6.5 Conclusion/Future directions ..................................................................................78

References ..........................................................................................................................80

 

 

http://archive.hshsl.umaryland.edu/handle/10713/2767

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