A COMPARATIVE STUDY ON THE INDUCTION OF MEDICATION OVERUSE HEADACHE IN MALE AND FEMALE RATS AND ITS RESULTANT TEMPORAL EFFECT ON PAG PROTEOME REGULATION

Abstract

 

Headache disorders can be categorized based on their underlying causes, frequency of occurrence, implicated mechanisms, and symptom presentation. Etiologically, headaches are commonly classified into three major types: vascular, tension-type, and those associated with traction and inflammation. Additionally, headaches are broadly divided into primary and secondary types. Secondary headaches result from systemic or localized illnesses [2].

Medication Overuse Headache (MOH) falls under the category of secondary headaches and is triggered by the prolonged use of acute headache medications—either according to prescription or due to misuse. The most frequently overused medications associated with MOH include triptans, opioids, and barbiturates [5].

In a study using male and female rat models of MOH, researchers assessed the severity, onset, and frequency of the condition. Throughout the post-infusion observation period, a higher number of male rats exhibited allodynia compared to females. Among the female groups, fewer animals displayed periorbital mechanical allodynia by day 10 compared to day 4 and day 7, a pattern consistently observed across different treatments. In both sexes, sumatriptan was associated with a higher incidence of periorbital allodynia than morphine, suggesting differing propensities for inducing MOH between the two drugs. However, further research is needed to confirm this due to the small sample size (n) in the morphine-treated group.

To explore molecular changes, Western blot analyses were conducted to measure CB1 receptor (CB1R) and diacylglycerol lipase alpha (DAGLα) expression. No significant differences in DAGLα levels were observed between naïve and saline-treated rats, nor between sexes. Similarly, CB1R levels did not differ significantly between naïve and saline groups overall or when analyzed by sex. Additionally, no significant differences in DAGLα or CB1R expression were found between treatment groups on day 4 or day 10.

Subsequent total and phospho-proteomic analyses were performed to investigate broader molecular changes in the endocannabinoid system (ECS) and overall function of the periaqueductal gray (PAG). In total proteomic comparisons between naïve and saline groups, DAVID analysis identified genomic pathways linked to addiction and dopaminergic systems. For day 7, pathways associated with addiction, dopamine signaling, and glutamate were observed. In the morphine group, glutamatergic and addiction-related pathways were prominent, while sumatriptan treatment revealed involvement of glutamate, endocannabinoid, and estrogen signaling pathways. Day 4 comparisons highlighted estrogen signaling, whereas day 10 results showed enrichment in pathways related to neurodegenerative diseases.

In phospho-proteomic analyses, comparisons between naïve and saline groups also revealed pathways involved in neurodegenerative conditions. Across saline-treated animals, both neurodegenerative and estrogen-related pathways were noted. Day 7 and day 4 phospho-proteomic profiles showed enrichment in neurodegeneration and estrogen signaling, with day 4 additionally linked to addiction. Day 10 phospho-proteomic analysis revealed connections to neurodegenerative and dopaminergic systems. Morphine-treated groups showed overlap with addiction and neurodegenerative pathways, while sumatriptan treatment was primarily associated with neurodegeneration.

Table of Contents

List of Figures

Figure 1: Female vs Male Rats Percentage with Periorbital Allodynia......................................................................................... 42

Figure 2: Female vs Male Rats Magnitude and Intensity of Periorbital Allodynia.......................................................................................... 43

Figure 3: Female Rats Light-Dark Box Test Results.............................................................................................. 44

Figure 4: Male Rats Light-Dark Box Test Results............................................................................................... 45

Figure 5: Male vs Female Light-Dark Box Test Results.............................................................................................. 46

Figure 6: DAGLα Naive vs Saline Western Blot Results.............................................................................................. 49

Figure 7 CB1R Naive vs Saline Western Blot Results.............................................................................................. 50

Figure 8: CB1R Across Day 4 Western Blot Results.............................................................................................. 51

Figure 9: DAGLα Across Day 4 Western Blot Results.............................................................................................. 52

Figure 10: DAGLα Across Day 10 Western Blot Results.............................................................................................. 53

Figure 11 Naive vs Saline Total Proteomic GO-Kegg Pathway............................................................................................ 58

Figure 12 Naive vs Saline Total Proteomic GO- Biological Processes.......................................................................................... 59

Figure 13 Naive vs Saline Total Proteomic GO- Cellular Component....................................................................................... 60

Figure 14 Naive vs Saline Total Proteomic GO- Molecular Function............................................................................................ 61

Figure 15 across Saline Total Proteomic GO-Kegg Pathway............................................................................................ 62

Figure 16 Across Saline Total Proteomic GO- Biological Processes.......................................................................................... 63

Figure 17 Across Saline Total Proteomic GO- Cellular Component....................................................................................... 64

Figure 18 Across Saline Total Proteomic GO- Molecular Function............................................................................................ 65

Figure 19 across Sumatriptan Total Proteomic GO-Kegg Pathway............................................................................................ 66

Figure 20 Across Sumatriptan Total Proteomic GO- Biological Processes........................................................................................... 67

Figure 21 Across Sumatriptan Total Proteomic GO- Cellular Component....................................................................................... 68

Figure 22 Across Sumatriptan Total Proteomic GO- Molecular Function........................................................................................... 69

Figure 23 Across Morphine Total Proteomic GO-Kegg Pathway........... 70

Figure 24 Across Morphine Total Proteomic GO- Biological Processes. 71

Figure 25 Across Morphine Total Proteomic GO- Cellular Component. 72

Figure 26 Across Morphine Total Proteomic GO- Molecular Function.. 73

Figure 27 Across Day 4 Total Proteomic GO-Kegg Pathway.................. 74

Figure 28 Across Day 4 Total Proteomic GO- Biological Processes....... 75

Figure 29 Across Day 4 Total Proteomic GO- Cellular Component........ 76

Figure 30 Across Day 4 Total Proteomic GO- Molecular Function........ 77

Figure 31 Across Day 7 Total Proteomic GO-Kegg Pathway.................. 78

Figure 32 Across Day 7 Total Proteomic GO- Biological Processes....... 79

Figure 33 Across Day 7 Total Proteomic GO- Cellular Component........ 80

Figure 34 Across Day 7 Total Proteomic GO- Molecular Function........ 81

Figure 35 Across Day 10 Total Proteomic GO-Kegg Pathway................ 82

Figure 36 Across Day 10 Total Proteomic GO- Biological Processes..... 83

Figure 37 Across Day 10 Total Proteomic GO- Cellular Component...... 84

Figure 38 Across Day 10 Total Proteomic GO- Molecular Function...... 85

Figure 39 Naive vs Saline Phospho-Proteomic GO-Kegg Pathway........ 95

Figure 40 Naive vs Saline Phospho-Proteomic GO- Biological Processes.................................................................................................. 96

Figure 41 Naive vs Saline Phospho-Proteomic GO- Cellular Component............................................................................................... 97

Figure 42 Naive vs Saline Phospho-Proteomic GO- Molecular Function................................................................................................... 98

Figure 43 across Saline Phospho-Proteomic GO-Kegg Pathway............ 99

Figure 44 Across Saline Phospho-Proteomic GO- Biological Processes................................................................................................ 100

Figure 45 Across Saline Phospho-Proteomic GO- Cellular Component 101

Figure 46 Across Saline Phospho-Proteomic GO- Molecular Function 102

Figure 47 across Sumatriptan Phospho-Proteomic GO-Kegg Pathway. 103

Figure 48 Across Sumatriptan Phospho-Proteomic GO- Biological Processes................................................................................................ 104

Figure 49 Across Sumatriptan Phospho-Proteomic GO- Cellular Component............................................................................................. 105

Figure 50 Across Sumatriptan Phospho-Proteomic GO- Molecular Function................................................................................................. 106

Figure 51 across Morphine Phospho-Proteomic GO-Kegg Pathway..... 107

Figure 52 Across Morphine Phospho-Proteomic GO- Biological Processes................................................................................................ 108

Figure 53 Across Morphine Phospho-Proteomic GO- Cellular Component............................................................................................. 109

Figure 54 Across Morphine Phospho-Proteomic GO- Molecular Function................................................................................................. 110

Figure 55 Across Day 4 Phospho-Proteomic GO-Kegg Pathway.......... 111

Figure 56 Across Day 4Phospho-Proteomic GO- Biological Processes 112

Figure 57 Across Day 4 Phospho-Proteomic GO- Cellular Component 113

Figure 58 Across Day 4 Phospho-Proteomic GO- Molecular Function. 114

Figure 59 Across Day 7 Phospho-Proteomic GO-Kegg Pathway.......... 115

Figure 60 Across Day 7 Phospho-Proteomic GO- Biological Processes................................................................................................ 116

Figure 61 Across Day 7 Phospho-Proteomic GO- Cellular Component 117

Figure 62 Across Day 7 Phospho-Proteomic GO- Molecular Function. 118

Figure 63 Across Day 10 Phospho-Proteomic GO-Kegg Pathway........ 119

Figure 64 Across Day 10 Phospho-Proteomic GO- Biological Processes................................................................................................ 120

Figure 65 Across Day 10 Phospho-Proteomic GO- Cellular Component............................................................................................. 121

Figure 66 Across Day 10 Phospho-Proteomic GO- Molecular Function................................................................................................. 122

List of Diagrams

Diagram 1: Trigeminal Nociceptive System and How it Communicates to the PAG................................................................................................... 22

Diagram 2: CGRP, -Gepants, CGRP Antibodies, Triptans Act With Each Other................................................................................................. 23

Diagram 3: The Synthesis and the Degradation of 2-AG and AEA.................................................................................................. 27

CHAPTER 1: BACKGROUND/INTRODUCTION

BACKGROUND

1.1  Headache

 There are three classifications of headache: vascular, tension-type, and traction and inflammatory headache. There are also primary and secondary headaches. Secondary headaches are caused by local or systemic illness [2]. A headache is considered chronic when it appears for at least 15 days a month and is estimated that 4% of people worldwide have chronic headaches. A migraine can occur with or without auras. Auras are visual disturbances that usually appear before the other headache symptoms. It is estimated that only 20% of migraine patients have auras. Diagnostic criteria for migraines the headache must last 4-72 hours. It must also have at least two of the following characteristics: unilateral location, pulsating quality, moderate to severe pain, aggravation or causing avoidance of routine physical activities. It must also at least include one of the following symptoms: nausea and/or vomiting, or photophobia and phonophobia.[3]

The trigeminovascular system (TGVS) is greatly recognized as a key part of migraine pathophysiology (Diagram 1). During a migraine attack there is sensitization of higher order neurons in the central nervous system causing nociceptive signaling. This is done by prolonged activation of the TGVS causing compromised meningeal trigeminal nerves and the vessels along the dural mast cells. There is also a positive feedback loop stimulating the TGVS. Within the TGVS the main migraine medicator is neuropeptide calcitonin gene-related peptide (GCRP). This promotes vasodilation and contributes to the sensitization of the nociceptive pathways. In total the three key meningeal structures are the nerves, vessels and dural mast cells.[7]

Stimulation of the TGVS causes a release of CGRP and substance P; during migraines only CGRP release is significantly elevated. The levels of CGRP are brought back to normal level after effective triptan intervention. During chronic migraine, levels of CGRP remain at a high level.

These data showing the importance of CGRP in migraines as well as the CGRP receptor led to CGRP antagonist drugs being discovered [22]; small molecule drugs are termed “-gepants”. It is thought that -gepants do not cross the blood brain barrier. In general, triptans are more effective but -gepants have less adverse effects and contraindications. Monoclonal antibodies targeting CGRP or its receptors were also developed. An advantage of these antibodies is that they have a long plasma half-life and are highly selective, which reduces the adverse effects and potential interactions with other medication.[23] (diagram 2)

Triptans selectively target 5-HT1 receptors. Binding to this site causes vasoconstriction. Since triptans cannot cross the blood brain barrier they have a peripheral mechanism of action. This means it affects cranial blood vessels, coronary arteries and the trigeminal ganglion. Triptans were found to decrease the levels of CGRP in the plasma, which suggest another mechanism other than vasoconstriction to reduce migraines.[23]

1.2  Medication overuse headache (MOH)

Medication overuse headache (MOH) is a secondary headache that is caused by chronic use of acute headache medication, including as prescribed/recommended and misused. The patient must also have an underlying headache disorder and usually have at least 15 days of headache per month. The most frequent drugs implicated in MOH are triptans, opioids and barbiturates.[5] MOH may be difficult to diagnose since the increase of headaches can be caused by overuse of medication or that primary headache condition is increasing in frequency. It is not required for diagnosis but one way to determine the difference is to discontinue medication use and record if headache frequency decreases.[5] In 2015, the Global Burden of Disease estimated that 59 million people globally have MOH. [5]

1.2.1  Risk factors

MOH is more prevalent in women than men which is expected since this trend is also seen in migraines.[5] While there are large systemic genome-wide association studies on people with migraine there are only small association studies done for those with MOH. The target genes that have been identified are modifications to ACE (encoding angiotensin-converting enzyme), mutation in BDNF (encoding brain-derived neurotrophic factor), and polymorphisms in COMT (encoding catechol-O-methyltransferase) and SLC6A4 (encoding serotonin transport). These factors are associated with abnormalities in the serotonergic and dopaminergic transmission, drug dependence and metabolic pathways. A behavioral and psychological risk factor found in some patients is substance use disorder. Maladaptive cognitive patterns such as obsessional drug taking behaviors is and additional risk factor for MOH.[5]

1.2.2  Comorbidities

There are psychiatric, medical and nonpsychiatric comorbidities associated with MOH. For psychiatric comorbidities these include depression and anxiety. Depression and anxiety are strong independent risk factors of headache chronification, predictors of poorer therapeutic outcomes and are associated with the negative effects and disabilities related to MOH. In adolescents, suicidal behavior is not correlated with MOH. However, in adults there was an association between MOH and risk of suicidal ideation and prior suicide attempt. Patients with MOH are more likely to have chronic musculoskeletal complaints, smoking, hypertension, insomnia, hypothyroidism, gastrointestinal problems and metabolic syndromes compared to the public. Chronic use of NSAIDS can be a reason for the increase in gastrointestinal problems due to adverse effects such as gastric ulcers. It is hypothesized that untreated psychological factors can aggravate or lead to the chronification of headaches.[5] When it comes to insomnia and migraine it has been found that insomnia increases pain intensity and chronification. It was also found that in those with chronic migraine, reducing insomnia reduces the frequency of a migraine. [25] It is reasonable to conclude that insomnia increases the risk of MOH as it increases the frequency of migraine resulting in more frequent use of analgesics.

1.2.3  Treatment

When it comes to prevention of MOH the most important aspect is educating medical providers and patients with primary headaches. Without proper education of the patient, they are not aware that chronic use of the medication can make their condition worse. Educating medical providers and pharmacists can allow for the prevention of providing enough medication per month to overuse. If the patient has a limited supply, then they cannot overuse triptans and opioids if they have chronic headaches. If a patient has MOH then the course of action is to discontinue or withdrawal the overused medication or replace treatment with a preventative medication. In a systemic review it was found that discontinuation and an added preventative medication was preferred over discontinuation alone. Another study found that complete discontinuation of medication was more effective than restricting use of the medication. When it comes to opioid overuse it is better to gradually tapered to avoid withdrawal symptoms. [5]

1.2.4  Mechanism

Since MOH is not reported in those without an underlying headache disorder the mechanism of MOH and results from anti-headache medication, exacerbation of headache mechanisms is likely. [5] Putative mechanisms implicated in MOH are discussed below.

1.2.4.1  CGRP

It has been found that if rats receive chronic exposure to triptans then there will be an increased number of trigeminal ganglion cell bodies expressing CGRP and an increase in expression of substance P. CGRP was also increased in unmyelinated C-fibers and myelinated afferents. It has also been seen that chronic used of morphine increases CGRP content in dorsal root ganglion neurons. [15] Since chronic use of common overused medication increases CGRP and within migraine there is an increase in CGRP it is a possibility that they share this pathology.

1.2.4.2  Serotonin System

Chronic use of analgesics has been found to alter the serotonin system. Chronic use of acetaminophen increased platelet serotonin concentrations. It also caused a downregulation of 5- HT2A receptors and upregulation of 5-HT transporter in the frontal cortex. With chronic acetaminophen use there is an increase cortical spreading depression susceptibility, but it was blocked with 5-HT2A receptor antagonist. It has also been found that there is an alteration of the 5-HT receptor and transporter expression in regions such as the PAG and the locus coeruleus after chronic triptan use. Decrease in the function of the 5-HT system can also affect the trigeminal nociception.[17] In animal models of MOH it was found that the expression of 5-HT 1b/1d receptors decreased. Decreased 5-HT levels and an upregulation of 5-HT2a receptors were also found in MOH models.[23]

1.2.4.3  Glutamate

Glutamate is the primary excitatory neurotransmitter in the central nervous system. It is also involved in migraine pathology in the cortical spreading depression, trigeminovascular activation and potentially linked to chronification of migraines. When chronic migraine patients who do not overuse triptans had high levels of glutamate in their CSF compared to control patients. However, when chronic migraine patients overuse triptans they showed lower levels of CSF glutamate compared to chronic migraine patients who do not overuse triptans. Although CSF glutamate levels in MOH patients were still higher than in control patients. There was no

significant difference in glutamate CSF levels in overuse of other analgesics. [19] This suggests that chronic use of analgesics has an effect on glutamate and potential pathology for MOH.

1.2.4.4  Trigeminal Nociceptive System

Peripheral sensitization is when peripheral nociceptors increase their response to a suprathreshold stimuli and will be responsive to it. Sensitization of nociceptors has several mediators released during tissue injury and inflammation, including CGRP, by neuronal protein phosphorylation. The phosphorylation of sensory neurons creates resistant sodium and calcium channels which will lower the threshold of nociceptor membrane. Ultimately this will lead to more nociceptors ready to fire and some inactive nociceptors to be turned on. This will allow low intensity stimuli to create a painful sensation. Chronic use of abortive medication will effect the trigeminal afferents. As mentioned before, chronic use of abortive medication increases levels of CGRP which can lead to a state of latent sensitization. It is also known that chronic use of triptans causes an increase in expression of neuronal nitric oxide synthase in trigeminal ganglionic cells innervating dura. The increase of nitric oxide can increase the sensitivity to stress and the susceptibility of a headache. Chronic use of morphine showed changes in the plasticity of central trigeminal neurons, which lead to the sensitization of central trigeminal neurons.[17]

1.2.4.5  Gray Matter Changes

Gray matter density was measured in MOH patients using fMRI. This study showed that there were gray matter changes in regions related to the pain network, reward system and the prefrontal area. There was increased gray matter in the bilateral striatum and ventral tegmental area (VTA)in those with MOH. Increased gray matter in the striatum can also be found in patients with cocaine abuse. The dysfunction of the reward system is considered a crucial pathology of drug abuse and addiction. This supports the idea that the reward system and drug dependance has a role within MOH; this is seen clinically as MOH patients show drug overuse behavior that is like substance abuse to other medication. fMRI has also shown dopamine system dysfunction on MOH patients and return to normal 6 months after removal of drugs.[14]

Gray matter atrophy was found in the prefrontal cortex, including orbitofrontal cortex (OFC), in MOH patients which may indicate neural degeneration or dysfunction. Abnormality of OFC can affect someone’s expectation and desires. It also weakens decision making capabilities which can play a role in MOH patient’s expectation from analgesic medication and the decision to chronically use medication. OFC is also associated with depression which is a comorbidity of MOH.[14]

Gray matter increased in regions related to the perception of pain, including the periaqueductal gray (PAG) in MOH patients. The PAG plays a role in the downward modulation of pain sensing and connects with the VTA. Increased gray matter in the pain pathway is also seen in migraine patients. The cerebellum is involved in pain perception and cognition. The cerebellum also had increased gray matter in MOH patients and reversed back to normal after drug withdrawal.[14] Additional work investigating global CNS dysfunction is warranted.

Diagram 1: This is a diagram of the Trigeminal Nociceptive System and how it communicates to the PAG. Starting at the meningeal dural blood vessels, neurons send signal to the trigeminal ganglion which then communicates to the SpV. The SPV then signals to the PAG.[10]

Diagram 2: This shows how CGRP, -Gepants, CGRP antibodies, triptans act with each other to treat headaches. Top) CGRP is released from the pre-synapse and binds to receptors on the post synapse which promotes post-synaptic activation. Bottom) Triptans bind to receptors on the pre- synapse preventing CGRP from being released. Triptans also bind to post-synaptic receptors which counteract the CGRP vasodilation. CGRP binds to CGRP while it is still in the synaptic cleft. -Gepants are taking the same post-synaptic receptors as CGRP, preventing it from binding. [22]

1.3  Endocannabinoid System

The endocannabinoid system is primarily responsible for homeostasis of the body and energy input and output. The ECS is also implicated as having a role in appetite, depression, anxiety, nervous function, emotional behavior, neurogenesis, cognition, memory, learning, neuroprotection, fertility, and pre-and post-natal development. Due to its involvement in many systems, it has become a potential target for various conditions.[6] More recently, the ECS has been implicated in headache, including MOH. In a previous study in MOH rat model, 2-AG and AEA levels were measured. They found that after chronic use of sumatriptan and morphine there was an increase of AEA in the cortex. After chronic use of sumatriptan and morphine caused a decrease of 2-AG levels within the PAG. This showed that chronic use of sumatriptan and morphine had an impact on the ECB. To further determine if the changes were from ECB metabolism DAGLα was pharmacologically inhibited (LEI106). When LEI106 was used it induced facial allodynia. This suggests that depletion of DAGLa reduces levels of 2-AG which induces facial allodynia; therefore loss of 2AG tone can be a potential pathology for MOH induction. [24]

The ECS is composed of receptors, ligands and enzymes. The three main receptor classes are G-Coupled protein receptors, ligand sensitive ion channels, and nuclear receptors. GPCR receptors include cannabinoid receptor type 1. Ligand sensitive ion channels include Transient Receptor Potential Vanilloid 1 (TRPV1). The endogenous ligands include anandamide or N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). The enzyme that synthesizes 2AG is DAGL. The enzyme that synthesizes AEA is NAPE-PLD. For degradation of AEA include enzyme FAAH and NAAA. For 2AG the degradation enzymes are ABHD6, ABHD12, and MAGL. [6 ] (diagram 3) 2-AG is the primary signaling ligand in the compared to AEA and is most abundant in the brain. CB2R is most abundant in the brain while CB1R is most abundant in peripheral tissue. Both 2AG and AEA can bind to CB1R and CB2R. [7]

Within the CNS and afferent neurons cannabinoids effects are medicated primarily by inhibitory CB1 receptors. The ECB acts as a retrograde messenger or synaptic modulators within both CNS and peripherally. Therefore, activation of CB1R within the eCB inhibits the release from presynaptic terminals of both inhibitory and excitatory neurotransmitters. Within the peripheral the ECB contributes to innate and adaptive immune responses. This acts as a preventative method against onset of pre-inflammatory responses. For immunosuppressive effects of the periphery CB2R is primarily responsible. Within the most severe or chronic forms of migraines more CB2R are made for activation. Within brain-residing immune cells such as microglia there are CB1R and CB2R hetero-receptor complexes. This is important to note since microglia are part of the pathogenesis of migraine with auras. The positive feedback loop found in cortical spreading depression can be distributed by CB1R agonist.[7]

1.3.1  ECB and Pain

Historically there has been records of cannabis being used for pain management. Which have been supported scientifically that the use of C. sativa L. and its secondary metabolite for pain management. This has opened more recent interest in the ECS possible mechanism in pain management. In previous studies it has been found that antagonizing CB2R has antinociceptive properties in inflammatory and nociceptive pain models. One of the theorized mechanisms of action of this property is the inhibition of AEA metabolism. It has also been found that cannabinoids and opioids can act synergistically. This has also been found in cannabinoid with steroidal anti-inflammatory drugs.[6] The ECS is also active in the stress responsive part of the central and peripheral nervous system. This results in reducing pain and inflammatory damage. [7]

As mentioned earlier, the three key parts associated with migraine and the TGVS is the nerves, vessels and dural mast cells. These three parts can be potential targets of action of the ECB when it comes to migraines. This is seen in previous studies that found AEA can reduce the dilation of dural blood vessels and neuronal pro-nociceptive pain activity. It is believed that CB1R can have anti-migraine action because it can stabilize the dural mast cells.[7] A previous study has also found that when rats are given a pharmacological inhibitor of MAGL and ABHD6 it prevents and reversed periorbital allodynia (headache like behavior) associated with CSD induction. They also found that when CSD is induced 2-AG levels in the PAG was reduced. CSD induction also increased ABHD6 and MAGL in the PAG. This suggests that a potential migraine pathology is 2-AG dysregulation in the PAG. [20]

1.3.2  ECB Sex Difference

Previously, a study was done to determine if there was sex difference within naive rat PAG tissue with proteomic analysis. This showed that there was significant higher regulation of the ECB in female than male tissue. There was also more MAGL and ABHD6 in female than male. This suggest that compared to male tissue, females have higher ECS metabolism in the PAG. [8] The stage of the estrous cycle in rats has been found to alter the levels of 2-AG and AEA. 2-AG in the hypothalamus was higher in the diestrus and estrus phase and, in the pituitary, it was higher in the proestrus phase. For AEA levels were highest in the proestrus phase in the pituitary and diestrus in the hypothalamus. [21

Diagram 3:

Diagram 3: This shows the synthesis and the degradation of 2-AG and AEA. By NAPE-PLD NAPE is turned in AEA. AEA is then degraded into AA EtNH₂ by FAAH. 2-AG is made from DAG by DAGLα. 2-AG is then metabolized by MAGL into AA Glycerol.

1.4  Proteomics and its Use

Proteins are made up of amino acids. The sequence and number of amino acids determine the characteristics of a protein. Amino acids are bonded together with peptide bonds. The product of these bonds are peptides. As the chain grows it becomes a polypeptide. Each polypeptide has a free amino group at one of its ends, which is called an N terminal. Polypeptide and protein are interchangeable terms. After translation, protein synthesis, most proteins are modified. This is known as post-translational modifications. There are many types and functions of proteins. Some examples are enzymes and hormones. Enzymes are catalysts in biochemical reactions. Hormones are chemical signaling molecules. [18]

The shape of a protein is crucial to its specific function. One of the potential structures a protein can have is a primary structure. Secondary structure arises when there is local folding to the polypeptide in some regions. This commonly creates a-helix and b-pleated sheet structures. Hydrogen bond holds these shapes. There is a three-dimensional structure called a tertiary structure that can also arise. It is caused by chemical interactions on the polypeptide chain. The last possible structure is a quaternary structure which arises when interaction of subunits from several polypeptide forms.[18]

A proteome is the entire set of proteins that a cell type produces. Proteomics is the study of the proteome. [18] Proteomics is used for the determination of the structure and the function of the complete set of proteins produced by the genome of an organism including co and posttranslational modifications. [1]

A genome can be transcribed into proteins which make up the proteome of a cell. The key difference between genomics and proteomics is that genomics gives information on what proteins can exist, but proteomics is about what proteins currently exist. Proteomics can also

provide information about protein alterations. This is important since biological activity of proteins is often controlled by these alterations. To determine changes in protein expression patterns, protein levels must be measured directly. One of the most common protein alterations is phosphorylation. This is where phosphor-proteomics comes in. [4]

Changes caused by diseases can be detected by analyzing samples for genes, proteins and small molecule changes. These specific changes are known as biomarkers. Proteomics is defined as a large-scale study of protein expression, structure and function in time and space. Changes in protein can be either changed in quantity or posttranslational modification. The most studied post- translational modification is glycosylation and phosphorylation. [9]What makes proteomics a complicated application for clinical use is that it is dynamic. This means a static set of genes may result in different proteomic phenotypes depending on the developmental stage of the organism and environmental factors. This means that there is a wide range of refence values for a healthy individual. Mass spectrometry-based approach to identify protein cleavage products is referred to as bottom-up proteomics. Bottom-up proteomics has some cons compared to top-up. This includes loss of information about protein isoforms and PTM. Although bottom-up proteomics requires less material, provides better peptide/protein identification and is the method of choice when it comes to biomarker discovery.[9] Protein are naturally unstable molecules which can make it harder to work with when compared to genomic analysis. [18] 

1.4.1Proteomics and Western Blots

Proteomics is used for the determination of the structure and the function of the complete set of proteins produced by the genome of an organism including co and posttranslational modifications. [1] While western blot is used for the detection and quantification of certain proteins of interest. Western blots use a housekeeping protein that should remain consistent across all treatments as a baseline for protein expression quantity. Through electrophoresis the