WO2018137028A1

WO2018137028A1 - Methods, compositions and kits for assessing brain inflammation in depression and related conditions

Info

Publication number: WO2018137028A1
Application number: PCT/CA2018/050081
Authority: WO
Inventors: Jeffrey H. MEYER; Elaine SETIAWAN
Original assignee: Centre for Addiction and Mental Health
Current assignee: Centre for Addiction and Mental Health
Priority date: 2017-01-24
Filing date: 2018-01-24
Publication date: 2018-08-02
Anticipated expiration: 2019-07-24

Abstract

Disclosed are methods, compositions and kits for assessing the level of brain inflammation in depressed subjects and assessing, preventing or inhibiting the chronological advancement of depression, including neuroprogression. The methods involve blood tests, comprising the quantification of blood prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), or a combination thereof; or positron emission tomography (PET) scans, comprising measuring translocator protein (TSPO) binding.

Description

Methods, Compositions and Kits for Assessing Brain Inflammation in Depression and

Related Conditions

FIELD OF THE INVENTION

[0001] The present invention relates to the state of brain inflammation in depressed subjects, and in particular the state of microglial activation in the brain of depressed subjects.

BACKGROUND OF THE INVENTION

[0002] According to the World Health Organization, maj or depressive disorder (MDD) is the leading cause of death and disability in moderate to high income countries like the United States⁽¹⁾. Key problems are that at least 50% of major depressive episodes (MDE) are treatment resistant⁽²⁾ and that the lifetime prevalence is over 15%⁽³⁾. MDD is highly impactful, often lifelong with recurrent episodes, and greatly increases the risk of suicide⁽¹'^4"6). MDD has several molecular subtypes ^(7"14) and there is increasing evidence that inflammation plays a role in MDD. Investigations frequently report greater plasma levels of the cytokines IL-6 and T F-a, and to some extent C-reactive protein during MDD, indicating that excessive autoimmune activity occurs in at least a subset of MDD^(15"44). Long term exposure to cytokines is associated with symptoms of depression as 20 to 50% of people receiving chronic interferon (IFN-a) treatment for infections or cancer have MDE⁽⁴⁵' ⁴⁶⁾. In addition, administration of cytokine inducing agents such as vaccinations, and lipopolysaccharide administration, can increase symptoms of depression⁽⁴⁷'⁴⁸⁾. Traumatic brain injury, which induces brain inflammation^(49"52), is frequently associated with depressed mood⁽⁵³⁾. Lifetime rates of MDE in systemic lupus erythematosis and multiple sclerosis, illnesses with brain inflammation, are about 50%, suggesting a link between inflammation and mood symptoms^(54"59). Some studies indicate that elevated levels of the inflammatory mediator, prostaglandin E2 (PGE2) are observed in the saliva, plasma, and CSF of depressed subjects^(78"81), and were thought to correlate with depression severity⁽⁸⁰'⁸¹⁾. These results are at odds with other findings, and no quantitative correlation has ever been established. [0003] Initial post-mortem studies of brain inflammation in MDD were not definitive, largely due to sample size and lack of subjects with a current major depressive episode (MDE). In a postmortem microarray study of the prefrontal cortex in 14 medication free MDE subjects and 14 healthy, Shelton et al. found increased transcription of cytokines that influence inflammation⁽¹¹⁾, which was interpreted as reflecting increased inflammatory stress in the prefrontal cortex⁽¹¹⁾ but several other microarray studies, most of which sampled adjacent regions of the prefrontal cortex, did not identify this result⁽⁶⁰' ⁶¹⁾. This study was underpowered to detect an effect of MDE diagnosis ⁽⁶²⁾. Van Otterloo et al.,⁽⁶³⁾ reported no difference in density of activated microglia, in the white matter of the orbitofrontal region in 10 MDD subjects. Dean et al. sampled 10 MDD subjects and found significantly increased levels of the transmembrane form of TNF in the dorsolateral prefrontal cortex but no difference in levels of this form of TNF in the anterior cingulate cortex and no difference in the soluble form of TNF in either region⁽⁶⁴⁾. Amongst investigations in suicide victims, one study reported greater HLA- DR staining, a marker of microglial activation, in the dorsolateral prefrontal and anterior cingulate cortex⁽⁶²⁾ and a second study reported greater levels of IL-6, TNF-a, and IL-Ιβ in BA10⁽⁶⁵⁾. Neither study of suicide found a relationship to MDD (or MDE) but there were less than 10 subjects with MDD in each study. The mixed results among these investigations in MDD have been attributed to issues of variation in brain regions sampled, inclusion of early and late onset MDD, comorbidity of other psychiatric disorders and addiction and, with the exception of the microarray studies, small sample size, although it is plausible that the lack of focus on sampling the state of MDE may be important for investigations of neuroinflammation.

[0004] Furthermore, prospective studies indicate that with greater length of illness,

MDD often transitions from infrequent solitary MDE with good inter-episode recovery towards increasingly persistent disease, with greater proportions of time spent in the midst of a MDE^(2a'^3a) a phenomenology strongly suggestive of neuropathological progression.

[0005] While neuroprogression, which may be defined as progressive damage of the brain with greater duration of illness, is firmly established at a neuropathological level in neuropsychiatric illnesses like Parkinson's disease and Alzheimer's disease, it is not well established in MDD, with few investigations demonstrating greater levels of pathology with longer duration of illness. Pathologies posited in the literature as potential components of neuroprogression in MDD include loss of astroglia and resultant reduced glutamate uptake > loss of somatostatin positive interneurons,^(5a) decreased neurogenesis'^ persistence of elevated MAO-A level, ^(7a) hippocampal volume loss^(8a), and chronic microglial activation,^(9a'^10a) however, only hippocampal volume loss has been demonstrated, primarily through cross sectional studies, to exhibit greater magnitude of effect with greater duration of MDD,^(8a'^lla) with mean reductions of 4% overall. ^{(8a>l la)} Since decreased hippocampal volume is mainly associated with reduced verbal recall, ^(8a) and MDD is an illness of multiple pathologies with a broad scope of symptoms, it is plausible that there are other pathologies besides hippocampal volume loss which progress with ongoing MDD. [0006] Chronic microglial activation is a process strongly implicated in neuroprogression due to its role in responding to accumulating tissue damage and the inherent feed-forward mechanisms from this process.^(9a'^12a'^13a) Microglial activation, an important aspect of neuroinflammation, occurs when microglia with small cell bodies and longer thinner dendrites, morphologically oriented towards detecting molecular patterns associated with damage or inflammation, shift from this monitoring role into an activated state in which their cell bodies enlarge and dendrites either become thicker or are removed entirely if the cell becomes amoeboid. In this activated state, microglia may produce cytokines, complement proteins, reactive oxygen species, and proteinases. The production of cytokines may have autocrine effects to recruit more activated microglia whereas the latter three processes may lead to cascades of neuronal damage and further inflammation. ^(13a) For example, neuronal damage as sequelae of reactive oxygen species and proteinases may also induce activation of microglia through microglial detection of damage associated molecular patterns with upregulation of tolllike receptors such as TLR3 and TLR4 receptors, the latter which was found in a sample of dorsolateral prefrontal cortex of MDD subjects. ^(14a) Given that chronic microglial activation is a well-accepted index of neuroprogression in neurodegenerative diseases, and given that greater microglial activation occurs in unmedicated MDE of MDD,^(10a'^15a) this process is a strong candidate for neuroprogression in MDD.

[0007] To measure neuroinflammation, positron emission tomography (PET) may be applied to measure translocator protein (TSPO) binding in vivo. TSPO is an 18 kDa protein located on outer mitochondrial membranes in microglia and increased expression of TSPO occurs when microglia are activated during neuroinflammation⁽⁶⁶⁾. Recently, a new generation of positron emission tomography radiotracers were developed with superior quantification of TSPO binding and among these, [¹⁸F]FEPPA has excellent properties including high, selective affinity for TSPO⁽⁶⁷⁾, increased binding during induced neuroinflammation⁽⁶⁸⁾ and a high ratio of specific binding relative to free and non-specific binding⁽⁶⁹⁾.

[0008] The first neuroimaging study applied [^UC] PBR28 PET to investigate TSPO levels in MDD, which was negative⁽⁷⁰⁾. This earlier study assessed whether TSPO levels were elevated in a sample of 10 MDD subjects (scanned once) under a variety of states (treated, untreated, symptomatic, partially symptomatic) hence, this study cannot be considered definitive for determining whether TSPO binding is elevated in MDE. More recently in a [¹⁸F]FEPPA PET study of 20 MDE secondary to MDD and 20 healthy subjects, it was discovered that TSPO VT, an index of translocator protein (TSPO) level, a marker of microglial activation, was elevated across cortical and subcortical regions in MDE (30% magnitude in prefrontal cortex, anterior cingulate cortex and insula; Fi5,23=4.5, p=0.001; JAMA Psych 2015)⁽¹⁴⁾. This latter study, which sampled early onset MDD during medication free MDE unbiased by concurrent active comorbid conditions is considered the present state of the art, and is supportive of neuroinflammation during MDE of MDD.

[0009] Since imaging the brain during a MDE is costly, technically challenging and impractical in a clinical setting, peripheral and/or proxy markers correlating to depression, MDE, MDD, and antidepressant treatment are needed. Further, there is also a need in the art for peripheral measures correlating to microglia activation in a subject. In WO2016112467, herein incorporated by reference in its entirety, translocator protein total distribution volume (TSPO VT), an index of TSPO density and brain inflammation, was measured applying [¹⁸F] FEPPA positron emission tomography scanning in 20 unmedicated MDE subjects. In this document, the concentration of serum peripheral prostaglandin E2, (PGE2) divided by the concentration of serum C-reactive protein (CRP), a measure of inflammation from peripheral tissues, was related to the levels of TSPO VT in the brain such that there was a curvilinear relationship: as PGE2/CRP ratio increased, so did regional brain levels of TSPO VT. Then as PGE2/CRP increased further there was a plateauing such that additional increases of PGE2/CRP were still associated with high levels of TSPO V_T, but the TSPO V_T levels did not increase further.

[0010] There is a need in the field to have measures of inflammation in blood that are predictive of brain inflammation in treated MDE subjects. At least 50% of MDE cases are associated with treatment resistance⁽²⁾ so there is interest in determining predictors of the neurobiology of MDE cases that do not respond to treatment. Under such circumstances, a predictor of brain inflammation could be applied to aid in the decision to initiate treatment with an anti-inflammatory treatment or an inflammatory modulating treatment. This would be a personalized medicine, or precision medicine approach where a marker of the neurobiology of the case of MDE is then applied to choose a matching treatment predicted to have a better response due to its higher likelihood of correct matching. Another reason to have measures of inflammation in blood predictive of brain inflammation is that they could be applied to predict when active disease is present. As defined herein, "active disease" is intended to mean the presence of a depressogenic brain change that is masked by use of an antidepressant which creates resiliency against the depressogenic brain change but does not reverse the depressogenic brain change itself. Approximately 50% of MDE cases are associated with recurrence over 2 to 3 years but this rate of recurrence is lowered when ongoing antidepressant treatment is administered⁽⁷¹⁾. Hence, if active disease is present, this would predict recurrence unless antidepressant treatment is continued. [0011] A problem in the clinical field is that many clinical trials of antidepressants do not yield significant results. This is a particular problem in America where the Food and Drug Administration (FDA) uses a low bar of 50% success rate for clinical trials of MDD. One issue that has been proposed is that inappropriate subjects enroll in these clinical trials. For example, healthy subjects seeking remuneration could decide to enroll in a study and pretend to have symptoms of depression. A marker that distinguishes healthy with no history of antidepressant use from treated depressed subjects would be useful. Similarly, there is a need for novel methods, compositions and kits to assess brain inflammation in depression and related conditions such MDD. SUMMARY OF THE INVENTION

[0012] In an embodiment of the present invention, there is provided a method of assessing a level of brain inflammation in a subject, the method comprising:

measuring a level of microglial activation in the brain of the subject,

wherein the level of microglial activation is a measure of brain inflammation.

[0013] In a further embodiment of the present invention, there is provided a method of assessing the chronological advancement of depression, including neuroprogression, in a depressed subject, the method comprising:

measuring a level of microglial activation in a brain of the subject at a first time point; and

measuring the level of microglial activation in the brain of the subject at a second time point,

wherein an increase in the level of microglial activation at the second time point compared to the first time point is indicative of the chronological advancement, including neuroprogression, of depression in the subject.

[0014] In a further embodiment of the methods described throughout, the subject has major depressive disorder (MDD), major depressive episode(s), melancholia or atypical depression, psychotic depression, antenatal and postnatal depression, bipolar disorder, cyclothymic disorder, persistent depressive disorder (dysthymia), seasonal affective disorder or a mood state that is characterized by one or more symptoms of depression.

[0015] In a further embodiment of the methods described throughout, the subject has major depressive disorder.

[0016] In a further embodiment of the methods described throughout, the subject is undergoing treatment for depression, is undergoing treatment for depression but is treatment- resistant or treatment-refractory, or is not undergoing treatment for depression. The subject may be diagnosed as having depression or the subject may be undiagnosed. In still a further embodiment, the subject is undiagnosed but exhibits one or more signs or symptoms of depression or MDD. [0017] In a further embodiment of the methods described throughout, microglial activation is measured by a blood test, positron emission tomography (PET) scan, or both.

[0018] In a further embodiment of the methods described throughout, the blood test comprises:

measuring one or more of blood prostaglandin E2 (PGE2) concentration ([PGE2]) and blood prostaglandin D2 (PGD2) concentration ([PGD2]), in a blood sample obtained from the subject,

wherein a decrease in blood [PGE2], blood [PGD2], or both, in the blood sample compared to a healthy control is predictive or indicative of a treated depressed subject or a treated depressed subject that is treatment-resistant or treatment-refractory.

[0019] In a further embodiment of the methods described throughout, the blood [PGE2] in the sample is below 1500pg/ml, or more preferably below 1400pg/ml, or still more preferably below 1300pg/ml, or still more preferably below 1200pg/ml, or still more preferably below HOOpg/ml, or still more preferably below 1050pg/ml, or still more preferably below lOOOpg/ml, or still more preferably below 950pg/ml, or still more preferably below 900pg/ml, or still more preferably below 850pg/ml, or still more preferably below 800pg/ml, or still more preferably below 750pg/ml, or still more preferably below 700pg/ml, or still more preferably below 650pg/ml, or still more preferably below 600pg/ml, or still more preferably below 550pg/ml, or most preferably below 500pg/ml. [0020] In a further embodiment of the methods described throughout, blood [PGD2] in the sample obtained from the test subject is below 23.5pg/ml.

[0021] In a further embodiment of the methods described throughout, the PET scan measures translocator protein (TSPO) binding.

[0022] In a further embodiment of the methods described throughout, the PET scan measures translocator protein total distribution volume.

[0023] In a further embodiment of the methods described throughout, the PET scan is performed on grey matter regions of the brain. [0024] In an embodiment of the present invention, there is provided a method of assessing a level of brain inflammation in a depressed subject, the method comprising:

measuring one or more of blood prostaglandin E2 (PGE2) concentration ([PGE2]) and blood prostaglandin D2 (PGD2) concentration ([PGD2]) in a blood sample obtained from the subject,

wherein a decrease in blood [PGE2], blood [PGD2], or both, in the blood sample compared to a healthy control is predictive of a treated depressed subject or a treated depressed subject that is treatment-resistant or treatment-refractory.

[0025] In an embodiment of the present invention, there is provided a kit for assessing a level of brain inflammation in a depressed subject, the kit comprising:

one or more diagnostic agents for quantifying PGE2, PGD2, or both in a blood sample of the depressed subject; and

one or more components, diluents or buffers for use with the above-identified diagnostic agents.

[0026] In an embodiment of the present invention, there is provided a method of preventing or inhibiting the chronological advancement of depression, including neuroprogression, in a subject, the method comprising:

measuring a level of microglial activation in a brain of the subject at a first time period; treating the subject for depression; and

wherein:

if the level of microglial activation at the second time point is equivalent or less than the level of microglial activation at the first time point, the treatment for depression is maintained, or

if the level of microglial activation at the second time point is greater than the level of microglial activation at the first time point, the treatment for depression is modified,

and wherein the step of treating may be performed prior to or in between the steps of measuring microglial activation. [0027] In a further embodiment of the present invention, there is provided a method of preventing or inhibiting the chronological advancement of depression, including neuroprogression, in a subject, the method comprising:

treating a subject for depression or selecting a subject being treated for depression; measuring a level of microglial activation in a brain of the subject at a first time period; measuring the level of microglial activation in the brain of the subject at a second time point,

wherein:

if the level of microglial activation at the second time point is greater than the level of microglial activation at the first time point, the treatment for depression is modified.

[0028] In a further embodiment of the methods described throughout, modifying the treatment for depression may comprise, for example, but not limited to, modifying a dose, or adding or removing the administration, of one or more medications in the treatment.

[0029] In a further embodiment of the methods described throughout, the method further comprises counseling and/or monitoring the subject.

[0030] In a further embodiment of the methods described throughout, the method further comprises:

measuring the level of microglial activation in the brain of the subject at one or more additional time points. The additional measurements may be taken over time until the subject exhibits no further increase in the level of brain inflammation as determined via measurement of microglial activation, or the measurements may be taken to monitor the subject periodically for changes to microglial activation.

[0031] In a further embodiment of the present invention, there is provided a method of determining a level of microglial activation in a subject and optionally treating the subject, the method comprising: quantifying a duration of untreated depression by subtracting the age at which treatment for depression began from the age of onset of depression and adding any treatment-free period, wherein a longer duration of untreated depression is indicative of an increased level of microglial activation. [0032] In a further embodiment of the methods described throughout, the any treatment- free period is over 1 month or greater, 2 months or greater, 3 months or greater, 4 months or greater, 5 months or greater, 6 months or greater, 9 months or greater, or 1 year or greater.

[0033] In a further embodiment of the methods described throughout, the method further comprises treating the subject. [0034] In a further embodiment of the methods described throughout, the method further comprises determining a second level of microglial activation by performing a blood test, PET scan, or both.

[0035] In a further embodiment of the methods described throughout, the blood test comprises:

wherein a decrease in blood [PGE2], blood [PGD2], or both, in the blood sample compared to a healthy control is predictive of a treated depressed subject or a treated depressed subj ect that may be treatment-resistant or treatment-refractory.

[0036] In a further embodiment of the methods described throughout, the PET scan measures translocator protein (TSPO) binding.

[0037] In a further embodiment of the methods described throughout, the PET scan measures translocator protein total distribution volume. [0038] In a further embodiment of the methods described throughout, the PET scan is performed on grey matter regions of the brain. [0039] In a further embodiment of the present invention, there is provided a method of determining a length of untreated depression in a depressed subject, the method comprising: measuring a level of microglial activation in the brain of the subject,

wherein every 14-18% increase in the level of microglial activation compared to a healthy subject is indicative of 10 years of untreated depression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0041] Figure 1. Prostaglandin E2 in Healthy Controls, Untreated and Treated Major Depressive Episodes. This graph illustrates that serum prostaglandin E2 (PGE2) level is lower in untreated major depressive episodes (NT-MDE) and lowest in treated major depressive episodes (T-MDE) as compared to healthy controls (HC).

[0042] Figure 2. Prostaglandin E2 and Prostaglandin D2 in Healthy Controls,

Untreated and Treated Major Depressive Episodes. This graph illustrates that serum prostaglandin E2 (PGE2) level is lower in treated major depressive episodes (T-MDE) as compared to untreated major depressive episodes (NT-MDE) or as compared to healthy controls (HC). It is also lower in treated major depressive episodes (T-MDE) as compared to untreated major depressive episodes (NT-MDE). This graph also illustrates that PGD2 was below the lower limit of detection in 6 of 11 healthy controls (HC), 12 of 16 untreated major depressive episode (NT-MDE) subjects and all (11 of 11) of the treated major depressive episode (T-MDE) subjects. Units of PGE2 are pg/ml. The minimum detection range for PGD2 in serum was 23.5pg/ml.

[0043] Figure 3. Relationship of Serum PGE2/PGF2a to Prefrontal Cortex (PFC)

TSPO VT. The TSPO VT is an index of translocator protein density, a marker of brain inflammation. PGE2 and PGF2a refer to prostaglandin E2 and prostaglandin F2alpha. In treated major depressive episodes not currently responsive to treatment, the PGE2/ PGF2a ratio is predictive of TSPO VT in the PFC (the PFC is a large region of brain tissue that may be representative of other brain regions). TSPO VT was scaled in those with a single nucleotide polymorphism that reduces binding of this and other second generation PET radiotracers to TSPO⁽⁷²' ⁷³⁾ so as to remove the effect of this nuisance variable (those with one copy of the allele are scaled up by a factor of 1.4 and homozygotes for the allele, which are rare are not measurable with the technique). When TSPO VT is elevated in the PFC, it is often elevated in other brain regions. The relationship of serum PGE2/PGF2a to the PFC TSPO V_T is similar to TSPO VT values in other brain regions. These data collectively might reflect that activated microglia behave in a specific manner in the treated state that can be detected by particular patterns of prostaglandin secretion. There was no difference in PGF2a between healthy/treated/untreated states for MDE subjects. [0044] Figure 4. Relationship between Regional Translocator Protein Distribution

Volume and Duration of Untreated Major Depressive Disorder. Analyses of covariance (ANCOVAs) with regional translocator protein distribution volume (TSPO VT) values as the dependent variable found that the combination of duration of untreated illness and rs6971 genotype these predictor variables accounted for approximately 50% of the variance in the three prioritized regions (duration of untreated illness Fi,₄₇ = 17 0 to 24· 1, P < 0· 001, overall model, F₂,47 = 24-6 to 32 0, P < 0 001, R² = 0 51 to 0 58, R² _Adj_usted = 0 49 to 0 56). As plotted, TSPO VT was associated with an increase of 0 15, 0 18, and 0 17 TSPO VT per year of untreated MDD in the respective regions. All second generation TSPO radioligands, such as [¹⁸F]FEPPA, show differential binding according to the single nucleotide polymorphism rs6971 of the TSPO gene resulting in high affinity binders (HAB) and mixed affinity binders (MAB). MABs have been multiplied by 1 -4 in this figure to adjust for the genotype difference.

[0045] Figure 5. Duration of Major Depressive Disorder and Antidepressant Exposure are Opposite Predictors of TSPO VT, But Similar in Magnitude. ANCOVAs with regional TSPO VT values as the dependent variable found that the combination of these predictor variables accounted for approximately 50% of the variance in the three prioritized regions (total duration of MDD, Fi,₄₆ = 9 · 4 to 15 · 4, P < 0 · 001 to P = 0 · 004; total duration of antidepressant exposure, Fi,₄₆ = 7-2 to 9 2, P = 0 010 to P = 0 008; overall model, F_3;46 = 14 0 to 17 9, P < 0 001, R² = 0-48 to 0-43, R² Adjusted = 0-44 to 0 51). As plotted, the slopes of the linear relationship between TSPO V_T and duration of MDD exposure were 0 17, 0 19, and 0 20, respectively for the prefrontal cortex, anterior cingulate cortex, and insula whereas the slopes of the linear relationship between TSPO V_T and duration of antidepressant exposure were - 0 13, -0 16, and -0· 13, respectively. TSPO VT presented is adjusted by genotype such that the TSPO V_T of mixed affinity binders of the rs6971 polymorphism, which influences binding of second generation PET radioligand for TSPO, were multiplied by 1 4. Also, in the top three graphs, a correction to reduce variance in TSPO V_T was applied to adjust values to correspond to an antidepressant exposure of 2 years: The difference between the duration of antidepressant exposure and 2 years multiplied by the slope of the linear predictor of the effect of antidepressant exposure on TSPO V_T was applied as a correction factor. Similarly, in the bottom three graphs, a correction to reduce variance in TSPO VT was applied to adjust values to correspond to a duration of MDD of 18 years. The difference between the duration of MDD and 18 years multiplied by the slope of the linear predictor of the effect of duration of MDD on TSPO VT was applied as a correction factor.

[0046] Figure 6. Translocator Protein Density Greater with More Years of Untreated

MDD Compared to Short Duration of Untreated MDD and Healthy Controls. Group (duration untreated > 10 years (N = 25), duration untreated < 10 years (N = 25), and healthy (N = 30)) and genotype were significant predictor variables in a multivariate analysis of variance with TSPO VT in the three primary regions of interest as the dependent variables (group, F₃,75 = 9 0, P < 0 001; genotype, F₃,7₄ = 12 68, P < 0 001). Additional comparisons based on the least significant difference test showed significant differences between a long duration of untreated MDD as compared to health (P < 0 · 001 for each region; differences of 31 · 2, 36 · 9, and 39 · 0% in the PFC, ACC, and INS respectively) and significant differences between long duration of untreated MDD and short duration of untreated MDD in all of these regions (P = 0 004, P = 0 002, P = 0 003; differences of 28 -98, 33 1%, and 31 0% in the PFC, ACC, and F S respectively). All second generation TSPO radioligands, such as [¹⁸F]FEPPA, show differential binding according to the single nucleotide polymorphism rs6971 of the TSPO gene resulting in high affinity binders (HAB) and mixed affinity binders (MAB). Red bars indicate means in each group. DETAILED DESCRIPTION

[0047] Described herein are embodiments illustrative of methods of assessing a level of brain inflammation and methods of assessing the chronological advancement of depression, including neuroprogression. All references to embodiments or examples throughout the disclosure should be considered a reference to an illustrative and non-limiting embodiment or an illustrative and non-limiting example.

[0048] According to an embodiment of the present invention, there is provided a method of assessing a level of brain inflammation in a subject, the method comprising measuring a level of microglial activation in the brain of the subject, wherein the level of microglial activation is a measure of brain inflammation.

[0049] According to a further embodiment of the present invention, there is provided a method of assessing chronological advancement of depression, including neuroprogression, in a subject. Chronological advancement of depression refers to the progressive worsening of depression symptoms over time, characterized by infrequent depressive episodes with good inter-episode recovery towards increasingly persistent depression, with greater proportions of time spent in the midst of a depressive episode. Neuroprogression is likely to be a significant component of the chronological advancement of depression. Specifically, microglial activation in the brain is implicated in neuroprogression. As is shown herein, there is a relationship between increasing duration of untreated depression and related illness and a greater level of microglial activation. This method comprises measuring a level of microglial activation in a brain of the subject at a first time point; and measuring the level of microglial activation in the brain of the subject at a second time point, wherein an increase in the level of microglial activation at the second time point compared to the first time point is indicative of the chronological advancement of depression, including neuroprogression, in the subject. [0050] In the context of the present invention, depression is meant to encompass any type of depression, ranging from mild to severe depression, any disorder that includes one or more symptoms of depression, or any mood state that is characterized by one or more symptoms of depression. In a particular embodiment, which is not meant to be limiting in any manner, the disorder is MDD. [0051] Symptoms of depression may include, as presented in the Diagnostic and

Statistical Manual of Mental Disorders, 4^th edition (DSM-IV), without limitation:

1. Depressed mood;

2. Markedly diminished interest or pleasure;

3. Significant weight loss, when not dieting, or weight gain, or decrease or increase in appetite;

4. Insomnia or hypersomnia nearly every day;

5. Psychomotor agitation or retardation nearly every day (observable by others, not merely subjective feelings or restlessness or being slowed down);

6. Fatigue or loss of energy;

7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delusional) nearly every day (not merely self-reproach or guilt about being sick);

8. Diminished ability to think or concentrate or indecisiveness nearly every day; and

9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation without a specific plan, or a suicide attempt or specific plan for committing suicide.

[0052] In the context of the present invention, a subject that has depression or is suspected of having depression may exhibit one or more of the symptoms listed above. Depression may be associated with a variety of disorders including, but not limited to major depressive disorder (MDD), major depressive episode (MDE), MDE secondary to MDD, melancholia and atypical depression, psychotic depression, antenatal and postnatal depression, bipolar disorder, cyclothymic disorder, persistent depressive disorder (dysthymia), or seasonal affective disorder. Depression is also meant to encompass a mood state that is characterized by one or more symptoms of depression. Depression may be clinically diagnosed, by use of DSM- IV or V, Hamilton Depression Rating Scale or another psychiatric rating scale, or may not be clinically diagnosed but suspected.

[0053] In one particular embodiment, the depression is MDD. MDD may vary from minor to severe, and is typically diagnosed by a qualified medical practitioner as a person having 5 or more of the above symptoms for a period of more than 2 weeks. [0054] It is also contemplated herein that the subject may be currently undergoing treatment for depression before practicing any of the methods as described herein. In a further embodiment, the subject is currently undergoing treatment for depression but is treatment- resistant or treatment-refractory. In still a further embodiment, the subject is not undergoing treatment for depression, or has not been diagnosed by a medical practitioner. In still a further embodiment, the subject is suspected of having depression or a depressive disorder, for example, but not limited to MDD.

[0055] As will be appreciated by a person of skill in the art, the term "treatment" may comprise the administration of any medication that is meant to stop, slow or decrease the severity of symptoms of depression, improve the symptoms of depression, modulate inflammation, or modulate microglial activity. Medications may include any active agent, therapeutic, or the like.

[0056] For example, without limitation, medication for depression includes antidepressants. Representative antidepressants include, without limitation, serotonin and norepinephrine reuptake inhibitors (S RIs), for example, but not limited to desvenlafaxine (Pristiq™, Khedezla™), duloxetine (Cymbalta™), levomilnacipran (Fetzima™), venlafaxine (Effexor XR™), and milnacipran (Savella™); selective serotonin reuptake inhibitors (SSRIs), for example, but not limited to sertraline (Zoloft™), fluoxetine (Prozac™, Sarafem™), citalopram (Celexa™), escitalopram (Lexapro™), paroxetine (Paxil™, Pexeva™, Brisdelle™), fluvoxamine (Luvox™), and vilazodone (Viibryd™); monoamine oxidase inhibitors (MAOIs), for example, but no limited to isocarboxazid (Marplan™), phenelzine (Nardil™), selegiline (Emsam™, Eldepryl™, Zelapar™), and tranylcypromine (Parnate™); tricyclic antidepressants (TCAs), for example, but not limited to amitriptyline (Elavil™, Vanatrip™), amoxapine (Asendin™), desipramine (Norpramin™), doxepin (Sinequan™, Silenor™), imipramine (Tofranil™), nortriptyline (Pamelor™), protriptyline (Vivactil™), trimipramine (Surmontil™); dopamine reuptake inhibitors, for example, but not limited to bupropion (Wellbutrin™, Zyban™, Budeprion™, Aplenzin™, Buproban™, Forfivo™), amineptine (Maneon™, Survector™), Altropane (0-587), Amfonelic acid (AFA), Benocyclidine (BTPC), DBL-58, difluoropine (O-620), fluorenol, GBR-12935, GYKI-52895, modafinil (Provigil™), RTI-229, RTI-55 (Iometopane™), and vanoxerine (GBR- 12909); monoamine receptor binders, for example, but not limited to trazodone, mirtazapine (Remeron™), ortiozetine, aripiprazole (Abilify™); and noradrenergic antagonists (NaSSAs), for example but not limited to, aptazapine (CGS-7525A), esmirtazapine (ORG-50,081), mianserin (Bolvidon™, Norval™, Tolvon™), mirtazapine (Remeron™, Avanza™, Zispin™), and setiptiline/teciptiline (Tecipul™).

[0057] In a further example, without limitation, medication for depression includes antiinflammatory agents, inflammation modulators, and microglial function modulators. Representative agents include, without limitation, prostaglandin synthesis inhibitors, for example, but not limited to nonsteroidal anti-inflammatory drugs (NSAIDs) such as COX-1 and COX-2 inhibitors (cyclooxygenase-1 or -2 inhibitors), for example, but not limited to acetylsalicylic acid (Aspirin™), ibuprofen (Advil™, Motrin™), naproxen (Aleve™, Anaprox™, Naprelan™, Naprosyn™), celecoxib (Celebrex™), diclofenac (Cambia™, Cataflam™, Voltaren™, Zipsor™, Zorvolex™), diflunisal (Dolobid™), etodolac (Lodine™), indomethacin (Indocin™), ketoprofen (Orudis™), ketorolac (Toradol™), nabumetone (Relafen™), oxaprozin (Daypro™), piroxicam (Feldene™), salsalate (Disalcid™, Salflex™), sulindac (Clinoril™), and tolmetin (Tolectin™); monoamine oxidase B inhibitors, for example, but not limited to selegiline (Eldepry™, Atapryl™, Carbex™, Deprenyl™, Emsam™, Zelapar™), rasagiline (Azilect™), and safinamide (Xadago™); microglial regulators, for example, but not limited to low dose naltrexone (LDN)(ReVia™, Vivitrol™), Dextro- naltrexone, Minocycline (Minocin™), dextromethorphan (Benylin™ DM, Mucinex™ DM, Camydex-20™, Robitussin™, NyQuil™, Dimetapp™, Vicks™, Coricidin™, Delsym™, TheraFlu™, Cheracol™ D), rifampin (Rifadin™), propentof lline, ceftriaxone (Rocephin™), glatiramer acetate (Copaxone™, Glatopa™), and ibidulast; and natural products, for example, but not limited to cucurmim, luteolin, resveratol, Gastodia Elata, obovotal, inflexin, Pipera kadsura, Ganoderma lucidum, Berberine, Isodon japonicas, Epimedium brevicornum, Stephania tetranda, stinging nettle, fisetin, pycnogenol, boswellia, and kratom.

[0058] Further, any other active agent, therapeutic, or the like, is contemplated in the treatment of depression. [0059] A subject undergoing treatment for depression but that is treatment-resistant or treatment-refractory is defined as responding inadequately, poorly, negligibly or not at all to the treatment.

[0060] In an embodiment of the present invention, microglial activation is measured by a blood test, positron emission tomography (PET) scan, or both. In a preferred embodiment, microglial activation is measured by a blood test.

[0061] One embodiment of the blood test comprises measuring one or more of blood prostaglandin E2 (PGE2) concentration ([PGE2]) and blood prostaglandin D2 (PGD2) concentration ([PGD2]) in a blood sample obtained from the subject. Other blood components may also be identified or measured.

[0062] In the blood test described above, [PGE2] and [PGD2] may be measured with diagnostic agents alone or via one or more suitable quantitative methods known in the art. The diagnostic agents may include anti-PGE2 or anti-PGD2 antibodies or fragments thereof which retain binding, anti-PGE2 or anti-PGD2 antibodies in conjunction with labeled antibody conjugates or secondary antibodies, antibody derivatives in conjunction with labeled antibody derivative conjugates or secondary antibodies, or any other labeled agent that is known to bind to PGE2 or PGD2. The diagnostic agents bound to PGE2 and PGD2 may be therefore quantified with any technique known in the art, for example, but not limited to, immunoassay (for example, but not limited to enzyme-linked immunosorbent assay (ELISA)), radioimmunoassay, mass spectrometry, high-performance liquid chromatography (HPLC), gas chromatography- mass spectrometry, or other chromatographic or non-chromatographic procedures.

[0063] In an embodiment, but without wishing to be limiting, PGE2 in serum can be measured using an ELISA kit involving a monoclonal anti-PGE2 antibody and horseradish peroxidase labelled PGE2 (rndsystems.com; Bio-Techne), Minneapolis, Minnesota, Prostaglandin E2 Assay, "Parameter" KGE004B, SKGE004B, PKGE004B). Without being limiting, PGD2 in serum can be measured using an ELISA kit involving a sandwich enzyme immunoassay technique (My Biosource.com, Southern California, San Diego, Human Prostaglandin D2 ELISA Kit, MBS700128). A variety of other techniques and reagents may be employed as would be understood by a person of skill in the art. [0064] In the blood test described above, the blood [PGE2] in the sample is preferably below 1500pg/ml, or more preferably below 1400pg/ml, or still more preferably below 1300pg/ml, or still more preferably below 1200pg/ml, or still more preferably below HOOpg/ml, or still more preferably below 1050pg/ml, or still more preferably below lOOOpg/ml, or still more preferably below 950pg/ml, or still more preferably below 900pg/ml, or still more preferably below 850pg/ml, or still more preferably below 800pg/ml, or still more preferably below 750pg/ml, or still more preferably below 700pg/ml, or still more preferably below 650pg/ml, or still more preferably below 600pg/ml, or still more preferably below 550pg/ml, or most preferably below 500pg/ml. [0065] In the blood test described above, the blood [PGD2] in the sample is below

23.5pg/ml.

[0066] In one embodiment, microglial activation is quantified by a PET scan that measures translocator protein (TSPO) binding. Increased expression of TSPO occurs when microglia are activated during neuroinflammation and with greater duration of untreated depression. In a further embodiment, the PET scan measures translocator protein total distribution volume (TSPO VT), an index of TSPO density.

[0067] The PET scan may be performed on one or more grey matter regions of the brain, for example, the prefrontal cortex, anterior cingulate cortex, or insula. Specific areas include, without limitation, the medial prefrontal cortex, ventrolateral prefrontal cortex, dorsolateral prefrontal cortex, orbitofrontal cortex, anterior cingulate cortex, insula, temporal cortex, parietal cortex, occipital cortex, hippocampus, thalamus, dorsal putamen, dorsal caudate, ventral striatum or any combination thereof.

[0068] One example of a PET radiotracer that has high, selective affinity for TSPO is

[¹⁸F]FEPPA. However, any other radiotracer that has selective affinity for TSPO is contemplated.

[0069] The present invention also provides a method of assessing a level of brain inflammation in a depressed subject, the method comprising measuring one or more of blood prostaglandin E2 (PGE2) concentration ([PGE2]) and blood prostaglandin D2 (PGD2) concentration ([PGD2]) in a blood sample obtained from the subject. A decrease in blood [PGE2], blood [PGD2], or both, in the blood sample compared to a healthy control is indicative of a depressed subject.

[0070] In a further embodiment of the present invention, the methods described above and throughout may be useful in the clinical staging of depression. The staging concept is practical as it can differentiate early, milder depression from progressed, chronic depression and enable the selection of different treatment approaches depending on the subject's stage of depression.

[0071] The present invention also provides for a kit for assessing a level of brain inflammation in a subject. In one embodiment, the kit comprises one or more diagnostic agents for quantifying PGE2, PGD2, or a combination thereof, in a blood sample of the subject and one or more components, diluents or buffers, for practicing a suitable quantitative method for determining the amount or concentration of the prostaglandin markers representative for microglial inflammation. Possible diagnostic agents are described above and throughout. [0072] According to a further embodiment of the present invention, there is a method of preventing or inhibiting the chronological advancement of depression, including neuroprogression, in a subject, the method comprising measuring microglial activation in a brain of the subject at a first time period, treating the subject for depression, and measuring microglial activation in the brain of the subject at a second time point, wherein:

if the level of microglial activation at the second time point is greater than the level of microglial activation at the first time point, the treatment for depression is modified. The step of treating may be performed prior to or in between the steps of measuring microglial activation

[0073] In the above method, if the level of microglial activation remains constant over the two time points, this may be an indication that the treatment for depression is preventing or inhibiting the chronological advancement of depression, including neuroprogression. Alternatively, if the level of microglial activation at the second time point is greater than the first measurement, then it is contemplated that the treatment be modified to reduce or halt the increase microglial activation, which is reflective of the chronological advancement of the disease.

[0074] The measurement at the second time point may be performed at any time after the first measurement at the first time point, for example, without limitation, 1 month, 6 months, 1 year, 5 years, 10 years, or at any time in between. For example, in the event that one or more medications is newly administered, increased in dose, increased in timing frequency or changed in another manner during the course of treating a subject, it is preferable that the time between measurements be sufficient so as to allow the new dosage regimens to take effect or normalize in the subject. Preferably this is two weeks, 4 weeks, 1 month or any other suitable time period.

[0075] In an embodiment of the invention, treating the subject comprises administering medication as described above and throughout.

[0076] Further, modifying a treatment for depression may comprise modifying the dose, or adding or removing the administration, of one or more medications in the treatment. Modifying the dose also may involve increasing the dose of the medication, for example if it is believed that the effect of a medication is unsuitable for a desired therapeutic effect, or decreasing the dose of a medication, for example if adverse side effects are being observed. Adding or removing medications may permit the selection of a more effective combination of medications for attenuating depression symptoms or treating disorders such as MDD. For example, the subject may be currently being administered one but may benefit more from a combination of medications, for example, but not limited to two, three or more medications.

[0077] It is further contemplated that any combination of the medications described above are contemplated in the context of modifying the treatment for depression.

[0078] In a separate embodiment or as a further embodiment of the method described above, the method comprises counseling and/or monitoring the subject. Counseling may be an important part of treatment and can aid in coping with feelings, solving problems and changing behavior patterns that may contribute to depression symptoms. Counseling may include, but without wishing to be limiting, behavioral therapy, cognitive therapy, cognitive-behavioral therapy, interpersonal therapy, and solution-focused therapy and may be offered by a psychiatrist, psychologist, social worker, counselor or other therapist. Monitoring may include, without limitation, follow-up meetings to monitor symptoms, or institutionalization should such measures be deemed necessary by a qualified medical practitioner. Monitoring may be performed by evaluating the subj ect's self-rated scales, such as the Patient Health Questionnaire (PHQ-9), Quick Inventory of Depressive Symptomatology - Self Report (QIDS-SR), or Beck's Depression Inventory (BDI), and tracking depressive symptoms, suicidality, treatment adherence and side effects from treatment. It can be determined if the current medications are still effective or if they are becoming less effective, requiring modification to the treatment. [0079] In an even further embodiment of the method, measuring the level of microglial activation in the brain of the subject at further time points is contemplated. This ensures that if an increase in the level of microglial activation is observed, the necessary measures can be taken to prevent a further increase, for example, modifying the treatment administered to the subject. [0080] According to an embodiment of the present invention, there is provided a method of determining a level of microglial activation in a subject and optionally treating the subject, the method comprising quantifying a duration of untreated depression by subtracting the age at which treatment for depression began from the age of onset of depression and adding any treatment-free period. A longer duration of untreated depression is indicative of an increased level of microglial activation.

[0081] In the context of the present invention, duration of untreated depression refers to a period of time that the subj ect is not undergoing treatment and is considered treatment-free.

[0082] In the context of the present invention, the age at which treatment for depression began includes the age at which the subject began a regular medication treatment regime, which can include the daily, bi-daily, or weekly intake of one or more antidepressants, antiinflammatory agents, inflammation modulators, or microglial function modulators. The regular medication treatment regime includes the occasional forgetfulness to take one or more doses of the one or more medication, for example, one, two, three or more doses of the one or more medication in a one-, two-, or more, week time frame. The treatment regime may be prescribed by a professional.

[0083] Age of onset includes the age at which the subject became depressed, ranging from mild to severe depression, for example, ranging from a mood disorder having one or more symptoms of depression to having major depressive disorder or the like. The depression may have been clinically diagnosed, or may not have been clinically diagnosed wherein the onset would be when the first symptoms of depression appeared.

[0084] In a further embodiment of the method, the any treatment-free period is 1 month or greater, 2 months or greater, 3 months or greater, 4 months or greater, 5 months or greater, 6 months or greater, 9 months or greater, or 1 year or greater.

[0085] The method may further comprise treating the subject, as described throughout.

[0086] In a further embodiment, the method further comprises determining a second level of microglial activation by performing a blood test, PET scan, or both.

[0087] The blood test comprises measuring one or more of [PGE2] and blood [PGD2] in a blood sample obtained from the subject, wherein a decrease in blood [PGE2], blood [PGD2], or both, compared to a healthy control is predictive of a treated depressed subject or a treated depressed subject that is treatment-resistant or treatment-refractory.

[0088] The PET scan comprises measuring translocator protein (TSPO) binding. In a further embodiment, the PET scan measures translocator protein total distribution volume. In yet a further embodiment, the PET scan is performed on grey matter regions of the brain.

[0089] The present invention further provides for a method of determining a length of untreated depression in a depressed subject, the method comprising measuring a level of microglial activation in the brain of the subject. Every 14-18% increase in the level of microglial activation compared to a healthy subject is indicative of 10 years of untreated depression.

[0090] To further illustrate the nature of the present invention, the following non- limiting examples are included. However, it will be readily apparent to a person skilled in the art that many solutions may be formulated, which nevertheless still fall within the scope of the present invention.

EXAMPLES

A) METHODS AND MATERIALS:

1) Determining Peripheral Prostaglandin Measures

[0091] Participants: There were three groups of participants. Group 1: Healthy

Controls (n=ll): Inclusion criteria are: (i) age 18-65; (ii) good physical health; (iii) non- cigarette smoking; (iv) negative urine pregnancy test at screening and scan days (for women); (v) negative urine screen for drugs of abuse. Exclusion criteria: (i) past or current diagnosis of axis I or axis II disorder as determined by the SCID I and SCID II for DSM IV⁽⁷⁴⁾; (ii) history of psychotropic medication use; (iii) history of neurological illness or autoimmune disorder. Group 2 (n=ll): Current major depressive episode (MDE) secondary to MDD receiving antidepressant treatment : Inclusion criteria are: (i) Age 18 to 65; (ii) good physical health with no active medical conditions; (iii) non-cigarette smoking; (iv) no past or current substance abuse or dependence with present substance abuse additionally ruled out with a negative drug screen; (v) negative urine pregnancy test at screening and scan days (for women); (vi) primary diagnosis of current major depressive episode (MDE) and major depressive disorder (MDD) verified by SCID for DSM IV⁽⁷⁴⁾; (vii) score greater than 19 on the 17 item HDRS at screening; (viii) Non-response to a clinical trial of at least one antidepressant given at appropriate clinical dose; (ix) presently taking an antidepressant at a standard clinical dose. Group 3 (n=16) Antidepressant Free MDE secondary to MDD: Inclusion criteria are: (i) DSM-IV diagnosis of current major depressive episode (MDE) and major depressive disorder (MDD) verified by SCID for DSM IV⁽⁷⁴⁾, and a psychiatric consultation (ii) early onset type MDD with first MDE prior to age 40 (iii) antidepressant free for at least six weeks (iv) score greater than 17 on the 17 item HDRS⁽⁷⁵⁾ at screening. Other exclusion criteria included concurrent active axis 1 disorder s⁽⁷⁴⁾, including current alcohol or substance dependence, MDE with psychotic symptoms, bipolar I or bipolar II disorder, and borderline or antisocial personality disorder.

[0092] Blood Prostaglandin Measurements: PGF2 in serum was measured using an

ELISA kit involving a monoclonal anti-PGF2 antibody and a PGF2-horseradish peroxidase conjugate (My Biosource.com, Southern California, San Diego, Human Prostaglandin F2 Elisa kit, MBS733881). PGE2 in serum was also measured using an ELISA kit involving a monoclonal anti-PGE2 antibody and horseradish peroxidase labelled PGE2 (rndsystems.com; Bio-Techne), Minneapolis, Minnesota, Prostaglandin E2 Assay, "Parameter" KGE004B, SKGE004B, PKGE004B). PGD2 in serum was measured using an ELISA kit involving a sandwich enzyme immunoassay technique (My Biosource.com, Southern California, San Diego, Human Prostaglandin D2 ELISA Kit, MBS700128). The detection range for PGD2 in serum with this latter kit is 23.5pg/ml to 1500pg/ml.

[0093] Brain Imaging: Each participant underwent one [¹⁸F]FEPPA PET scan conducted at the Research Imaging Centre at the Centre for Addiction and Mental Health, Toronto, Canada. For this, intravenous [¹⁸F]FEPPA⁽⁷⁶⁾ was administered as a bolus (mean ± SD, 180.5 ± 14.5 MBq or 4.88 ± 0.4 mCi). [¹⁸F]FEPPA was of high radiochemical purity (>96%) and high specific activity (119 ± 125 TBq/mmol). Manual and automatic (ABSS, Model #PBS-101 from Veenstra Instruments, Joure, the Netherlands) arterial blood samples were obtained to determine the ratio of radioactivity in whole blood to radioactivity in plasma, and the unmetabolized radioligand in plasma needed to create the input function for the kinetic analysis. ⁽⁶⁹⁾ The scan duration was 125 minutes following the injection of [¹⁸F]FEPPA. The PET images were obtained using 3D HRRT brain tomography (CPS/Siemens, Knoxville, TN, USA). All PET images were corrected for attenuation using a single photon point source, ¹³⁷Cs (Ti/2= 30.2 years, Eg = 662 keV) and were reconstructed by filtered back projection algorithm, with a HANN filter at Nyquist cutoff frequency. ⁽⁷²⁾

[0094] Each subject underwent a 2D axial proton density magnetic resonance scan acquired with a General Electric (Milwaukee, WI, USA) Signa 1.5 T magnetic resonance image scanner (slice thickness = 2mm, repetition time > 5 300 ms, echo time = 13 ms, flip angle = 90 degree, number of excitations = 2, acquisition matrix = 256 x 256, and field of view = 22cm). Regions of interest were automatically generated using the in-house software, ROMI, as previously described. ⁽⁷⁷⁾ Time activity curves were used to estimate TSPO VT using a two- tissue compartment model, which has been shown previously to be an optimal model to quantitate TSPO V_T with [¹⁸F]FEPPA PET.⁽⁶⁹⁾ [0095] The binding affinity of the second generation of radiotracers for TSPO, including [¹⁸F]FEPPA, is known to be affected by a co-dominantly expressed single nucleotide polymorphism (rs6971, C→T) in exon 4 of the TSPO gene.⁽⁷²' ⁷³⁾ High affinity binders (HAB, Alal47/Alal47) and mixed affinity binders (MAB, Alal47/Thrl47) account for >90% of the population in North America.⁽⁷²⁾ The polymorphism rs6971 was genotyped as described previously. ⁽⁷²⁾ To nullify the effect of genotype, TSPO VT in subjects with the MAB genotype was multiplied by a factor of 1.4, a proportion which results in equivalent values between subjects with MAB and HAB genotypes.

2. Determining TSPO VT Measures [0096] Participants: Fifty-one subjects with a current MDE secondary to MDD

(hereafter termed MDE subjects) and 30 healthy participants completed the study. One participant could not provide reliable information about previous medication use and was excluded resulting in 50 MDE subjects for analysis. Participants were recruited from the Toronto area community and a tertiary care psychiatric hospital (Centre for Addiction and Mental Health, Toronto, Canada). TSPO VT values from 20 MDE subjects and 25 healthy controls are from previously published data sets.^10a'^16a All were aged 18-72 (MDE participants, range = 18-68 years, Healthy controls, range = 19-72 years), non-smoking and in good physical health. None had a history of autoimmune disease nor reported any recent medical illness. MDE subjects had early onset MDD (first MDE prior to age 50). Health or MDE was confirmed using the Structured Clinical Interview for DSM-IV and consultation from a psychiatrist (JHM). Exclusion criteria for all subjects included being pregnant, breastfeeding, and history of neurological illness or injury. All participants underwent urine drug screening and women received a urine pregnancy test on the PET scan day. All subjects provided written informed consent after all procedures were fully explained. The protocol and informed consent forms were approved by the Centre for Addiction and Mental Health Research Ethics Board, Toronto, Canada.

[0097] Participants with MDE were administered the 17-item Hamilton Depression

Rating Scale (HDRS) at enrollment and on the PET scan day. For enrollment, a minimum score of 17 on the 17-item HDRS was required. MDE subjects that were medication-free reported not having taken anti-depressant medications for at least six weeks prior to the PET scan day and had a negative urine screen. MDE subjects taking medication reported receiving a stable dose of medication for at least four weeks prior to the PET scan day. Other exclusion criteria included concurrent alcohol or substance dependence, MDE with psychotic symptoms, bipolar disorder (type I or II), and borderline or antisocial personality disorder. All were free of acute medical illnesses for the previous two weeks and none had history of neurological illness, autoimmune disorder, severe hepatic or renal disease, gastrointestinal disease, ischemic heart disease, cerebrovascular disorder, or congestive heart failure. Years of untreated MDD were calculated as the age at which antidepressant medication was taken regularly minus the age of onset plus any medication-free period greater than six months which occurred after antidepressant medication was taken regularly. Total duration of MDD was the age of onset minus age at which PET scanning took place. Duration of antidepressant treatment was the total cumulative duration of antidepressant treatment. Additional measures taken are listed in Table 1.

[0098] Image Acquisition and Analysis: Each participant underwent one [¹⁸F]FEPPA PET scan conducted at the Research Imaging Centre at the Centre for Addiction and Mental Health, Toronto, Canada. For this, intravenous [¹⁸F]FEPPA^23a was administered as a bolus (mean ± SD, 184 7 ± 12 4 MBq). [¹⁸F]FEPPA was of high radiochemical purity (> 96%) and high specific activity (107· 8 ± 104· 8 TBq/mmol). Manual and automatic (ABSS, Model #PBS- 101 from Veenstra Instruments, Joure, The Netherlands) arterial blood samples were obtained to determine the ratio of radioactivity in whole blood to plasma, and to determine the unmetabolized radioligand in plasma for the input function for the kinetic analysis. The scan duration was 125 minutes following the injection of [¹⁸F]FEPPA. The PET images were obtained using 3D HRRT brain tomography (CPS/Siemens, Knoxville, TN, USA). All PET images were corrected for attenuation using a single photon point source, ¹³⁷Cs (Ti/₂ = 30 2 years, Eg = 662 keV) and were reconstructed by filtered back projection algorithm, with a HANN filter at Nyquist cutoff frequency.

[0099] Regions of interest were generated using the in-house software, ROMI, as previously described.^10a'^16a Time activity curves were used to measure TSPO VT using a two- tissue compartment model, which is an optimal model to quantitate TSPO VT with [¹⁸F]FEPPA PET. For the anatomical delineation of regions of interest (ROIs), all MDE subjects and 19 healthy underwent 2-dimensional axial proton density magnetic resonance imaging (MRI) acquired with a General Electric (Milwaukee, WI, USA) Signa 3-T MRI scanner (section thickness, 2 mm; repetition time, 6000 ms; echo time, 8 ms; flip angle, 90°; number of excitations, 1; acquisition matrix, 256 x 192; and field of view, 16 5 mm). 11 healthy participants underwent 2-dimensional axial proton density MRI acquired with a General Electric (Milwaukee, WI, USA) Signa 1 - 5-T MRI scanner (section thickness, 2 mm; repetition time, >5300 ms; echo time, 13 ms; flip angle, 90°; number of excitations, 2; acquisition matrix, 256 x 256; and field of view, 22 mm). A subset of subjects were scanned with both MRI machines to rule out bias due to delineation with ROM! The differences in MRI acquisitions resulted in near identical TSPO VT values and regional volumes in a sample undergoing both MRI (N = 12, R > 0-97, P < 0 001)^16a.

[00100] DNA Extraction and Polymorphism Genotyping: The binding affinity of the second generation of radiotracers for TSPO, including [¹⁸F]FEPPA, is known to be affected by a co-dominantly expressed single nucleotide polymorphism (rs6971, C→T) in exon 4 of the TSPO gene.^24a High affinity binders (HAB, Alal47/Alal47) and mixed affinity binders (MAB, Alal47/Thrl47) account for 90% to 95% of the population in North America. The polymorphism rs6971 was genotyped as described previously.^10a'^16a This genotype was included in the statistical analyses to account for variance related to its influence on [¹⁸F]FEPPA binding to TSPO. Low affinity binders (LAB, Thrl47/Thrl47) were excluded. [00101] Statistical Analysis: TSPO VT data were analyzed by multivariate analysis of covariance (MANCOVA) with TSPO Vrin PFC, ACC, and INS as the dependent variables and years of untreated MDE and genotype as independent predictor variables. A second MANCOVA model for the same dependent variables applied a different set of predictor variables which included duration of MDE, years of antidepressant exposure, and genotype. For both models an additional stepwise approach was taken to additionally assess the effect of age as an independent variable. Overall main effects were considered significant at the conventional P < 0 05. As secondary analyses, for each set of predictor variables in the aforementioned two models (i.e. total duration of untreated illness; total illness duration and duration of antidepressant exposure) analyses of covariance (ANCOVA) were also applied with TSPO VT in every brain region sampled as the dependent variables. [00102] In addition, years of untreated MDE was converted to a categorical variable by dividing at the median resulting in two MDE groups and the healthy controls. A multivariate analysis of variance was applied with TSPO VT in PFC, ACC, and INS as the dependent variables and categorization across the three groups (MDE untreated for long duration, MDE untreated for shorter duration, healthy) and genotype as the independent variables. To specifically assess whether regional TSPO VT was elevated in the MDE untreated for long duration versus the healthy controls, additional comparisons were carried out using the least significant difference procedure. In addition the effect of group on TSPO VT was assessed within every brain region applying analyses of variance with group and genotype as the predictor variables.

B. RESULTS:

1) Determining Peripheral Prostaglandin Measures

[00103] Serum Prostaglandin Levels Are Reduced in Treatment Resistant MDE Subjects

[00104] Previous data show that TSPO VT, an index of activated microglia is elevated in MDE. Microglial activation refers to a change in morphology in microglial cells such that their cell bodies enlarge, their dendrites thicken and/or they change into a shape like an amoeba. Without wishing to be limited by theory, during microglial activation, microglia might alter how they secrete cytokines and prostaglandins and that this could also be influenced by antidepressant treatment. Figure 1 shows that PGE2 levels in serum are reduced during MDE, and further reduced during antidepressant treated MDE. Figure 2 shows further that serum PGE2 levels are reduced and that serum PGD2 levels are further reduced. PGD2 levels are demonstrated as often being reduced because they were often below the level of detectability with the PGD2 assay.

[00105] Accordingly, the results herein suggest it is possible to differentiate some subjects with MDE, and subjects with MDE receiving antidepressant treatment from healthy controls by measuring PGE2, PGD2, or combinations of both in blood. For example, serum PGE2 levels below 1500pg/ml, 1400pg/ml, 1300pg/ml, 1200pg/ml, l lOOpg/ml, 1050pg/ml, lOOOpg/ml, 950pg/ml, 900pg/ml, 850pg/ml, 800pg/ml, 750pg/ml, 700pg/ml, are more likely to occur in untreated MDE as compared to healthy subjects and serum PGE2 levels below 1500pg/ml, 1400pg/ml, 1300pg/ml, 1200pg/ml, l lOOpg/ml, 1050pg/ml, lOOOpg/ml, 950pg/ml, 900pg/ml, 850pg/ml, 800pg/ml, 750pg/ml, 700pg/ml, 650pg/ml, 600pg/ml, 550pg/ml and 500pg/ml are more likely to occur in antidepressant treated MDE. Similarly PGD2 levels below 23.5pg/ml are more likely to occur in untreated MDE as compared to healthy and in treated MDE as compared to healthy. Hence serum PGE2 and/or PGD2 or other combinations of prostaglandin secretion may be applied as a method to differentiate MDE from healthy subjects or treated MDE from healthy subjects. For example, if subjects are being screened for enrolment in an antidepressant trial and a requirement is to have a diagnosis of treatment resistant MDE, a useful screening tool to verify the interview based information would be to measure prostaglandin levels such as PGE2 and PGD2 in blood and determine if they are low and similar to what is observed in treatment resistant MDE. Similarly, as a component of a diagnostic panel, measurement of prostaglandins in blood such as PGE2 and PGD2, could be included to aid in the diagnosis of MDE.

[00106] Microglia secrete prostaglandins and data presented here show that this is altered during MDD and is further altered during treatment of MDD. Serum level of both PGE2 and PGD2 are reduced in MDD and further reduced during treatment of MDD. PGD2 is often undetectable in the treated depressed subjects as compared to the healthy controls. Figure 2 shows data of both serum PGE2 levels and PGD2 levels (the latter as detectable or not).

[00107] The Ratio of PGE2 to PGF2a Is Predictive of Brain Inflammation in Treatment Resistant MDE Subjects

[00108] When microglia are activated, their morphology changes and their propensity to secrete cytokines and prostaglandins changes. Activated microglia, as measured by elevated TSPO VT, may occur in treatment resistant MDE. Microglia secrete prostaglandins. Figure 3 is a graph sampling depressed subjects currently receiving antidepressant treatment (people most likely eligible to receive an additional anti-inflammatory medication). The results suggest the ratio of serum PGE2 to PGF2alpha is predictive/indicative of brain inflammation (TSPO VT). A theoretical explanation could be that there is an effect of both disease and antidepressants on the relative secretion of prostaglandins and that the PET imaging measures activated microglia, a key source of such prostaglandins in the midst of both. [00109] The ratio of PGE2 to PGF2alpha measured in blood at values of 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 8.0, 10, 11, 12, 13, 14, 15, 20 may have practical uses since values around or above these numbers are frequently observed in those with greater levels of TSPO V_T. For example, subjects undergoing antidepressant treatment such as monoamine raising medications, such as serotonin and norepinephrine reuptake inhibitors, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, tricyclic antidepressants, dopamine reuptake inhibitors, monoamine receptor binding treatments (such as, but not limited to trazodone, mirtazapine, Vortioxetine, aripiprazole), who wish to know whether they have evidence of brain inflammation, could measure the ratio of blood PGE2 to PGF2alpha to ascertain whether this is likely or not. This information could be used as a predictor/indicator of treatment response to treatment interventions that are anti-inflammatory, such as a treatment that reduces microglial activation, or an inflammatory modulating treatment, which induces microglia or other inflammatory cells to function in a more curative or restorative manner, or a treatment that influences the downstream effects of inflammation, such as a prostaglandin synthesis inhibitor (like a COX-1 or COX-2 inhibitor), or monoamine oxidase B inhibitor. Measures of blood PGE2 to PGF2alpha might also be used as a monitoring approach to determine whether continuation of an antidepressant treatment would be helpful; since a high ratio of PGE2 to PGF2alpha indicates brain inflammation, it would indicate that active disease is present and that it would be important to continue such treatment. Antidepressant treatments could include monoamine raising medications, such as: serotonin and norepinephrine reuptake inhibitors, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, tricyclic antidepressants, dopamine reuptake inhibitors, monoamine receptor binding treatments (such as, but not limited to trazodone, mirtazapine, Vortioxetine, aripiprazole); or anti -inflammatory or inflammation modulating treatments that are intended to improve depressed mood.

2. Determining Neuroprogression in Depression

[00110] Demographics [00111] The MDE group was divided into those with 10 or more years of untreated MDD and those with 9 or less years of untreated MDD given the median of 10 years of untreated MDE, resulting in three groups as described in Table 1 below. There were no significant differences in sex (P = 0-39), genotype (P = 0 28), or BMI (P = 0 47) among the three groups. Within the two MDE groups, there were no significant differences in age at enrollment (P = 0-25), severity of depression (HAMD, P = 0 33), age of first MDE (P = 0 13), or number of MDEs (P = 0 51). Within the two MDE groups, those with longer durations of untreated depression had significantly lesser durations of anti-depressant treatment (P = 0 003). Most of the antidepressant exposure was to serotonin reuptake inhibitors as 30 of 31 people taking antidepressants were taking SRIs at the time of scanning.

[00112] Years of Untreated Illness as a Predictor of Regional TSPO VT

[00113] A MANCOVA with TSPO V_Tin PFC, ACC, and INS as the dependent variables found that years of untreated MDE, and genotype were highly significant independent predictor variables (years of untreated MDE, F_3;45 = 8- 5, P < 0 001; genotype, F_3;45 = 9 1, P < 0 001). ANCOVAs with regional TSPO VT values as the dependent variable found that the combination of these predictor variables accounted for approximately 50% of the variance in the three prioritized regions (duration of untreated illness Fi,₄₇ = 17 0 to 24· 1, P < 0· 001, overall model, F_2; 7 = 24-6 to 32 0, P < 0 001, R² = 0 51 to 0 58, R² _Adju_Sted = 0 49 to 0 56; see Figure 4) and that years of untreated MDD and genotype significantly predicted TSPO VT across all the grey matter regions sampled (see Table 2 below). Addition of age as a predictor variable to the models tested found it to be consistently non-significant.

[00114] Total Duration of Illness and Total Duration of Antidepressant Exposure as Predictors of Regional TSPO VT

[00115] Multivariate analysis of covariance (MANCOVA) with TSPO V_Tin PFC, ACC, and INS as the dependent variables found that duration of MDD, duration of antidepressant exposure, and genotype were highly significant independent predictor variables (duration of MDD, F_3;44 = 5 - 8, P = 0 002; duration of antidepressant exposure, F_3;44 = 3 1, P = 0 037; genotype, F_3;44 = 8-9, P < 0 001. ANCOVAs with regional TSPO V_T values as the dependent variable found that the combination of these predictor variables accounted for approximately TABLE 1. Demographic Characteristics of Participants

Depressed Depressed Healthy Effect of

(< 10 years untreated) (> 10 years untreated) Controls Group

Characteristics (N = 25) (N = 25) (N = 30)

N % N % N % P

Female 16 64 15 60 14 47 0-39^d

TSPO Genotype^a 0-28^e

HAB 16 64 21 84 22 73

MAB 9 36 4 16 8 27

Current AD Treatment¹³ 20 80 11 44 Na na 0 009^d

Mean SD Mean SD Mean SD P

Age, year 31 - 8 10 3 37 1 11 0 33 -2 13 -2 0-25^e

BMI 25 -3 4-9 23 -9 4- 8 24-9 2-9 0-47^e

HDRS Score 21 -6 3 -4 20-5^c 4.4c Na na 0-33^f

Age of First MDE, year 17 0 8 1 14 0 5 0 Na na 0 13^f

MDEs, number 6-2 6-6 7-2 3 0 Na na 0- 51^f

Previous AD Treatment, year 9- 8 8-7 3 -3 5 -7 Na na 0 003^f

Years of Untreated MDD, year 4-2 2- 5 18-6 9 0 Na na <0 001^f ^a Single nucleotide polymorphism rs6971 of the TSPO gene known to influence [¹⁸F]FEPPA binding: HAB, high affinity binders; MAB, mixed affinity binders.

b All medicated subjects, except one, were taking a serotonin reuptake inhibitor.

c 17-item Hamilton Depression Rating Scale (HDRS); scores derived on the day of scanning. Missing data in one subject.

d Pearson chi-squared test.

e Analysis of variance.

f T-test between two groups.

Abbreviations: AD, antidepressant; BMI, body mass index; MDD, major depressive disorder; MDE, major depressive episode; N, number; na, not applicable; SD, standard deviation; TSPO, translocator protein.

TABLE 2. Duration of Untreated Major Depressive Disorder is Associated with TSPO VT across Grey Matter Regions

Effect (ANCOVA)

Duration of Untreated MDD Genotype R²

Region of Interest Fl,47 P Fl,47 P Unadjusted Adjusted

PFC 16-96 <0001 2306 <0 001 051 0-49

MPFC 22-28 <0001 27-49 <0 001 0-57 0-55

VLPFC 14-55 <0001 24-93 <0 001 051 0-49

DLPFC 13-42 0001 20-51 <0 001 0-47 0-45

OFC 13-48 0001 29-57 <0 001 0-53 051

ACC 2413 <0001 27-79 <0 001 0-58 0-56

Insula 21-92 <0001 25-98 <0 001 0-56 0-54

Temporal Cortex 23-84 <0001 31 15 <0 001 0-59 0-57

Parietal Cortex 21-51 <0001 29-59 <0 001 0-57 0-55

Occipital Cortex 20-60 <0001 33-32 <0 001 0-59 0-57

Hippocampus 7-69 0008 18-33 <0 001 0-40 0-38

Thalamus 18-54 <0001 26-26 <0 001 0-54 0-52

Dorsal Putamen 21-60 <0001 33-76 <0 001 0-59 0-57

Dorsal Caudate 15-47 <0001 29-39 <0 001 0-54 0-52

Ventral Striatum 11-50 0001 18-40 <0 001 0-44 0-42

For each region, analysis of covariance was conducted applying TSPO VT as the dependent variable, and duration of untreated MDD and the rs6971 genotype that influences binding of second generation radiotracers for TSPO as the predictor variables. R² refers to the proportion of variance attributable to the model, one adjusted for number of predictors.

Abbreviations: ACC, anterior cingulate cortex; ANCOVA, analysis of covariance; DLPFC, dorsolateral prefrontal cortex; HAB, high-affinity binding; MAB, mixed-affinity binding; MDD, major depressive disorder; MPFC, medial prefrontal cortex; OFC, orbitofrontal cortex; PFC, prefrontal cortex; TSPO VT, translocator protein density measured by distribution volume; VLPFC, ventrolateral PFC.

50% of the variance in the three prioritized regions (total duration of MDD, F_{1 ;4}6 = 9 4 to 15 4, P < 0 001 to P = 0 004; total duration of antidepressant exposure, Fi,₄₆ = 7-2 to 9 2, P = 0 004 to P = 0 01; overall model, F_3;46 = 14 0 to 17-9, P < 0 001, R² = 0 48 to 0 - 54, R²Adj_usted = 0 44 to 0 51) and that duration of MDD, duration of antidepressant exposure, and genotype significantly predicted TSPO V_T across all the grey matter regions sampled (see Table 3 below). The relationship between TSPO VT in the PFC, ACC, and INS with duration of antidepressant exposure is illustrated in Figure 5 in which both are linear predictors of similar magnitude, but opposite effect. Addition of age as a predictor variable to the models tested found it to be consistently non-significant. [00116] Comparison of MDD with Long Duration of Untreated Illness, MDD with Short

Duration of Untreated Illness and Healthy Subjects

[00117] MDD subjects were subdivided into those with duration of untreated MDD greater than or equal to 10 years and those less than or equal to 9 years duration of untreated MDD. With the sample of healthy subjects, this created three groups: long duration of untreated MDD, shorter duration of untreated MDD, and health. A MANOVA with TSPO Vr in the three primary regions of interest as the dependent variables with group and genotype as the predictor variables showed a significant effect of each (group, F_3;75 = 9 0, P < 0 001; genotype, F_3;74 = 12-7, P < 0 001; also see Figure 3). Additional comparisons based on the least significant difference test showed significant differences between a long duration of untreated MDD as compared to health (group, F₃,₅₀ = 7 · 1 , P < 0 · 001 ; genotype, F₃,₅₀ = 7 · 2, P < 0 · 001 ; differences of 31 -2, 36-9 and 39 0% in the PFC, ACC, and INS respectively) and significant differences between long duration of untreated MDD and short duration of untreated MDD in all of these regions (F ₇ = 9-3, Fi,₄₇ = 11 2, Fi,₄₇ = 10 1; P = 0 004, P = 0 002, P = 0 003; differences of 28-8%, 33 1%, and 31 0% in the PFC, ACC, and INS respectively). ANOVAs with regional TSPO VT as the dependent variable in each of the grey matter regions sampled also found group to be a significant predictor, and that the least significant difference test indicated that the differences in TSPO VT between the long duration group and the other two groups were typically significant (see Figure 6, and Table 4 below). TABLE 3: Total Duration of Major Depressive Disorder and Antidepressant Exposure are Associated with TSPO VT Across Grey Matter Regions tilled (A L'UVA)

Duration of Antidepressant

Total Duration of MDD Exposure Genotype

Region of Interest Fl P Fl P Fl P Unadjusted Adjusted

PFC 9-36 0 004 7 16 0 ^■010 22-01 <0 001 0 ^■48 0 ^■44

MPFC 14-22 <0 001 10-53 0 ^■002 26-73 <0 001 0 ^■55 0 ^■52

VLPFC 7-35 0 009 7 06 0 ^■on 23-33 <0 001 0 ^■48 0 ^■44

DLPFC 7-92 0 007 6 02 0 ^■018 19-91 <0 001 0 ^■45 0 ^■41

OFC 8 16 0 006 8-82 0 ^■005 28 05 <0 001 0 ^■52 0 ^■49

ACC 15-4 <0 001 7-73 0 008 27-64 <0 001 0 ^■54 0 ^■51

Insula 11 -25 0 002 9-45 0 004 24-22 <0 001 0 ^■51 0 ^■48

Temporal cortex 12-64 0 001 10 00 0 003 28-97 <0 001 0 ^■55 0 ^■52

Parietal cortex 11 -22 0 002 12-17 0 001 27-31 <0 001 0 ^■55 0 ^■52

Occipital cortex 10-32 0 002 9-44 0 004 30-62 <0 001 0 ^■55 0 ^■52

Hippocampus 1 -60 0-212 5-63 0 ^■022 16 13 <0 001 0 ^■38 0 ^■34

Thalamus 8-22 0 006 9-37 0 004 23-82 <0 001 0 ^■50 0 ^■47

Dorsal putamen 10-64 0 002 11 -86 0 001 30-77 <0 001 0 ^■56 0 ^■54

Dorsal caudate 10-99 0 002 10-94 0 002 26-65 <0 001 0 ^■55 0 ^■52

Ventral striatum 10-99 0 002 10-94 0 002 28-65 <0 001 0 ^■55 0 ^■52

For each region, analysis of covariance was conducted applying TSPO VT as the dependent variable, and total duration of MDD, duration of antidepressant exposure and the rs6971 genotype that influences binding of second generation radiotracers for TSPO as the predictor variables. R² refers to the proportion of variance attributable to the model, one adjusted for number of predictors.

Abbreviations: ACC, anterior cingulate cortex; ANCOVA, analysis of covariance; DLPFC, dorsolateral prefrontal cortex; HAB, high-affinity binding; MAB, mixed-affinity binding; MDD, major depressive disorder; MPFC, medial PFC; OFC, orbitofrontal cortex; TSPO VT, translocator protein density measured by distribution volume; VLPFC, ventrolateral PFC.

TABLE 4. Regional TSPO VT Values Grouped by Years of Being Untreated and Diagnosis¹

Duration

Depressed Depressed ong

Healthy Controls Effect Short

Effect of Group °ί Uτntreated

(<10 untreated)(>10 untreated) N = 30 Genotype

Healthy

N = 25 N = 25 Untreated

Region of Interest Mean SD Mean SD Mean SD P P P P

PFC 10-2 2-5 131 3-3 100 2-8 <0001 <0001 <0001 0004

MPFC 9-9 2-6 130 3-3 9-4 2-6

<0001 <0001 <0001 0002

VLPFC 11-2 2-7 13-8 3-2 10-7 30 0001 <0001 0001 0011

DLPFC 101 2-4 130 3-3 101 2-9

0001 <0001 <0001 0005

OFC 10-6 30 135 3-3 10-3 2-9

<0001 <0001 <0001 0008

ACC 9-7 2-5 130 3-4 9-5 2-8

<0001 <0001 <0001 0002

Insula 10-3 2-6 135 3-5 9-7 30

<0001 <0001 <0001 0003

Temporal Cortex 10-2 2-5 13-6 3-4 10-3 31

<0001 <0001 <0001 <0001

Parietal Cortex 10-8 2-7 14-2 3-4 10-9 30

<0001 <0001 <0001 0001

Occipital Cortex 10-3 2-5 13-7 3-7 10-4 3-2

<0001 <0001 0001 0001

Hippocampus 9-6 3-2 11-7 3-2 91 31

0016 <0001 0005 0103

Thalamus 121 3-4 15-9 4-3 116 3-8

<0001 <0001 <0001 0005

Dorsal Putamen 8-6 2-3 118 31 8-3 2-5

<0001 <0001 <0001 <0001

Dorsal Caudate 80 2-1 10-3 2-7 7-6 2-4

<0001 <0001 <0001 0005

Ventral Striatum 91 2-9 11-7 3-3 8-6 2-7 0001 <0001 <0001 0021

aValues are expressed as mean (SD). Analyses of variance with regional TSPO VT as the dependent variable was done and the least significant difference test was applied towards differences in TSPO VT between the long duration group and the other two groups.

Abbreviations: ACC, anterior cingulate cortex; DLPFC, dorsolateral prefrontal cortex; MPFC, medial prefrontal cortex; SD, standard deviation; TSPO, translocator protein; TSPO VT, translocator protein density; OFC, orbitofrontal cortex; VLPFC, ventrolateral prefrontal cortex; yrs, years. HAB, high affinity binders (Alal47/Alal47); MAB, mixed affinity binders (Alal47/Thrl47) of the single nucleotide polymorphism rs6971 of the TSPO gene.

3. Anti-inflammatory administration to depressed subjects

[00118] In a study, 2 out of 11 subjects with very low TSPO V_T (<11.5) had a response to celecoxib, a COX2 inhibitor, versus 5 out of 10 subjects who have a TSPO VT of 11.5 or greater had a clinically meaningful response to celecoxib. In the present context, a clinically meaningful response refers to as an increase in to the number of points on the Hamilton Depression Rating Scale, for example, 1, 2, 3, 4, 5 or more points.

[00119] Discussion

[00120] It has been found that greater TSPO VT in the grey matter regions sampled, including prefrontal cortex, anterior cingulate cortex, and insular cortex is associated with greater duration of untreated MDD. The second main finding was that total duration of illness predicted greater TSPO VT and duration of antidepressant exposure was a similar magnitude negative predictor of TSPO VT throughout the grey matter regions sampled. This demonstration of progressively abnormal neuropathology with more advanced illness has major implications for conceptualizing neuroprogression in MDD and the relationship of TSPO VT to antidepressant exposure is important for understanding the impact of antidepressants on microglial activation in the clinical setting.

[00121] A compelling issue is that there is great potential for more precise staging of MDD given the increase in TSPO V_T of 14% to 18% per decade. The DSM V addresses progression by differentiating between single and multiple episode. However, duration of untreated MDD may reflect a more precise measure since elevated TSPO VT is best interpreted as reflecting greater levels of microglial activation. The increased TSPO expression in mammalian brain after diverse paradigms like stroke, neurotoxins, and lipopolysaccharide administration,^25a'^26a has a temporal course that closely matches the increased expression of other markers of microglial activation rather than astroglial activation. Microglial activation is a well-established quantitative response to brain injury in neurodegenerative conditions. In addition, induction of microglial activation itself is implicated in the generation of depressive behaviors in humans and rodents through mechanisms such as the diversion of tryptophan metabolism to kynurenine, stimulation of the hypothalamic-pituitary-adrenal axis and glucocorticoid receptor resistance.^27a Thus, given that microglial activation is a marker of advancing disease and is implicated in depressive symptoms it may be advantageous to clinically investigate and categorize chronologically advanced MDD differently.

[00122] The data presented herein indicates that the yearly increase of TSPO VT is halted by antidepressants. Most of the antidepressant exposure was SRI which provides a context for interpreting literature reports that SRI reduce induction of microglial activation in rodents and in cell culture.^18a'^19a Such findings in rodent and in vitro models might have been interpreted to indicate that short durations of SRI exposure reduce microglial activation vigorously and rapidly, but this study demonstrates that the yearly accumulation of greater TSPO VT merely stops: duration of antidepressant exposure is only a negative predictor in the model when total duration of MDD is applied, rather than when duration of untreated MDD is applied. Moreover, in the model inclusive of both total duration of MDD and antidepressant exposure, the linear predictors are of similar magnitude in the opposite direction. It may be advantageous to develop superior strategies to target microglial activation such as minocycline administration or vagal nerve stimulation for which rapid effects are reported in rodents,^28a'^29a although presently it is not yet known whether these interventions, once modified for dose and method of administration, would effectively reduce microglial activation in humans.

[00123] Additional nuances and limitations should be considered in the context of these findings. First, this study is cross sectional in design and does not indicate the etiology of the greater TSPO VT with duration of untreated illness, so rather than being a marker of progressively worse disease as it is typically interpreted in investigations of neurodegenerative diseases,^30a it is theoretically possible that those with the most persistent illness had the highest TSPO VT from the inception of illness. Even so, the clinical predictors represent useful indicators of elevated TSPO VT. Second, in regards to therapeutic strategies for greater TSPO VT, although microglial activation is associated with depressive behaviors, microglial activation also spans a broad range of function of which some may be useful, hence modulating microglial function rather than reducing microglial activation may be the best therapeutic strategy. Third, investigations of the role of TSPO are ongoing, hence, while it is clear that increased microglial activation is most strongly associated with greater TSPO expression, ' minor contributions from astroglial activation are possible and in the future, other mechanisms that influence TSPO expression may be identified.

[00124] In summary, this is the first study to evaluate a marker of microglial activation in relation to neuroprogression in major depressive disorder. There a strong relationship between increasing duration of untreated illness and greater TSPO V_T, indicating that at the level of this marker, chronologically advanced illness is extremely different from the early phase of this disease. Duration of antidepressant treatment was associated with cessation of the ongoing elevation in TSPO V_T, indicating that commonly prescribed antidepressants may halt, but may not reduce microglial activation in clinical settings.

[00125] All citations are hereby incorporated by reference.

[00126] Various embodiments of methods of assessing a level of brain inflammation and methods of assessing the chronological advancement of depression have been described. The above-described embodiments are intended to be examples, and alterations and modifications may be effected thereto by those of ordinary skill in the art without departing from the spirit and scope of the teachings.

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Claims

What is claimed is:

1. A method of assessing a level of brain inflammation in a subject, the method comprising: measuring a level of microglial activation in the brain of the subject,

wherein the level of microglial activation is a measure of brain inflammation.

2. A method of assessing the chronological advancement of depression, including neuroprogression, in a depressed subject, the method comprising:

measuring a level of microglial activation in a brain of the subject at a first time point; and measuring the level of microglial activation in the brain of the subject at a second time point,

wherein an increase in the level of microglial activation at the second time point compared to the first time point is indicative of the chronological advancement of depression, including neuroprogression, in the subject.

3. The method of claim 2, wherein the subject has major depressive disorder (MDD), major depressive episode(s), or a mood state that is characterized by one or more symptoms of depression.

4. The method of claim 3, wherein the subject is undergoing treatment for depression, is undergoing treatment for depression but is treatment-resistant or treatment-refractory, or is not undergoing treatment for depression.

5. The method of claim 1, wherein microglial activation is measured by a blood test, positron emission tomography (PET) scan, or both.

6. The method of claim 5, wherein the blood test comprises:

wherein a decrease in blood [PGE2], blood [PGD2], or both, in the blood sample compared to a healthy control is indicative of a depressed subject.

7. The method of claim 6, wherein the blood [PGE2] in the sample is below 1500pg/ml, or more preferably below 1400pg/ml, or still more preferably below 1300pg/ml, or still more preferably below 1200pg/ml, or still more preferably below HOOpg/ml, or still more preferably below 1050pg/ml, or still more preferably below lOOOpg/ml, or still more preferably below 950pg/ml, or s till more preferably below 900pg/ml, or still more preferably below 850pg/ml, or still more preferably below 800pg/ml, or still more preferably below 750pg/ml, or still more preferably below 700pg/ml, or still more preferably below 650pg/ml, or still more preferably below 600pg/ml, or still more pref cerably below 550pg/ml, or most preferably below 500pg/ml.

8. The method of claim 7, wherein blood [PGD2] in the sample obtained from the test subject is below 23.5pg/ml.

9. Akit for assessing a level of brain inflammation in a depressed subject, the kit comprising: one or more diagnostic agents for quantifying PGE2, PGD2 or a combination thereof in a blood sample of the depressed subject; and

one or more components, diluents or buffers for use of the diagnostic agents or for practicing any analytical assay to quantify PGE2, PGD2 or a combination thereof.

10. A method of preventing or inhibiting the chronological advancement of depression, including neuroprogression, in a subject, the method comprising:

wherein:

if the level of microglial activation at the second time point is equivalent or less than the level of microglial activation at the first time point, the treatment for depression is maintained, or if the level of microglial activation at the second time point is greater than the level of microglial activation at the first time point, the treatment for depression is modified,

and wherein the step of treating may be performed prior to or in between the steps of measuring microglial activation.

11. The method of claim 10, wherein microglial activation is measured by a blood test, positron emission tomography (PET) scan, or both.

12. The method of claim 10, wherein modifying the treatment for depression comprises modifying the dose, or adding or removing the administration, of one or more medications in the treatment.

13. The method of claim 10, wherein the method further comprises counseling and/or monitoring the subject.

14. The method of claim 10, wherein the method further comprises:

measuring the level of microglial activation in the brain of the subject at one or more additional time points.

15. A method of determining a level of microglial activation and optionally treating a subject, the method comprising:

quantifying a duration of untreated depression by subtracting the age at which treatment for depression began from the age of onset of depression and adding any treatment-free period, wherein a longer duration of untreated depression is indicative of an increased level of microglial activation, and optionally further treating the subject.