1. Introduction
1.1 Preface
Pain can be extremely painful and debilitating with no time limit, which is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” – International Association for the Study of Pain (IASP).
According to American Pain Society, 45% of Americans suffer from persistent pain at some point in their lives. Pain is the most common reason Americans seek for medical care. With improvement of medical care, reliability requirement for pain manager has become more critical.
Therefore, in order to manage unpleasant feeling caused by pain, the modulation of the pain inhibition has to be widely investigated. Among all, endogenous opioid peptides play a crucial role as the most effective autoregulated analgesics.
In recent years, in vivo functional neuroimaging, such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) providing intact whole-brain functional responses, are frequently used to investigate the antinociception effect in brain. Using these imaging modalities, it has been proved that peripheral noxious stimulation would increase regional cerebral blood flow (rCBF) and cerebral metabolic rate of glucose (CMRglc) in the brain. However, the delicate relationship between pain and endogenous analgesic is still ambiguous, and especially affects the interpretation of imaging data.
1.2 Outline of brain opioid systems
Opioids are the most widely used and extremely effective analgesic drugs to treat moderate to severe pain. Endogenous opioid system plays a critical role in modulating a large number of sensory, motivational, emotional, and cognitive functions. There is an abundance of evidence to suggest the existence of several families of opioid peptides in the mammalian central nervous system (Zadina et al., 1997; Darland et al., 1998). The most commonly recognized endogenous opioids are dynorphins, enkephalins and endorphins, which are all post-transcriptionally generated by their precursors (Table. 1-1-1). For example, Pro-opiomelanocortin’s (POMC) is the pro-peptide of endorpine.
After the post-translational cleavage of the proenkephalin (PENK), PENK can generate four copy of methionine-enkephalin (M-Enk) and one copy of leucine-enkephalin (L-Enk) (Hughes et al., 1975).
The analgesic ability of an opioid must works by binding to opioid receptors. Opioid receptors are a group of G-protein coupled receptors, separating into three main types: µ, κ and δ. Each of these receptors is differentially distributed in several neuronal circuits (Table. 1-1-2). The µ receptors (MOR) are widely distributed in the rat brain, and mainly located in amygdala (Amyg), hippocampus (HIP), ventral dentate gyrus, presubiculum, nucleus accumbens (NAs), caudate putamen (CPu), thalamus (Th), habenula, interpeduncular nucleus, pars compacta of the substantia nigra (SN), superior and inferior colliculi, and raphe nuclei. The Kappa binding test showed not widely spread but densely distributed in the Amyg, olfactory tubercle (OT), NAs, CPu, medial preoptic area, hypothalamus (HT), median eminence, periventricular thalamus, and interpeduncular nucleus. Besides, the binding of δ was restricted only to few cerebral areas, such as anterior cingulate cortex (ACC), neocortex, Amyg, OT, NAs, and CPu.
coupling, in most cases leading to the decrease of cAMP concentration in target cells. The downstream signaling will promote the opening of K+ channels and inhibit the opening of voltage gated Ca 2+ channels, and then decrease the firing of target cells, mostly GABAergic interneurons.
Although three types of opioid receptors are all considered to involve in analgesia, based on pharmacological and clinical observation, MOR are generally recognized as the major site of interaction in clinical analgesics, such as morphine (Appendix 1: the structure of morphine). MOR mediation of much morphine-induced analgesia often company with reduced blood pressure, nausea, euphoria, and decreased respiration.
Pro-pepetide Peptide(s)
POMC Endorpine
Endormophine-1 Endormophine-2 PENK Met-enkephalin
Leu-enkephalin
PDYN Dynorphin A
Dynorphin B Pro-Orphanin FQ Orphanin FQ
Table 1-1-1. Endogenous opioid peptides and its precursor
NC-IUPHAE Recommended opioid receptors Presumed Endogenous Ligands
μ, mu, MOR β-Endorpine
Endormophine-1 Endormophine-2 Enkephalin
δ, delta, DOR β-Endorpine
Enkephalin
κ, kappa, KOR Dynorphin A
Dynorphin B α-Neoendorpine
Table 1-1-2. Types of opioid receptors and its endogenous ligands
1.3 Opioid systems and dopaminergic neuron
As reported by Choi et al., stimulating the dopamine D1/D5 systems causes cerebral vasodilation, whereas stimulating the D2/D3 systems, in contrast, causes vasoconstriction (Choi et al., 2006). In another aspect, while opioid exerts its effect directly from the binding of opioid receptors, still there are numerous secondary interaction thought to be mediated by its target—
GABAergic cells (Johnson and North, 1992). Acting on cerebral dopamine neurotransmitter systems, opioids are considered to modulate both the nigrostriatal and the mesolimbic dopaminergic neuronal transmission (Fig. 1-2-1). Several pharmacological studies have shown that selective agonism of MOR using morphine and methadone increase turnover and metabolism of dopamine in brain (Clouet and Ratner 1970). After morphine administration, both the concentration of dopamine precursor DOPA and corresponding metabolites are accumulated (Moleman et al., 1984). Most of the studies suggested that µ-opioid agonists activate dopaminergic neurons via inhibiting the GABAergic neurons that serve as an inhibitory control in the firing of dopaminergic neurons (Henry et al., 1992; Johnson and North, 1992).
The opioid-enhanced of dopamine release in the mesolimbic dopaminergic pathway is widely recognized. Studies conducted by in vivo microdialysis indicate that binding of MOR increases synaptic dopamine levels in the nucleus accumbens as well as in the prefrontal cortex in animals (Di Chiara and Imperato, 1988; Spanagel et al., 1990; Devine et al., 1993). Electrophysiological evidences on local drug injections have shown that peripherally administered µ-opioid agonists generally enhance the firing frequency of dopaminergic neurons in the ventral mesencephalon (Gysling and Wang, 1983; Hommer and Pert, 1983). Furthermore, the infusion of an enkephalinase
However, the evidence for opioid-induced dopamine release effect in the nigrostriatal pathway is not as clear as in the mesolimbic pathway. Some claimed that the application of MOR agonists evaluated the dopamine release in the CPu (Di Chiara and Imperato, 1988); while, other experiments contrarily reported that the activation of MOR might have an inhibitory effect on dopamine release in the CPu (Piepponen et al., 1999). The specific mechanism and regulation is still not well understood.
Figure 1-2-1. Cerebral dopaminergic neurotransmitter system: (1) The nigrostriatal pathway:
originating from the substantia nigra (SN) to the caudate and putamen (CPu) and (2) The mesolimbic pathway: originating from the ventral tegmental area (VTA) to the nucleus accumbens (NAs) and then ascending to the prefrontal cortex.
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1.4 Pain and functional imaging
In traditional pain research, scientists often apply electrophysiology such as single-unit recording and field potential recording to record signals of neuronal activity. It involves measurements of voltage change or electrical current flow on neurons, and particularly action potential activity. A major limitation for traditional neuronal signal recording techniques in small animals is the restricted recording areas, the undetectable depth, and the limited signal sensitivity.
Also, it is not easy to simultaneously obtain the global signals induced by painful stimuli among a large number of nuclei in different positions. These traditional electrophysiological methods may have a better temporal resolution but do not have the capability to record the whole brain signals.
Functional images, such as PET and fMRI, can provide intact functional signals by using brain mapping techniques. These imaging techniques provide non-invasive, in vivo measurement of the cerebral hemodynamics as well as molecular processes (Phelps, 2000; Logothetis et al., 2001;
Heeger and Ress, 2002; Jacobs et al., 2003; Zanzonico, 2004; Heiss and Herholz, 2006).
In 1990, Ogawa proposed the blood-oxygenation-level-dependent (BOLD) fMRI method based on cerebral hemodynamics (Ogawa et al., 1990). And then in 1992, Kwong applied this theory to depict the activation of visual and motor cortex (Kwong et al., 1992). Several groups also used the BOLD theory to illustrate the brain function (Bandettini et al., 1992; Frahm et al., 1992; Ogawa et al., 1992). Since then it comes a brand-new era of neuroscience in fMRI. The principle of BOLD is based on the existence of paramagnetic substance, such as deoxy-hemoglobin, which could interfere with magnetic field and further reduce the intensity of signal. Generally speaking, the local cellular activity is supported by blood flow, and the consumption of glucose is directly related to synaptic activity (Schwartz et al., 1979; Fox et al., 1988; Chugani et al., 1991; Sibson et al., 1998). Therefore, these mechanisms could roughly infer that the blood flow increase is related to the activation of neurons (Branston, 1995; Cohen et al., 1996). The activation of neurons would further induce arteriolar vasodilatation and over-compensate the regional cerebral blood flow (CBF) to bring more
oxygen and glucose. Hence, it causes the ratio of paramagnetic deoxy-hemoglobin in blood to be reduced when the neural activity increases in brain, so more intensive signal can be obtained in the activated regions (Hyder et al., 2001; Logothetis et al., 2001; Heeger and Ress, 2002).
PET is another noninvasive imaging technique which is one of the important tools in clinical diagnosis and research. The image of PET is formed by detecting the distribution of positron emitter.
Positrons are given off during the decay of the nuclei of specific radioisotopes. When a positron annihilate with an electron, after a succession of collision and deceleration , two gamma rays having the same 511keV energy, at an angle of 180 ± 0.25 degree will be produced. The gamma rays leave the body and are detected by the PET scanner. The information is then reconstructed by a mathematic algorithm and fed into a computer to be converted into a complex picture of the activities. Since microPET is designed for small animals which could conduct preclinical studies and facilitate basic researches, it can eliminate the need of sacrificing animals by enabling noninvasive, longitudinal, and serial studies. As noted, microPET can be used for serial assessment of metabolic function of individual, awake rats with a minimal degree of invasiveness, and therefore, has the potential for use in the study of brain disorders and repair. To conduct the scan, a short-lived radioactive tracer isotope, which has been chemically incorporated into a metabolically active molecule, is injected into the living subject. Therefore, the function being studied during a PET scan determines which radiopharmaceutical is used. Recently, the molecule most commonly used in the observation of the brain’s glucose metabolism is fluorodeoxyglucose (18F-FDG). Since incorporation of deoxyglucose reflects the metabolic activity of the brain (Sokoloff et al., 1977), microPET studies permit assessment of brain metabolic activity in conscious animals, followed by scanning in sedated animals.
1.5 Objectives
Although nociception studies have widely used in vivo functional imaging to track neuronal activities, whether the endogenous neurotransmitters would affect the function signals under nociceptive stimulation is still obscure. It has been commonly known that extracellular dopamine concentration is regulated by opioids, which are highly implicated in the antinociceptive response.
Also, it has been reported that different dopamine subtypes would cause vascular coupling effect and affect the functional signal in rat brain. Therefore, it is reasonable to speculate the possibility that opioids may modulate dopamine systems and affect the imaging signals.
The goal of the present thesis was to map the opioids intervention in functional imaging of pain.
First, microPET is used to examine whole brain metabolic responses as well as antinociceptive effect of opioids. Further assessments were performed by phMRI to address the effect of opioid system in the generation of fMRI signals. With the aid of multimodality imaging, the results could extensively interpret neurovascular modulations under antinoceptive regulation of opioids.
2. Whole Brain Imaging of Morphine Antinociception Effect in Rats using
18F-FDG microPET
The present experiment used high resolution microPET to evaluate brain glucose metabolism following peripheral noxious stimuli. 18F-FDG was used as a radiotracer and was intravenously injected following 5% formalin stimuli at the left hindpaw. Animals were then returned to their cage for 45 min uptake during the conscious state. The results demonstrated that an injection of formalin into the hindpaw resulted in significant activation (p<0.05) in the bilateral ACC, motor cortex (M), primary somatorysensory cortex (S1), secondary somatorysensory cortex (S2), insular cortex (IC), CPu, HIP, periaqueductal gray (PAG), Th, contralateral auditory cortex (AC),
visual cortex
(VC), Amyg, and HT. Among the measured areas, clear lateralization (p<0.05) was observed in S1, VC, and HT. Compared to the formalin group, pretreatment of lidocaine (4 mg/kg, i.v.) significantly decreased 18F-FDG uptake in bilateral ACC, M, S1, S2, IC, AC, CPu, HIP, PAG, Th, HT, and ipsilateral Amyg. In addition, pretreatment of morphine (10 mg/kg, i.v.) suppressed 18F-FDG uptake in bilateral ACC, M, S1, S2, IC, AC, CPu, HIP, PAG, Th, HT, ipsilateral VC, and Amyg. The present protocol allowed identification of the brain levels involving in pain modulation, and also provide evidence of each brain area under antinociceptive control of morphine and lidocaine.2.1 Introduction
Exploring the nociceptive responses in brain is an important issue. In recent years, new pain-relief drugs and technologies have been sequentially developed to alleviate human suffering of pain. These advancements were indispensable to the animal testing stage and thus reinforce the importance of the pain study in small animals. The mechanisms and circuitries underlying nociception in the brain are extremely complex, involving not only the sensory responses to noxious stimuli, but also cognitive and emotional factors (McMahon and Koltzenburg, 2005). This makes it difficult to conclusively identify the brain areas that specifically process and respond to nociceptive signals. Imaging approaches such as PET and fMRI can provide intact functional mapping of the brain and allow measurement of the responses in different brain areas simultaneously (Phelps et al., 1979; Ogawa et al., 1990).
In the studies of nociception, formalin is one of the most commonly used chemical stimulants for generating nociceptive responses since it evokes a chemical pain without the influence of other sensory modalities (Tjolsen et al., 1992). However, during fMRI experiments, anesthesia is usually needed to achieve animal sedation and reduce motion artefacts. This tackles a very difficult technical obstacle that is to image nociceptive responses in anesthetized animal. Therefore, the present study aimed to use high resolution microPET with the aid of MRI to investigate the rat brain nociceptive response to formalin stimuli. In order to accurately illustrate the brain nociceptive maps, anesthesia was not applied during the uptake period, thus minimizing the possible confounding which may influence the induced nociception. Compared to the previous small animal nociceptive imaging studies, the glucose metabolism instead of indirect hemodynamic compensation was measured (Morrow et al., 1998; Tuor et al., 2000; Shah et al., 2005; Shih et al., 2008b; Shih et al., 2008e). In addition, the effect of pain-relief drugs, morphine and lidocaine, were examined under formalin induced nociception. The present study establishes a non-invasive glucose metabolism measuring protocol which can be used for illustrating the nociceptive responses in the conscious rat
brain. Moreover, antinociceptive effects reflected to neuronal activation in each cerebral region could be acquired for further investigation.
2.2 Materials and methods
2.2.1 Subjects
Nineteen adult male Wistar rats (8–10 weeks old; weighing approximately 250–300 g;
National Laboratory Animal Center, Taiwan) were used in the present study. The animals were housed in a well-controlled environment with a 12:12-hour light:dark cycle and constant humidity and temperature. Rats were housed in plastic cages at three animals per cage with free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee, National Taiwan University, College of Medicine.
2.2.2 Imaging experiments
Seven rats were used to produce the formalin-induced nociceptive maps. The 18F-FDG was used as a radiotracer to reveal brain glucose metabolism. Each rat was initially lightly anesthetized using ether, and 0.5 ml of 18F-FDG with an activity of 1.0–1.2 mCi was administered intravenously via the tail lateral vein, after which the rat was returned to its cage in a quiet environment for 45 min uptake in the conscious state. Following the uptake, the rat was lightly anesthetized using 1.5%
isoflurane and fixed in a custom-built stereotaxic head holder by two ear bars and an incisor fixer so as to minimize motion artifacts (Shih et al., 2007). The body temperature was maintained using a warming lamp whose light field was restricted to avoid additional visual stimulation. MicroPET imaging (R4, Concorde Microsystems/Siemens, Knoxville, TN, USA) was performed for 30 min, with the images reconstructed using the MAP algorithm (Qi et al., 1998). After 1 week, 50 µl of 5%
formalin was injected into the left hindpaw prior to 18F-FDG injection. The imaging procedures were identical to those described above.
Another two groups containing six rats each were used to evaluate the effects of lidocaine and morphine. The drugs were given prior to the formalin stimulation, followed by an identical imaging protocol. Rats were intravenously injected with 4 mg/kg lidocaine in one group and 10 mg/kg
morphine in the other group.
MRI anatomical images were captured using a 4.7-T Biospec 47/40 spectrometer to define the brain margin. A 72-mm volume coil was used as the RF transmitter, and a 2-cm quadrature surface coil placed on the head was used as the receiver. A T2-weighted scout image was taken in the mid-sagittal plane to localize the anatomical position by identifying the anterior commissure (bregma –0.8 mm). T2-weighted template images were then acquired using RARE sequence with a repetition time of 4000 ms, echo time of 80 ms, field of view of 2.56 cm, slice thickness of 1.2 mm, number of excitation of 2, and an acquisition matrix of 256×128 (zero-filled to 256×256).
2.2.3 Data analysis
Images were analyzed using PMOD (PMOD Technologies, Adliswil, Switzerland) and a custom-built ISPMER system (Shih et al., 2007). MicroPET images were initially coregistered among the subjects using a mutual-information algorithm and then averaged to generate the incidence images. A pixel value in incidence images represents the averaged percentage injected dose per gram (%ID/g) of an experimental group, where a higher pixel value indicates a greater number of rat responses consistent with the given task (Fig.2-2-1).
The statistical analysis was based on the %ID/g values sampled from different brain structures of each rat. Repeated-measures ANOVAs were used to examine whether formalin stimulation induced metabolic changes in the corresponding brain regions in both hemispheres, with the significance level set at P<0.05. Factorial ANOVAs were used to assess differences in 18F-FDG uptake among the groups with formalin stimulation alone, formalin stimulation with lidocaine
Fig. 2-2-1. 18F-FDG-microPET and T2-weighted MR images were registered and fused with the rat atlas by custom-built ISPMER system. These templates provide anatomical alignment and corresponding ROI. The locations of the three images are (A) bregma +0.7 mm, (B) -1.8 mm, and (C) -7.8 mm.
2.3 Results
2.3.1 Formalin-induced nociceptive maps
The present study used 18F-FDG microPET to elucidate the nociception-induced glucose metabolic changes in the brains of conscious rats. In order to further improve the accuracy with which anatomical locations were determined, microPET and MRI images were coregistered and fused with a digital atlas of the rat brain (Paxinos and Watson, 1998). This method allowed regions of interest to be selected based on clear spatial references (Fig. 2-3-1). Averaged formalin-induced metabolic maps overlaid on the MRI images are shown in Fig. 2-2-1. The averaged 18F-FDG uptake in the hindlimb area of the S1 (S1HL) was higher on the contralateral side than on the ipsilateral side. Statistical comparisons of the 18F-FDG uptake in the control and formalin groups are shown in Fig. 2-3-2. Repeated-measures ANOVAs with Fisher’s post-hoc tests indicated the presence of significant activations in the bilateral ACC, M, S1, S2, IC, VC, CPu, HIP, PAG, Amyg, Th, and HT, whereas no changes were evident in muscle tissue (Mu). In addition, clear lateralization was only observed in S1 and HT (Fig. 2-3-2).
2.3.2 Antinociceptive effects of lidocaine and morphine
The effects of antinociceptive drugs are shown in Fig. 2-3-3. These reduced the brain 18F-FDG uptake, with no clear lateralized differences evident in the sensory cortices. Factorial ANOVAs with Fisher’s post-hoc tests showed that 18F-FDG uptake in the bilateral ACC, M, S1, S2, IC, VC, CPu, HIP, PAG, Th, Amyg, and HT was lower for pretreatment with lidocaine and morphine than for formalin stimulation alone. Among the measured areas, 18F-FDG uptake in the CPu was higher for morphine treatment than for lidocaine treatment.
Fig. 2-3-1. Incidence 18F-FDG microPET maps overlaid on the MRI images showing the cerebral metabolic changes in three groups of rats, quantified as the averaged %ID/g. (A) Averaged response of seven rats subjected to left hindpaw formalin stimulation alone. (B) Averaged response of six rats subjected to left hindpaw formalin stimulation with lidocaine pretreatment. (C) Averaged response of six rats subjected to left hindpaw formalin stimulation with morphine pretreatment. Clear lateralization is only evident in (A), in which the 18F-FDG uptake is highest in the contralateral S1 and the responsive region match with that of the hindlimb region defined by the rat brain atlas. The image position was 1.8 mm posterior to the bregma.
Fig. 2-3-2. Formalin-induced metabolic increases and cerebral laterality. Repeated-measures
Fig. 2-3-2. Formalin-induced metabolic increases and cerebral laterality. Repeated-measures