Role of phospholipase a2 in orofacial pain and synaptic transmission 2

Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.4. Discussion This part of the study aimed to examine sPLA2-III expression profile in the rat CNS and its effects on exocytosis to elucidate its role in nociceptive transmission. Expression of sPLA2-III was analyzed in entire regions of the rat brain including olfactory bulb, striatum, cortex, hippocampus, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar spinal segments using real-time RT-PCR. Among these regions, sPLA2-III showed the highest level of mRNA expression in the brainstem and cervical, thoracic and lumbar spinal segments suggesting that this sPLA2-III plays a role in the ascending pain pathway. Similarly, using Western blot analysis, protein expression sPLA2-III was observed to be at the highest level in homogenates of the spinal segments, consistent with the findings in the Chapter 2. The immunoreactivity of sPLA2-III was also observed to be high in the spinal trigeminal nucleus and dorsal horn of the spinal segments. This protein was localized to dendrites, the postsynaptic terminal in neurons, reflecting the possible role of sPLA2-III in nociception. These findings are supported by previous studies which showed that sPLA2-III was expressed in the neuronal cells, such as peripheral neuronal fibres, spinal dorsal root ganglia neurons and cerebellar Purkinje cells (Masuda et al. 2008). The findings above supported the results in the Chapter which showed an increased sPLA2-III mRNA expression in the CM region after peripheral inflammation induced by facial CA injection, indicating the role of this isozyme in nociception. sPLA2-III has also been associated to various diseases such as 101 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS atherosclerosis and cancer (Murakami et al. 2005; Sato et al. 2008) thus leading it to be a likely target for drugs. sPLA2-III could regulate nociception in mammals as subcutaneous injection of PLA2-related peptide isolated from the bee venom led to nociceptive paw flinches (Chen et al. 2006; Chen and Lariviere 2010). Both sPLA2-III that was expressed in mouse skin and Tg mice overexpressing human sPLA2-III could lead to spontaneous development of inflammation in the skin. This was accompanied by entry of neutrophils and macrophages and augmented levels of pro-inflammatory cytokines, chemokines and PGE2 were observed (Sato et al. 2009). Studies have also shown the association of sPLA2-III with microvascular endothelium in human tissues after inflammation and ischemic injury as sPLA2-III expression is induced by pro-inflammatory cytokines (Murakami et al. 2005). Moreover, the enzyme transcription was upregulated by the production of cytokines such as IL-1 α, TNF- α and interferon-γ by immune cells at the site of inflammation (Farroqui et al. 2002). sPLA2-III when injected into cervical dorsolateral funiculus, it showed dose-dependent demyelination, and axonopathy, thus indicating its role in pain transmission (Titsworth et al. 2007). After the N- and C-terminal of sPLA2-III are proteolytically cleaved, it allows for the production of sPLA2 only domain in almost all cell types (Murakami et al. 2005). sPLA2 domain alone is adequate to conduct catalytic activity and generate PGE2 in various cell types (Murakami et al. 2005), contributing to inflammatory pathway. However, the mechanism by which this enzyme is proteolytically processed in cells is still not known (Murakami and Kudo 2004). Even so, it has been suggested that the proteolytic cleavage of the enzyme is 102 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS likely to occur prior to the secretion of the sPLA2 domain of the sPLA2-III into the extracellular space (Murakami et al. 2005). The presence of either C or N or both terminals of the sPLA2-III results in the cytoplasmic localization of the enzyme, whereas in the absence of both, the enzyme was found to be distributed mainly on the plasma membrane of the cell (Murakami et al. 2003). Upon the release of the enzyme into the extracellular space, the sPLA2-III will act upon plasma membrane of neighboring cells, giving rise to various catalytic products of membrane phospholipids, possibly suggesting its function in nociceptive transmission. In this study, it was observed in PC-12 cells that there was an increase in capacitance measurement indicating exocytosis, under voltage clamp conditions after addition of sPLA2-III in this study. Exocytosis induced by sPLA2-III was attenuated by pretreatment with MBCD suggesting dependence on integrity of lipid rafts on the cell membrane which are membrane domains in which neurotransmitter signaling could take place via clustering of receptors and components of receptor-activated signaling cascade (Allen et al. 2007). Moreover, treatment of cells with thapsigargin and recording in zero Ca2+ conditions, or treatment of cells with lanthanum chloride and recording in external solution containing Ca2+, resulted in attenuation of sPLA2-III induced exocytosis.sPLA2-III also induced rise in [Ca2+]i, and this effect was abolished in cells which had been pre-incubated with MBCD, or cells that were pre-treated with lanthanum chloride and recorded in external solution containing Ca2+. Together, the results indicated that neurotransmitter release triggered by sPLA2III could possibly be dependent on the integrity of cholesterol rich lipid domains 103 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS on cellular membranes and a rise in [Ca2+]i concentration, through influx via Ca2+ channels. Various studies have also shown the involvement of bee venom in exocytosis in tissues and PC-12 cells (Kurihara et al. 1986; Ray et al. 1997; You et al. 2008). Evidence from previous study showed that application of PLA2 activators, melittin and mastoparan on rat anterior pituitary gland cells induced hormonal release (Kurihara et al. 1986). The bee venom test, using bee venom or components of bee venom is also a well established experimental animal model for pain to show its role in nociceptive transmission (Mogil et al. 1999; Lariviere and Melzack 2000; Lariviere et al. 2002). Peripheral bee venom injection induced changes in synaptic transmission of the anterior cingulate cortex which plays an important role in the affective dimension of pain (Gong et al. 2010), indicating the important role of bee venom in nociceptive transmission. The findings in this section of the study, together with the results from the Chapter 2, it suggests the role of sPLA2-III in nociceptive transmission. Besides sPLA2-III, sPLA2-IIA also participated in neurotransmission in the hippocampal cultured neurons and PC-12 cells (Wei et al. 2003). Therefore in the next chapter, the role of sPLA2-IIA in nociception by elucidating its expression and localization in the CNS will be further explored. 104 Chapter Role of Group IIA sPLA2 in nociception CHAPTER ROLE OF GROUP IIA sPLA2 IN NOCICEPTION 105 Chapter Role of Group IIA sPLA2 in nociception 4.1. Introduction Mammalian sPLA2 consists of enzymes which have low molecular masses (13–19 kDa) (Yang et al. 2009) and their require mM range of Ca2+ for their activities (Six and Dennis 2000; Valentin and Lambeau 2000; Farooqui and Horrocks 2004). These isozymes include sPLA2-IB, IIA, IIC, V and X and are involved in multiple physiological and pathological processes via release of AA from membrane phospholipids or specific binding to membrane receptors (Suzuki et al. 2000). Total sPLA2 activity is highest in the medulla oblongata, pons, and hippocampus, moderate in the hypothalamus, thalamus, and cerebral cortex, and low in the cerebellum and olfactory bulb (Thwin et al. 2003). sPLA2 has a well-established role in inflammation and inflammatory diseases (Nevalainen et al. 2000) and thus, inhibition of sPLA2 would prevent the formation of inflammatory eicosanoids prior to the COX reaction in which PLA2 is the rate limiting precursor in AA production (Schaefers et al. 1996). Therefore its blockade should eliminate the need for COX-1 versus COX-2 specificity in antiinflammatory therapeutics. sPLA2 activity is elevated in several body fluids of patients with acute pancreatitis (Makela et al. 1990). Synovial fluid from arthritic joints of rheumatic patients contains sPLA2-IIA (Kramer et al. 1989; Seilhamer et al. 1989) while the total PLA2 activity and sPLA2-IIA is also enhanced in bronchoalveolar lavage fluids from patients with adult respiratory distress syndrome (Kim et al. 1995a). TNFα, IL-1, and LPS were shown to induce sPLA2IIA production in cultured astrocytes and direct injection of LPS into brain increased sPLA2-IIA mRNA (Oka and Arita 1991). 106 Chapter Role of Group IIA sPLA2 in nociception sPLA2-IB mRNA is present in the human brain and its distribution is mainly neuronal. It is highly expressed in the cerebral cortex and hippocampus. sPLA2IB mRNA has been identified in rat cerebral neurons, cells of neurodermal origin (Kolko et al. 2005; Kolko et al. 2007) and the normal spinal cord by quantitative PCR (Lucas et al. 2005). sPLA2-IIA is ubiquitously expressed in the rat brain (Molloy et al. 1998) and spinal cord (Lucas et al. 2005). sPLA2-IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata (Shirai and Ito 2004). Increased sPLA2-IIA mRNA and immunoreactivity is present in the rat brain after cerebral ischemia and in Alzheimer’s disease (Lauritzen et al. 1994; Lin et al. 2004; Moses et al. 2006; Adibhatla and Hatcher 2007). Moreover, sPLA2-IIA is upregulated by cytokines including TNF-α and IL1α/β (Adibhatla and Hatcher 2007). sPLA2-IIC mRNA expression is low in peripheral tissues but is found in all parts of the brain (Molloy et al. 1998) and spinal cord (Lucas et al. 2005). sPLA2-V mRNA is expressed in the cerebral cortex and hippocampus while present at low levels in most areas of the brain (Molloy et al. 1998; Kolko et al. 2006) and is also identified in the rat cerebellum by immunostaining and in situ hybridization histochemistry and localized in Bergmann glia cells (Shirai and Ito 2004). sPLA2-V is also present in the rat spinal cord as shown by quantitative PCR and Western blot analysis (Lucas et al. 2005; Svensson et al. 2005). sPLA2-X is expressed in the rat brain and in primary neuronal cell cultures, and is expressed at low levels in the cerebral cortex (Kolko et al. 2006). Having shown in the previous chapters that sPLA2-III was the particular isozyme which was significantly increased after orofacial pain induced 107 Chapter Role of Group IIA sPLA2 in nociception by facial CA, the important role of sPLA2 isoforms with strong secretory signals in nociceptive transmission could not be neglected especially when it has been shown that addition of sPLA2-IIA which has strong secretory signal was able to induce exocytosis in hippocampal neurons (Wei et al. 2003). In this part of the study the expression profile of multiple sPLA2 isoforms in the rat CNS was elucidated with focus on sPLA2-IIA in the brainstem and spinal cord. 108 Chapter Role of Group IIA sPLA2 in nociception 4.2. Materials and methods 4.2.1. Real-time RT-PCR sPLA2 isoforms with strong secretory signal (P > 0.6), and which could be released as a neuromodulator were selected for analyses using SignalP 3.0 Server, which predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms. These included sPLA2-IB (P = 0.82), -IIA (P = 0.67), -IIC (P = 0.7), and -X (P = 0.63). sPLA2-V (P = 0.4) was also analysed for comparison. Four uninjected adult male Wistar rats weighing approximately 200 g each were used for this portion of the study. The rats were anesthetized with an intraperitoneal injection of ketamine and xylazine cocktail and killed by decapitation. Adequate measures were taken to minimize pain and discomfort, and procedures involving rats were approved by the Institutional Animal Care and Use Committee. Various regions of the brain including olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar spinal segments were quickly removed and immersed in RNAlater (Ambion, TX,USA), snap frozen in liquid nitrogen and kept at -80 oC till analyses. Total RNA was extracted and isolated using TRizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNeasy1 Mini Kit (Qiagen, Inc., CA, USA) was used to purify the RNA. The samples were then reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, CA, USA). Real-time PCR amplification was then carried out in the 7500 Real time PCR system using TaqMan1 109 Chapter Role of Group IIA sPLA2 in nociception Universal PCR Master Mix, and sPLA2-IB (Applied Biosystems ID Rn00580896_m1), sPLA2-IIA (Rn00580999_m1), sPLA2-IIC (Rn01520676_m1), sPLA2-V (Rn00567782_m1), sPLA2-X (Rn00691350_m1) or rat β-actin probes, according to the manufacturer’s instructions. All reactions were carried out in triplicate. The amplified transcripts were quantified using the comparative CT method (Livak and Schmittgen 2001), with the formula for relative fold change = 2ΔΔCT. The fold change for each sPLA2 subgroup expression in different parts of the brain was observed. 4.2.2. Western blot analysis Four uninjected Wistar rats of 200 g each were used for this portion of the study. The animals were anesthetized and killed as described above. The olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic, and lumbar spinal segments were dissected out and homogenized in 10 volumes of ice-cold buffer containing 0.32 M sucrose, mM Tris–HCl, pH 7.4, mM EDTA, and 0.25 mM dithiothreitol. After centrifugation at 1000 g for 30 min, the supernatant was collected and protein concentrations in the preparation measured using the BioRadprotein assay kit (Bio-Rad Laboratories). Total proteins were resolved in 10% SDS polyacrylamide gels under reducing conditions and electrotransferred to a PVDF membrane (Amersham Pharmacia Biotech). Nonspecific binding sites on the PVDF membrane were blocked by incubation with 5% non-fat milk for h. The PVDF membrane was then 110 Chapter Role of Group IIA sPLA2 in nociception incubated overnight with sPLA2-IIA antibody diluted 1:5000 in TTBS at oC. The antibody was prepared by immunizing a rabbit with purified recombinant rat sPLA2-IIA as described earlier (Nyman et al. 2000). After washing with TTBS, the membrane was incubated with horseradish peroxidase conjugated anti-rabbit immunoglobulin IgG (Amersham) for h at room temperature. The protein was visualized with an enhanced chemiluminescence kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. 4.2.3. Immunohistochemistry A further three uninjected male Wistar rats were used for this portion of the study. The animals were anesthetized as described above and perfused through the left ventricle with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and sectioned coronally at 100 mm using a Vibratome. The sections were washed for h in PBS. They were then incubated overnight with sPLA2-IIA antibody (diluted to 1:500 in 0.15 M NaCl in 0.05 M Tris buffer, pH 8.6, and containing 1% bovine serum albumin). This antibody has been characterized previously, and used for immunohistochemical labeling of sPLA2-IIA in the rat ileum (Nyman et al. 2000). The sections were then washed in PBS and incubated for h at room temperature in a 1:200 dilution of biotinylated horse anti-rabbit IgG (Vector). The sections were reacted for h at room temperature with an avidin-biotinylated horseradish peroxidase complex, and visualized by treatment for in 0.05% DAB solution in TBS 0.05% hydrogen peroxide. The color reaction was stopped 111 Chapter Role of Group IIA sPLA2 in nociception with several washes of TBS. Some sections were mounted on glass slides and lightly counterstained with methyl green before coverslipping. The remaining sections were processed for electron microscopy. Control sections were incubated with preimmune rabbit serum (1:500 dilution) instead of primary antibody. 4.2.4. Electron microscopy Electron microscopy was carried out by subdissecting the immunolabeled spinal cord sections into smaller portions that included the dorsal or ventral horn. The sections were soaked in PBS overnight at oC before processing. Following which, the sections were post fixed in 1% osmium tetroxide, pH 7.4 for h at room temperature. This was followed by washing the sections with PBS two times for 5-10 at room temperature. The sections were then dehydrated through a series of ascending ethanol series at room temperature. Finally the sections were embedded in araldite and left to polymerise at 60 oC for 24 h. Semi-thin sections were obtained from the first µm of the sections, mounted on Formvar-coated copper grids, and stained with lead citrate. They were viewed using a Jeol 1010EX electron microscope. 112 Chapter Role of Group IIA sPLA2 in nociception 4.3. Results 4.3.1. Real-time RT-PCR (Fig. 2.4.1., Table 2.4.1.) The expression of various sPLA2 isoforms were normalized to the lowest level of message among the isoforms in the different brain regions (apart from sPLA2-X, which was nearly undetectable), i.e., the value for sPLA2-IB in the cerebral neocortex, to give an indication of relative expression in different parts of the brain and spinal cord. sPLA2-IB expression was low throughout the CNS, sPLA2-IIC expression was relatively high in the cerebral neocortex, hippocampus and thalamus/hypothalamus; sPLA2-V showed greatest expression in the olfactory bulb and cerebellum, and moderate expression in the spinal cord, and sPLA2-X was expressed at very low levels in the CNS. sPLA2-IIA showed greatest expression in the brainstem and spinal cord; the brainstem and spinal cord also contained highest levels of this isoform (Fig. 2.4.1., Table 2.4.1.). 113 Chapter Role of Group IIA sPLA2 in nociception Olfactory Bulb Hippocampus Thalamus/hypothalamus Brainstem Thoracic 50 Cortex Striatum Cerebellum Cervical Lumbar Relative Expression 40 30 20 10 A sPLA2-IB sPLA2-IIA sPLA2-IIC sPLA2-V sPLA2-X Fig.2.4.1. A: Real-time RT-PCR analysis of differentially expressed sPLA2 subgroups in the CNS. The values were normalized to the lowest level of message among the isoforms in the different brain regions, i.e., the value for sPLA2-IB in the cerebral neocortex, to give an indication of relative expression in different parts of the brain and spinal cord. sPLA2-IB expression was low throughout the CNS, sPLA2-IIC expression was relatively high in the cerebral neocortex, hippocampus and thalamus/hypothalamus; sPLA2-V showed greatest expression in the olfactory bulb and cerebellum, and moderate expression in the spinal cord, and sPLA2-X was expressed at very low levels in the CNS (not visible in this scale). sPLA2-IIA showed greatest expression in the brainstem and spinal cord; conversely, the brainstem and spinal cord contained highest levels of this isoform. Data represent the mean and standard deviation of four rats. 114 Chapter Role of Group IIA sPLA2 in nociception 10 Relative Expression B ry to c lfa O bu lb C x te or H c po ip pu am s Th us am l a m tu rt ia S C e er llu be m a Br in em st C l ca vi r e or Th ic ac ba m Lu r Fig.3.4.1. B: Real-time RT-PCR analysis of sPLA2-IIA in the various regions of CNS. The values were normalized to the lowest level of message in the different brain regions, i.e., the value for sPLA2-IIA in the striatum, to give an indication of relative expression in different parts of the brain and spinal segments. sPLA2-IIA showed highest level of expression in the hindbrain. Data represent the mean and standard deviation of four rats. 115 Chapter Role of Group IIA sPLA2 in nociception Table 2.4.1. Comparison of sPLA2 isoforms mRNA and protein expression in rat CNS between previous reports and current findings. sPLA2 isoforms sPLA2-IB sPLA2-IIA sPLA2-IIC sPLA2-V sPLA2-X Olfactory Bulb Cortex Hippocampus Striatum Thalamus/ hypothalamus Cerebellum Brainstem Spinal cord Detected Expression (mRNA/protein) References N.S. + N.S. + +++ + N.S. + +++ + N.S. + N.S. + N.S. + N.S. + N.S. + +++ + N.S. + N.S. + N.S. + N.S. N.S. +++ + mRNA, protein mRNA mRNA mRNA Kolko et al., 2007 Molloy et al., 1998 Lucas et al., 2005 Current data N.S. N.S. N.S. + + N.S. N.S. + + N.S. N.S. + + N.S. N.S. + + N.S. N.S. + + N.S. N.S. + +++ N.S. N.S. ++ N.S. +++ +++ +++ mRNA mRNA protein mRNA, protein Molloy et al., 1998 Lucas et al., 2005 Svensson et al., 200 Current data N.S. N.S. + +++ N.S. +++ + N.S. +++ +++ N.S. + ++ N.S. +++ + N.S. + + N.S. + N.S. +++ + mRNA mRNA mRNA Molloy et al., 1998 Lucas et al., 2005 Current data + N.S. N.S. N.S. +++ + N.S. N.S. +++ + +++ N.S. N.S. +++ + + N.S. N.S. N.S. + + N.S. N.S. N.S. + + N.S. N.S. N.S. +++ + N.S. N.S. N.S. + N.S. +++ +++ N.S. ++ mRNA mRNA protein mRNA, protein mRNA Molloy et al., 1998 Lucas et al., 2005 Svensson et al., 200 Kolko et al., 2006 Current data N.S. + + + ++ + N.S. + N.S. + N.S. + N.S. + N.S. + mRNA, protein mRNA Kolko et al., 2006 Current data +++, high expression; ++, moderate expression; +, low expression; N.S. not studied. 116 Chapter Role of Group IIA sPLA2 in nociception 4.3.2. Western blot analysis (Fig. 2.4.2.) The antibody to sPLA2-IIA detected a band at 14 kDa in homogenates from brainstem and various parts of spinal cord consistent with the expected molecular weight of sPLA2-IIA. A dimerised band of sPLA2-IIA with molecular weight of approximately 30 kDa was also detected in the cervical, thoracic and lumbar spinal segments. High level of expression was detected in the brainstem, cervical, thoracic and lumbar spinal segments whilst low level of expression was found in the olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus and hypothalamus and cerebellum (Fig. 2.4.2.). β-actin ~30kDa 14kD a 10 Fig.2.4.2. Western blot analyses of sPLA2-IIA protein expression in different parts of the rat CNS. The antibody to sPLA2-IIA detects a band at 14 kDa in homogenates from brainstem and various parts of spinal cord consistent with the expected molecular weight of the active form of sPLA2-IIA. A dimerised band of sPLA2-IIA with molecular weight of approximately 30 kDa is also detected in the cervical, thoracic and lumbar spinal segments. High level of expression was detected in the brainstem (lane 7), cervical (lane 8), thoracic (lane 9) and lumbar (lane 10) spinal segments. In contrast, low level of expression was found in the olfactory bulb (lane 1), cerebral neocortex (lane 2), hippocampus (lane 3), striatum (lane 4), thalamus and hypothalamus (lane 5) and cerebellum (lane 6). 117 Chapter Role of Group IIA sPLA2 in nociception 4.3.3. Immunohistochemistry (Figs. 2.4.3., 2.4.4. and 2.4.5.) Sections incubated with sPLA2-IIA antibody showed very little labeling in the forebrain including the cerebral neocortex (Fig. 2.4.3A) and hippocampus (Fig. 2.4.3B). Little labeling was observed in thalamus (Fig. 2.4.3C) and light labeling was observed in Purkinje cells in the cerebellar cortex (Fig. 2.4.3D). CX HC B A CCX TH C D Fig.2.4.3. Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS. Very little labeling is observed in the cerebral neocortex (A) and hippocampus (B). Little labeling was observed in thalamus (C) and light labeling was observed in Purkinje cells in the cerebellar cortex (D). Abbreviations: CX, cerebral neocortex; HC, hippocampus; TH, thalamus; CCX, cerebellar cortex. Scale: 200 μm. 118 Chapter Role of Group IIA sPLA2 in nociception In contrast to the forebrain, dense labeling for sPLA2-IIA was detected in the brainstem and spinal cord. Dense labeling was observed in neurons of the central grey matter in the brainstem (Fig. 2.4.4A), spinal trigeminal nucleus (Fig. 2.4.4B) and facial motor nucleus (Fig. 2.4.4C). Dense sPLA2-IIA labeling was also observed in the neurons in the dorsal- (Fig. 2.4.4D) and ventral horns of the spinal cord (Fig. 2.4.4E). Control sections incubated with preimmune rabbit serum showed only background labeling (Fig. 2.4.4F). 119 Chapter Role of Group IIA sPLA2 in nociception V CG * * B A * VII * D C VH E DH * VH (C) * F Fig.2.4.4. Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS. Dense staining is observed in the central gray (A, asterisk), spinal trigeminal nucleus (B, asterisk), facial motor nucleus (C, asterisk), dorsal horn of the cervical spinal cord (D, asterisk), ventral horn of the lumbar spinal cord (E, asterisk). (F) Section through the ventral horn of lumbar spinal segment, incubated with preimmune rabbit serum, showing background labeling (asterisk). Abbreviations: CG, central gray matter; V, spinal trigeminal nucleus; VII, facial motor nucleus; DH, dorsal horn of spinal cord; VH, ventral horn of spinal cord; VH (C), ventral horn of spinal cord (control). Scale: 200 μm. 120 [...]... an indication of relative expression in different parts of the brain and spinal segments sPLA2-IIA showed highest level of expression in the hindbrain Data represent the mean and standard deviation of four rats 115 Chapter 4 Role of Group IIA sPLA2 in nociception Table 2. 4.1 Comparison of sPLA2 isoforms mRNA and protein expression in rat CNS between previous reports and current findings sPLA2 isoforms... Scale: 20 0 μm 118 Chapter 4 Role of Group IIA sPLA2 in nociception In contrast to the forebrain, dense labeling for sPLA2-IIA was detected in the brainstem and spinal cord Dense labeling was observed in neurons of the central grey matter in the brainstem (Fig 2. 4.4A), spinal trigeminal nucleus (Fig 2. 4.4B) and facial motor nucleus (Fig 2. 4.4C) Dense sPLA2-IIA labeling was also observed in the neurons in. .. expression in the olfactory bulb and cerebellum, and moderate expression in the spinal cord, and sPLA2-X was expressed at very low levels in the CNS (not visible in this scale) sPLA2-IIA showed greatest expression in the brainstem and spinal cord; conversely, the brainstem and spinal cord contained highest levels of this isoform Data represent the mean and standard deviation of four rats 114 Chapter 4 Role of. .. highest levels of this isoform (Fig 2. 4.1., Table 2. 4.1.) 113 Chapter 4 Role of Group IIA sPLA2 in nociception Olfactory Bulb Hippocampus Thalamus/hypothalamus Brainstem Thoracic 50 Cortex Striatum Cerebellum Cervical Lumbar Relative Expression 40 30 20 10 0 A sPLA2-IB sPLA2-IIA sPLA2-IIC sPLA2-V sPLA2-X Fig .2. 4.1 A: Real-time RT-PCR analysis of differentially expressed sPLA2 subgroups in the CNS The... CNS, sPLA2-IIC expression was relatively high in the cerebral neocortex, hippocampus and thalamus/hypothalamus; sPLA2-V showed greatest expression in the olfactory bulb and cerebellum, and moderate expression in the spinal cord, and sPLA2-X was expressed at very low levels in the CNS sPLA2-IIA showed greatest expression in the brainstem and spinal cord; the brainstem and spinal cord also contained highest... level of expression was found in the olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus and hypothalamus and cerebellum (Fig 2. 4 .2. ) β-actin ~30kDa 14kD a 1 2 3 4 5 6 7 8 9 10 Fig .2. 4 .2 Western blot analyses of sPLA2-IIA protein expression in different parts of the rat CNS The antibody to sPLA2-IIA detects a band at 14 kDa in homogenates from brainstem and various parts of spinal cord... (lane 2) , hippocampus (lane 3), striatum (lane 4), thalamus and hypothalamus (lane 5) and cerebellum (lane 6) 117 Chapter 4 Role of Group IIA sPLA2 in nociception 4.3.3 Immunohistochemistry (Figs 2. 4.3., 2. 4.4 and 2. 4.5.) Sections incubated with sPLA2-IIA antibody showed very little labeling in the forebrain including the cerebral neocortex (Fig 2. 4.3A) and hippocampus (Fig 2. 4.3B) Little labeling was... Real-time RT-PCR (Fig 2. 4.1., Table 2. 4.1.) The expression of various sPLA2 isoforms were normalized to the lowest level of message among the isoforms in the different brain regions (apart from sPLA2-X, which was nearly undetectable), i.e., the value for sPLA2-IB in the cerebral neocortex, to give an indication of relative expression in different parts of the brain and spinal cord sPLA2-IB expression was... sPLA2-IIA detected a band at 14 kDa in homogenates from brainstem and various parts of spinal cord consistent with the expected molecular weight of sPLA2-IIA A dimerised band of sPLA2-IIA with molecular weight of approximately 30 kDa was also detected in the cervical, thoracic and lumbar spinal segments High level of expression was detected in the brainstem, cervical, thoracic and lumbar spinal segments whilst... observed in thalamus (Fig 2. 4.3C) and light labeling was observed in Purkinje cells in the cerebellar cortex (Fig 2. 4.3D) CX HC B A CCX TH C D Fig .2. 4.3 Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS Very little labeling is observed in the cerebral neocortex (A) and hippocampus (B) Little labeling was observed in thalamus (C) and light labeling was observed in Purkinje cells in . range of Ca 2+ for their activities (Six and Dennis 20 00; Valentin and Lambeau 20 00; Farooqui and Horrocks 20 04). These isozymes include sPLA 2 -IB, IIA, IIC, V and X and are involved in multiple. after orofacial pain induced Chapter 4 Role of Group IIA sPLA 2 in nociception 108 by facial CA, the important role of sPLA 2 isoforms with strong secretory signals in nociceptive transmission. contained highest levels of this isoform. Data represent the mean and standard deviation of four rats. 0 10 20 30 40 50 sPLA2-IB sPLA2-IIA sPLA2-IIC sPLA2-V sPLA2-X Relative Expression Olfactory