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Modulation of nociceptive signalling on a spinal cord level under normal and pathological conditions
Pontearso, Monica ; Špicarová, Diana (advisor) ; Uchytilová, Eva (referee) ; Krůšek, Jan (referee)
Present pain therapies are often insufficient and poorly managed, mainly for neuropathic pain treatment. In the past years, research has provided a body of evidence on the association of nerve injury and inflammation with mobilized immune reactions. Many cytokines and hormones are released during injury or inflammation, and these factors regulate neuronal encoding of painful sensations. For example, in pre-clinical models, inflammatory cytokines cause or amplify nociception by enhancing neuron excitability. Besides increased excitability via direct neuronal sensitization, it has also been demonstrated that a reduction of synaptic inhibition (disinhibition) occurs naturally in the course of neuropathic and inflammatory pain states. The spinal cord dorsal horn (SCDH) is the first relay station of the ascending pain pathway and a crucial modulatory site of painful stimuli transmission. Three main aims characterize this doctoral thesis; each focused on the modulation of spinal nociceptive transmission by different molecules fundamental for pain processing. The first aim was to study the role of proinflammatory cytokine macrophage migration inhibitory factor (MIF) in nociceptive signalling following peripheral neuropathy induced by chronic constriction injury (CCI) of the sciatic nerve. The second aim...
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The role of SGIP1 protein interaction with cannabinoid receptor 1 in nociception under pathological conditions.
Dresslerová, Denisa ; Špicarová, Diana (advisor) ; Zímová, Lucie (referee)
The cannabinoid receptor type 1 (CB1) is a component of the endocannabinoid system and is abundantly expressed in the central nervous system. CB1 receptor has been extensively studied primarily due to its inhibitory role in neurotransmitter release from presynaptic terminals, and this process subsequently modulates postsynaptic currents and postsynaptic neuron excitation. Its interaction with the Src homology domain 3 growth factor 2 receptor binding protein (SGIP1) has recently been investigated in the context of nociception modulation (Hájková et al., 2016b). However, the role of this CB1 regulatory protein, SGIP1, in pathological pain conditions has not yet been described. Clarification of the role of SGIP1 interaction with CB1 receptor may help future research based on the use of cannabinoids as potential therapeutics. The thesis is structured into two parts, theoretical and practical. In the theoretical part of the thesis, knowledge of the given topic is summarized. First, pain is described from an anatomical-physiological perspective, followed by a description of the endocannabinoid system, where a part is dedicated to the CB1 cannabinoid receptor itself, and then another part is dedicated to the SGIP1 protein and its interaction with the CB1 receptor. In the practical part, the role of SGIP1...
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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee) ; Obšilová, Veronika (referee)
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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The role of regulatory protein SGIP1 in nociceptive synaptic transmission at the spinal cord level.
Mužík, David ; Špicarová, Diana (advisor) ; Lindovský, Jiří (referee)
Cannabinoid receptor 1 (CB1), abundantly expressed in the CNS, is a promising target for the pharmacological treatment of pathological pain conditions due to its function as an inhibitor of neurotransmitter release from presynaptic neurons. Recently, the SGIP1 protein has been found to interact with the CB1 receptor and participates in the modulation of nociception. However, whether SGIP1 modulates spinal CB1 receptor signaling at the spinal cord level is unknown. To answer this question, we used the patch-clamp method in the superficial spinal cord dorsal horn neurons of wild-type (WT) and knockout (KO) SGIP1 mice to measure spontaneous (s) and miniature (m) excitatory postsynaptic currents (EPSCs). Results of naive mice and mice with carrageenan-induced peripheral inflammation were compared. The results show that the efficacy of the CB1 receptor agonist WIN 55,212-2 at the first spinal nociceptive synapse is identical in naive mice for both SGIP1 WT and KO phenotypes. The control frequencies of both groups of neurons did not differ in naïve conditions or the peripheral inflammation model. On the contrary, the WIN 55,212-2 application was more effective in SGIP1 KO mice during peripheral inflammation. This study further addressed how CB1 receptor activation affects spinal inhibitory synaptic...
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The role of nociceptive synaptic transmission modulation
Heleš, Mário ; Paleček, Jiří (advisor) ; Rokyta, Richard (referee) ; Krůšek, Jan (referee)
Pain represents a major symptom in a multitude of medical conditions and can often become the main negative factor in a patient's low quality of life. The complex issue of pain management is further underscored by the reduced efficacy of conventional analgesics in conditions such as neuropathic pain. Neuropathic pain, unlike acute nociceptive pain, originates from damage to the peripheral or central nervous system and often develops into chronic pain syndrome. Most analgesics available today provide only limited and unsatisfactory analgesia in chronic neuropathic pain and are often associated with severe adverse effects. Modulation of nociceptive transmission in spinal cord dorsal horn (SCDH) stands out in recent research as a pivotal mechanism, especially in chronic pain development and maintenance. The major aim of this doctoral thesis was to investigate how pain-associated processes interfere with opioid-induced analgesia, with the main focus on the interaction between chemokine (C-C motif) ligand 2 (CCL2), transient receptor potential vanilloid type 1 (TRPV1), and μ-opioid receptor (MOR). To achieve a better insight into opioid signaling in SCDH we studied the following issues: (I.) How does CCL2 modulate MOR-mediated effects on nociceptive synaptic transmission in SCDH neurons and in vivo...
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Pathological pain states, the role of synaptic modulation at spinal cord level
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...
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Joint configuration changes pressure pain threshold-centered and decentered position
Jevič, Filip ; Čech, Zdeněk (advisor) ; Vaculín, Šimon (referee)
The term "joint centration" is recently perceived primarily in its biomechanical context. This paper suggests a possible neurophysiological point of view and examines the effect of centered or decentered position on pressure pain treshold (PPT) of three muscles (m. temporalis, m. tibialis anterior, m. interosseus dorsalis I) in sitting and lying position, researched on healthy volunteers. PPT was measured in four randomly ordered positions with 49 people (21 women, 28 men). Significantly higher PPT was detected in all three muscles (p=0,001; 0,0016; 0,00009). In percent the PPT change resembles some of studies working with therapeutical techniques (mobilization, joint manipulation, exercise). Influence of higher postural position (sitting compared to lying down) on PPT was not proved. Cenetered joint position increases the PPT of healthy young adults compared to decentered position. This newly proved fact opens further possibilities of conceiving the key physiotherapeutical notion of "joint centration".
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The role of nociceptive synaptic transmission modulation at the spinal cord level in different pain states
Adámek, Pavel ; Paleček, Jiří (advisor) ; Vaculín, Šimon (referee) ; Vlachová, Viktorie (referee)
Pain is a common symptom of many clinical syndromes and diseases. In particular, the treatment of neuropathic pain represents a serious public health issue because currently available analgesia is ineffective in many cases or it has adverse effects. Treatment of pain-related suffering requires knowledge of how pain signals are initially generated and subsequently transmitted by the nervous system. A nociceptive system plays a key role in this process of encoding and transmission of pain signals. Modulation of the nociceptive synaptic transmission in the spinal cord dorsal horn represents an important mechanism in the development and maintenance of different pathological pain states. This doctoral thesis has aimed to investigate and clarify some of the mechanisms involved in the modulation of the spinal nociceptive processing in different pain states. The main attention was paid to study the following issues: (I.) Which is the role of Transient Receptor Potential Vanilloid type 1 channels (TRPV1), Toll-Like Receptors 4 (TLR4), and phosphatidylinositol 3-kinase (PI3K) in the development of neuropathic pain induced by paclitaxel (PAC) chemotherapy in acute in vitro, and subchronic in vivo murine model of PAC-induced peripheral neuropathy (PIPN)? (II.) How is affected spinal inhibitory synaptic control...
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A study of the temperature sensitivity of the human TRPA1 channel
Barvíková, Kristýna ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Transient receptor potential (TRP) ion channels play important physiological roles in the detection of environmental stimuli that occur primarily at the peripheral terminals of specialized sensory neurons. The recently resolved cryo-electron microscopy structures and molecular biological techniques have provided new tools that enable to study these channels in relation to their function, and thus to understand more deeply their pharmacology and physiology. The aim of this bachelor thesis is to give an overview of the current status of research on the ankyrin TRP channel subtype 1 (TRPA1), a channel activated by diverse irritant chemical stimuli but also by temperature changes. The experimental part is focused on the elucidation of the role of the sensor domain in thermal sensitivity of the TRPA1 channel. Using whole-cell patch-clamp electrophysiological technique, the presented results demonstrate that the sensor is an important determinant of voltage-dependent gating. Mutation of the conserved tyrosine in the center of the sensor resulted in channels with clearly different activation kinetics and increased chemical responses upon increasing the temperature from 25 řC to 35 řC. Key words: TRP ion channel, ankyrin receptor, nociception, structure-function, carvacrol
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The role of synaptic modulation in pain states.
Adámek, Pavel ; Paleček, Jiří (advisor) ; Moravec, Jan (referee)
Everybody has experienced pain. Pain by definition is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. In the peripheral tissues acute painful stimuli activate specialized endings of afferent neurons called nociceptors. The information about tissue damage is then transmitted to the cell bodies of these dorsal root ganglion neurons by unmyelinated or thinly myelinated axons (C and A fibers, respectively). The central branches of these neurons form synapses with superficial dorsal horn neurons in the spinal cord. The information is conveyed at the synaptic connections by neurotransmitters such as glutamate and many others neuromodulators. Important is the subsequent activation of projection neurons that transmit the information to supraspinal brain areas. Activity of excitatory and inhibitory interneurons, glial cells and descending pathways from the CNS are also important for the modulation of nociceptive information at the spinal cord level. After peripheral tissue damage and in other pathological states, increased sensitivity to peripheral stimuli may develop. As results of this change innocuous stimuli are perceived as painful (alodynia) and increased pain is perceived after noxious stimuli (hyperalgesia). The underlying mechanisms of these changes may be...
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