Developing neuroimaging biomarker for sustained experimental and clinical pain

Lee, Jaejoong Sungkyunkwan University 2023 국내박사

Understanding neurobiological mechanisms of pain has enormous implications for basic science and clinical practices. However, the subjectivity and ambiguity of the measurement of pain have hampered its progress. This dissertation presents a series of attempts to develop brain-based biomarkers that can quantitatively and objectively measure subjective pain using functional magnetic resonance imaging (fMRI), with particular focus on the sustained pain which is a hallmark of clinical pain disorders. In Chapter 1, we reviewed the emergence of predictive modeling and graph-theoretical approach from the history of pain research, and highlighted the translational potential of experimentally-induced sustained pain. In Chapter 2 and 3, we introduced the actual application of these approaches based on fMRI data. In the Chapter 2, we used whole-brain functional connectivity to develop a biomarker that can predict ongoing sustained pain induced by orofacial capsaicin. This biomarker not only showed high sensitivity and specificity across multiple capsaicin pain fMRI datasets but also could be generalized onto clinical back pain fMRI datasets as well, suggesting the shared neural mechanisms across sustained experimental and clinical pain. In the Chapter 3, we used graph-theoretical analyses to examine how functional brain architecture was reconfigured over the experience of sustained pain induced by orofacial capsaicin. The characteristic features of dynamic brain reconfiguration were then used to develop biomarkers predictive of sustained pain, which also demonstrated significant sensitivity and specificity. Overall, these results provide new insight into the brain mechanisms of sustained pain, and ultimately contribute to the development of clinically useful objective biomarkers of pain. 만성 통증 연구 및 치료는 그 막대한 중요성과 사회적 함의에도 불구하고 측정 대상이 되는 통증 자체의 주관성과 모호함 때문에 많은 어려움이 있어왔다. 본 연구에서는 실험적으로 유발된 지속적 통증과, 임상에서 진단된 만성 통증 질환을 대상으로 뇌 기능 영상을 통해 신경생물학적 기전을 규명하고 통증을 객관적으로 측정할 수 있는 생물학적 표지자를 개발하고자 한다. 1장에서는 통증 연구의 역사적인 흐름에서, 전체 뇌의 분산된 기능을 반영하는 기법으로서의 예측 모델링과 그래프 이론적 접근의 출현을 검토하고, 실험적으로 유도된 만성 통증의 임상적 중개 가능성을 서술한다. 이어지는 2장과 3장에서는 위의 두 가지 방법론의 뇌 기능 영상 데이터에서의 실제적 적용을 다룬다. 2장에서는 전체 뇌의 기능적 연결성을 사용하여 캡사이신에 의해 유도된 지속적인 구강안면 통증을 예측할 수 있는 바이오마커를 개발했다. 이 바이오마커는 여러 캡사이신 통증 데이터셋에 걸쳐 높은 민감도와 특이도를 보여줬을 뿐만 아니라 임상적 허리 통증을 예측하는 데에도 우수한 성능을 나타냈으며, 이는 본 바이오마커가 실험적 및 임상적 통증에 공통적인 신경생물학적 기전을 반영함을 시사한다. 3장에서는 그래프 이론을 사용하여 캡사이신에 의해 유발된 지속적 통증에 따른 기능적 뇌 네트워크의 동적인 재구성을 확인했다. 또한, 통증 변화에 따른 뇌 네트워크의 특징적인 변화의 패턴을 기반으로 지속적 통증의 세기를 예측하는 바이오마커를 개발하였으며 이 바이오마커는 높은 민감도와 특이도를 보여주었다. 본 결과는 실험적 및 임상적 만성 통증의 뇌 메커니즘에 대한 새로운 통찰을 제공하며, 궁극적으로 임상적으로 유용한, 객관적인 통증 바이오마커 개발에 기여할 것으로 기대된다.

Unlocking Novel Therapeutic Targets and Molecular Mechanisms of Inflammatory Pain

미틸레쉬쿠마르자 경북대학교 대학원 2015 국내박사

본 연구의 목적은 염증성 통증에서 새로운 치료 표적으로서 pyruvate dehydrogenase kinase (PDK)-pyruvate dehydrogenase (PDH)-lactic acid axis와 리포칼린2 (LCN2)의 작용기전을 밝히는 것이다. PDK (PDK1-4)는 pyruvate가 호기적 상태에서 acetyl-CoA로 또는 혐기적 상태에서는 lactate로 변환되는데 중요하게 작용하는 PDH의 활성을 억제하는 미토콘드리아의 대사 조절자이다. 대사장애와 염증반응은 여러 병리생리학적 조건에서 동시에 일어난다. Metabolic shift로 인한 lactic acid의 축적은 다양한 통증 상태에 나타나는 것으로 알려져 있다. 본 연구에서는 만성 염증성 통증의 병리에서 PDK-PDH-lactic acid axis의 역할을 조사하였다. Pdk2,Pdk4각각, 또는 두 유전자가 (Pdk2/4)동시에 결여(knockout) 된 생쥐에서는 complete Freund’s adjuvant (CFA)에 의해서 유도되는 통증 과민반응이 완화되었다. 행동반응과 유사하게, Pdk2/4DKO쥐에 CFA를 발바닥에 주사했을 때 말초/중추신경의 염증반응과 발바닥에서 lactic acid의 축적이 감소되었다. Invitro연구를 통하여 PDK2/4가 대식세포 (macrophage)의 염증반응을 유도하는 것을 확인하였다. 그리고 PDK 또는 lactic acid 생성의 약리적 억제는 CFA에 의해 유도되는 염증 반응과 통증 과민성을 감소시켰다. PDK-PDH-lactic acid axis는 염증반응에 의한 만성 통증을 매개하는 것으로 확인하였고 따라서 염증성 통증의 병리기전에서 대사반응이 밀접하게 관련되어 있음을 시사한다. 리포칼린2는 다양한 뇌질환에서 나타나는 글리아 활성, 케모카인의 유도, 염증영역으로 면역세포의 침투 및 염증성 신호전달에서 중요한 역할을 한다고 알려져 있다. LPS, TNF-α, IFN-g, IL-1β와 같은 다양한 염증성 자극은 neutrophil, 대식세포와 같은 다양한 세포에서 리포칼린2의 발현과 분비를 유도할 수 있다. 리포칼린2는 또한 중요한 paracrine chemoattractant로서 작용하고 염증상황에서 neutrophil의 기능을 위한 필수 요소로 알려져 있다. 이전 연구결과를 토대로 우리는 만성 염증성 통증의 발병에서 리포칼린2가 관련되어 있을 것으로 추측하였다. 본 연구에서 만성 염증성 통증의 동물 모델을 사용하여 리포칼린2가 염증반응이 일어나는 말초신경에서 염증성 사이토카인과 케모카인의 발현, neutrophils과 대식세포의 침투를 조절하고 뿐만 아니라 척수의 dorsal horn영역에서 글리아 활성을 조절하여 통증 발병에서 중요한 역할을 하는 것으로 확인하였다. 이 결과는 리포칼린2가 만성 염증성 통증의 치료를 위한 신규 약물표적임을 제시한다. The main aim of the studies described in this thesis was to investigate the novel therapeutic targets and the molecular mechanisms of inflammatory pain with an insight into the pivotal roles played by a pyruvate dehydrogenase kinase (PDK)–pyruvate dehydrogenase (PDH)–lactic acid axis and an acute phase protein lipocalin-2 (LCN2). PDKs (PDK1–4) are mitochondrial metabolic regulators that serve as decision makers via modulation of PDH activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of a PDK–PDH–lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of Pdk2 and/or Pdk4 in mice attenuated complete Freund’s adjuvant (CFA)-induced pain hypersensitivities. Likewise, Pdk2/4 deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammations following intraplantar administration of CFA. In vitro studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK–PDH–lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain. Emerging evidence advocates the crucial role of LCN2 in modulation of glial phenotypes, induction of chemokines, regulation of immune cell recruitment to sites of inflammation, and proinflammatory signaling in diverse neurological disorders. Diverse inflammatory stimuli, such as LPS, TNF-α, IFN-γ and IL-1β, can markedly induce LCN2 expression and secretion from neutrophils, macrophages, and several other cell types. LCN2 has also been acknowledged as an important paracrine chemoattractant and an obligatory factor for neutrophil function in inflammation. These previous findings led us to investigate the involvement of LCN2 in the pathogenesis of chronic inflammatory pain. In the present study, by using the preclinical model for chronic inflammatory pain, we show that LCN2 may have important roles in the amplification of the deleterious effects of proinflammatory cytokines and chemokines at the sites of inflammation, in the recruitment of proalgesic neutrophils and macrophages, in glial activation in the spinal cord dorsal horn, and ultimately in nociception. These findings identify LCN2 as a potential target for the treatment of chronic inflammatory pain.

내인성 단백질 기반 말초신경계 내 통증 조절 기전 연구 : GLP-1에 의한 TRPV1 억제와 c-Met 수용체 매개 이온 채널 활성 변화

고은진 가천대학교 메디컬캠퍼스 일반대학원 2025 국내박사

Pain is generally controlled through intrinsic pain modulation systems that maintain balance under normal physiological conditions. However, in pathological scenarios such as neuropathic pain, inflammation, or diabetes-induced nerve damage, these systems can become compromised. This impairment leads to an overactivation of pain pathways and the onset of chronic pain. This study aims to explore key mechanisms involved in pain modulation, with a focus on innovative strategies targeting two distinct pathways to address chronic pain conditions. Chapter 1 investigates the role of glucagon-like peptide-1 (GLP-1) and its derivatives in modulating nociceptive signaling. It highlights how GLP-1-based peptides, particularly exendin 9–39 and exendin 20–29, provide effective relief from acute and chronic pain. These peptides are shown to act on TRPV1 channels, offering significant analgesic effects without triggering thermoregulatory disturbances, thereby presenting a safer alternative for pain management. Chapter 2 explores the c-Met receptor pathway, focusing on the NK1 splice variant of hepatocyte growth factor (HGF). This variant selectively activates c-Met, modulating neuronal excitability through the regulation of voltage-gated sodium channels. The findings underscore its potential in reducing chronic pain while avoiding excessive cell proliferation. By leveraging this pathway, a targeted therapeutic approach can be employed to address chronic pain effectively. Together, these studies shed light on intrinsic pain regulation mechanisms and present innovative therapeutic options for managing chronic pain, providing valuable insights into improving patient outcomes.

Pain Management Among Dominican Patients with Advanced Osteoarthritis: A Qualitative Study

Yu, Amy Harvard University ProQuest Dissertations & Theses 2018 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Background: Advanced osteoarthritis and total joint replacement (TJR) recovery are painful experiences and often prompt opioid use in developed countries. Physicians participating in the philanthropic medical mission Operation Walk Boston (OpWalk) to the Dominican Republic have observed that Dominican patients require substantially less opioid medication following TJR than US patients. We conducted a qualitative study to investigate approaches to pain management and expectations for postoperative recovery in patients with advanced arthritis undergoing TJR in the Dominican Republic.Methods: We interviewed 20 patients before TJR about their pain coping mechanisms and expectations for postoperative pain management and recovery. Interviews were conducted in Spanish, translated, and analyzed in English using content analysis.Results: Patients reported modest use of pain medications and limited knowledge of opioids, and many relied on non-pharmacologic therapies and family support to cope with pain. They held strong religious beliefs that offered them strength to cope with chronic arthritis pain and prepare for acute pain following surgery. Patients exhibited a great deal of trust in powerful others, expecting God and doctors to cure their pain through surgery.Conclusion: We note the importance of understanding a patient’s individual pain coping mechanisms and identifying strategies to support these coping behaviors in pain management. Such an approach has the potential to reduce the burden of chronic arthritis pain while limiting reliance on opioids, particularly for patients who do not traditionally utilize powerful analgesics.

Pain Management in Nineteenth-Century American Literature

Utphall, Jamie Anna The Ohio State University ProQuest Dissertations & 2023 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Pain Management in Nineteenth-Century American Literature explores how three writers, Emily Dickinson, Walt Whitman, and Charles Chesnutt, imagine and theorize pain and pain relief during a period of burgeoning medical and political upheaval in the nineteenth-century United States. To situate these writers within their historic moment, I trace a trajectory of the cultural and medical attitudes toward pain. In theorizing human suffering, each of these three writers actively revises the cartesian division between the body and mind that still persists in writing about pain today, as demonstrated by the dominance of Elaine Scarry's 1985 argument that pain actively destroys language. This dissertation fundamentally revises Scarry's claim by illustrating how literature from my three case studies works to redefine the relationship between the phenomenology of pain and each writer's respective representational projects. Overall, I offer a way out of Scarry's insistence on the inexpressibility of pain and embrace these authors' success in rendering pain and in fashioning literary strategies for pain management and relief. For Dickinson, I situate her poetics of the sublime within the context of the newly developed technology of anesthesia. I argue that Dickinson portrays sublime encounters with art as analogous to the pain relief provided by anesthesia, which demonstrates her poetry's ability to oscillate between metaphorical and literal pain management methods. To the field of Whitman studies, I provide a new investigation of Whitman's rendering of soldiers' pain through a cluster of his earliest Civil War poems. These poems, I argue, demonstrate Whitman's attempt to find the most precise language possible to depict the immediacy and immanence of soldiers' pain. In my third chapter, I contribute a new way of reading Charles Chesnutt's Uncle Julius tales, in which Chesnutt demonstrates how the act of storytelling is similar to Black conjure magic in its ability to help mitigate slavery's lasting traumas while preserving the struggles experienced and fortitude expressed by slavery's victims and ancestors. Overall, this study reveals how each of these authors engages with pain not only as a destructive and destabilizing force but also as a creative one, primarily through each author's ability to generate literary language depicting pain that oscillates between the literal and figurative, which I comprehend as a critical part of nineteenth-century United States writing.

Effects of Acetaminophen on Pain Response among Overweight or Obese Women Exposed to Weight Stigmatization

Landers, Jacob David The Ohio State University ProQuest Dissertations & 2021 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Pain is widely prevalent across the world, and individuals with excess weight are disproportionally affected by it relative to those with normal weight. Research suggests that obesity is associated with biological indicators of systemic inflammation (e.g., C-reactive protein), which may contribute to pain. Individuals with excess weight, especially women, also are at greater risk of experiencing the emotional pain of weight stigma, which may in turn exacerbate physical pain. Recent studies have shown that brain areas involved in the processing of physical pain (dorsal anterior cingulate cortex, anterior insula) are the same areas associated with emotional pain, and that analgesics (e.g., acetaminophen) used for reducing physical pain may also dampen extremity of emotional responses. However, analgesic effects have not been tested in the context of individuals with excess weight experiencing weight stigmatization.The current study was designed to 1) evaluate the influence of a weight stigma induction and acute dose of acetaminophen on pain, distress, and affect among women with overweight or obesity; and 2) evaluate the relationship between body mass index (BMI) and pain, the degree to which this relationship is mediated by weight stigma, and the degree to which acetaminophen moderate the weight stigma-pain relationship.One hundred sixty women with overweight or obesity (78% Caucasian; average age 41.7¡¾17.4 years; average BMI 32.4¡¾6.4) were recruited from the Columbus and surrounding communities. Prospective participants were screened for eligibility including BMI 25 or greater, female sex, and age 18 years or greater. The sample of eligible participants was stratified by age (18-40 years vs. over 40 years) and BMI (overweight vs. obese) for randomization into one of four study conditions: acetaminophen and weight stigma induction; placebo and weight stigma induction; acetaminophen and control condition; and placebo and control condition.Participants attended one, 120-minute session that began with anthropometric measurements, as well as collection of blood and saliva samples to measure inflammation. Participants were then administered either acetaminophen or placebo according to their randomization. Participants completed self-report questionnaires for sixty minutes while waiting for sufficient drug absorption, after which they were presented with the induction to which they were randomly assigned. Participants next completed a second set of self-report measures to assess the influence of the induction and they provided a second saliva sample to assess acute change in inflammation.Data analyses primarily included Pearson correlations; analyses of variance and covariance; and mediation and moderation analyses using PROCESS (Hayes, 2013). Results indicated that higher BMI was associated with greater endorsement of weight stigma, worse weight-related and physical quality of life, and greater pain. Higher baseline inflammation (C-reactive protein) was associated with higher BMI and greater perceived weight stigma. Group comparisons revealed more negative affect and distress among those exposed to weight stigmatization after ingesting acetaminophen compared to the two control condition groups. Post-hoc analyses revealed greater anxiety and distress in the acetaminophen and weight stigma group compared to the other three groups. Although weight stigma did not mediate the relationship of BMI to pain, post-hoc analyses indicated serial mediation of the BMI-pain relationship through both greater endorsement of weight stigma and greater distress. In contrast to study hypotheses, greater internalized weight stigma and greater BMI in the presence of acetaminophen was associated with greater post-induction pain.Results of the study conflict with prior published research suggesting a blunting effect of acetaminophen on emotional pain response but are consistent with newer, unpublished data demonstrating exacerbated emotional response with acetaminophen ingestion. These results support the relevance of physical and emotional pain for women with overweight or obesity, and suggest the need for further investigation into the effects of acetaminophen on emotional response among adults with overweight or obesity. Additionally, study results highlight the relevance of distress when considering the role of weight stigma in the BMI-pain relationship.

한방병원에 내원하는 파킨슨병 환자의 임상양상 및 통증에 관한 고찰 : 후향적 차트 리뷰

정혜선 경희대학교 대학원 2020 국내석사

Background : Parkinson's disease (PD) is a heterogeneous , progressive and neurodegenerative disease . Pain is one of frequently reported nonmotor symptoms in PD and inappropriately controlled pain can cause depression, diminished quality of life. There is a lack of attention to pain in PD because pain is relatively subjective symptom and main treatment of PD is focused on managing motor symptoms. There are no clear pain management guideline as well as each therapeutic realities have some limitations. While Study of pain in PD has been reported more and more, those studies were poorly documented in Korea. Objectives : To investigate prevalence of pain, pain characteristics, association between clinical features and pain, efficacy of Korean medical treatment for PD with pain. Methods : We performed a retrospective review of the medical records for patients diagnosed with Parkinson's disease between 2012 and 2019 at Gangdong kyunghee university korean medicine hosptal in South Korea. Results : Of 172 PD patients, 147 patients(85.5%) reported pain. Female PD patients more frequently reported pain than male(P=0.03). Sixty nine point three percent(69.3%) of patients complaining about musculoskeletal pain, and musculoskeletal pain show significant difference depending on the PD motor subtype(P=0.039). Pain was located mainly in the leg(57.8%) or back(34.7%). 22 patients with KPPS scores at least twice were analysed for evaluating efficacy of Korean medicine on pain in PD. Mean total scores before and after korean medical treatment were 15.23±11.01 and 9.2±8.7, respectively, and mean difference between before/after total scores was 6.0±5.8(P<0.001). Specifically, score of radicular pain was significantly decreased(P=0.048). Conclusions : In comparision with general population, PD patients has high prevelence of pain. This study suggests Korean Medicine could be beneficial for PD by reducing pain. For clinical efficacy, further study as a large-sample cohort study, RCTs should be done to clarify pathological pain relief mechanism and analgesic effect of Korean Medicine.

Decoding spontaneous ongoing pain from brain cellular calcium activities using a deep learning in mice

윤희라 경희대학교 대학원 2020 국내박사

The objective measurement of spontaneous pain is a longstanding challenging issue in the fields of pain and neuroscience. The pain processing involves multiple brain regions, including the primary somatosensory (S1) cortex which plays a key role in discrimination of bodily sensation. In the present study, I hypothesized that neuronal activity patterns in the mouse S1 cortex are distinct between spontaneous pain and non-pain conditions, and that this discrepancy can be used for quantitatively measuring spontaneous pain and then evaluating the analgesic effects of pain killers. To explore this hypothesis, I performed in vivo two-photon calcium imaging in the layer 2/3 S1 neurons of awake, head-fixed mice with or without formalin-induced spontaneous pain. I then applied a deep learning algorithm, AI-bRNN (Average training, Individual test-bidirectional Recurrent Neural Network), that decodes spontaneous pain information from brain cellular calcium in awake head-fixed mice. The deep learning model successfully predicted the intensity of pain induced by various doses of formalin injection. Furthermore, the model could estimate the efficacies of analgesic drugs, such as ketoprofen and lidocaine, on formalin-induced pain. Interestingly, the deep learning model could also discriminate other types of pain, such as acute pain induced by topical capsaicin, inflammatory pain induced by complete Freund’s adjuvant (CFA), and neuropathic pain induced by sciatic nerve injury, even though the model was trained using formalin-induced pain group data. Further, the AI-bRNN could discriminate itch from pain, suggesting its broad applicability. These results show that spontaneous pain could be measured objectively from brain cellular calcium activities using AI-bRNN and provide a deep insight into the pain processing of animals. This method is expected to be widely used for screening new analgesics in the medical and pharmaceutical industries. 지속적 자발 통증의 객관적인 측정은 통증 및 신경 과학 분야에서 오랫동안 해결하지 못한 도전적인 문제이다. 통증 처리는 통증 인식과 관련된 1차 체성감각피질을 포함하여 다수의 뇌 영역을 포함한다. 본 연구에서는 생쥐 1차 체성감각피질에서 신경활동의 패턴을 이용하여 지속적 통증과 비 통증을 구별할 수 있고, 이를 활용하여 지속적 통증을 정량적으로 측정 가능하며, 진통제 효능 평가에도 사용할 수 있다고 가설을 세웠다. 이 가설을 탐구하기 위해, 깨어 있으면서 머리가 고정되어 있는 생쥐의 1차 체성감각피질 신경세포에서 생체 내 이광자 칼슘 이미징을 수행하였다. 다음으로, 생쥐의 뇌 세포 칼슘에서 지속적 통증 정보를 해독하기 위해서 딥러닝 알고리즘인 AI-bRNN을 적용하였다. AI-bRNN은 다양한 농도의 포르말린 주사에 의해 유발되는 통증의 강도를 성공적으로 예측하였다. 또한, 이 딥러닝 모델은 케토프로펜 및 리도카인과 같은 진통제가 포르말린 유발 지속적 통증을 억제하는 효과를 평가할 수 있었다. 흥미롭게도, 이 딥러닝 모델은 포르말린 유발 통증 그룹 데이터를 사용하여 훈련되었음에도 불구하고, 캡사이신 유발 통증, CFA 염증성 통증 및 신경 손상 유발 신경 병증성 통증과 같은 다른 유형의 지속적 통증을 측정할 수 있었다. 또한, 거시적인 해부학적 신경 경로가 동일한 두 감각인 통증과 가려움증을 뚜렷하게 구별할 수 있었다. 이러한 결과는 지속적 자발 통증이 뇌 세포 칼슘 활동으로부터 딥러닝을 사용하여 객관적으로 측정될 수 있고, 다양한 통증 모델 및 다른 종류의 감각을 측정하는 데 광범위하게 적용될 수 있음을 보여준다. 나아가 이 방법은 만성 난치성 통증의 뇌신경 기전을 규명하는 연구에 활용될 수 있을 뿐만 아니라, 의료 및 제약 산업에서 임상 상황과 더욱 밀접하게 연결된 새로운 진통제를 선별하는 데 널리 사용될 수 있을 것으로 기대된다.

Development of polymeric nanoparticles for microglia-specific gene delivery to treat neuropathic pain

최부민 서울대학교 대학원 2022 국내박사

Pain is a sensation that allows us to notice danger and avoid it as fast as possible or to recognize disease. However, in the context of neuropathy, non-noxious stimuli can also cause pain. Cancer, diabetes, infection, and nerve injury can produce neuropathic pain, in which aberrant pain signals are continuously transmitted. Non-nociceptive stimuli, such as touch, vibration, and pressure, can also transmit pain signals via activating spinal cord microglia. In an animal model of nerve injury, microglia in the spinal cord are excessively activated by various stimuli due to damaged nerves. Microglia in this context secrete neurotrophic factors to induce abnormal activity in spinal dorsal horn neurons. Therefore, controlling the activity of microglia is a promising strategy for treating neuropathic pain. Nanoparticles have been actively studied as a gene delivery system for their multiple advantages over virus-based systems in that the probabilities of inflammatory response, insertional mutations, and carcinogenesis are very low. Furthermore, nanoparticles can exhibit a wide range of properties due to differences in materials, sizes, and surface modifications. Therefore, they can be developed for specific cell-targeting applications. The goal of this study is to create polymeric nanoparticles for microglia-specific gene delivery and to alleviate neuropathic pain via anti-inflammatory gene delivery. In Chapter 1 of this paper, a study conducted to discover a new nanoparticle for microglia-specific gene delivery is described. Polyethyleneimine (PEI) was applied, which is typically utilized for gene transfer. While maintaining the DNA transfection ability of PEI, triethylene glycol dimethacrylate (TG) was introduced to improve biodegradability, as this polymer incorporates ester linkage. These TG-PEI nanoparticles were preferentially delivered to the microglia than astrocytes in the primary culture, and were exclusively delivered to the microglia in the spinal cord and the brain in vivo. Transfecting primary mixed glia was effectively performed using this nanoparticle, while showing lower cytotoxicity than treating PEI only. In Chapter 2, a Food and Drug Administration-approved nanoparticle, poly(lactide-co-glycolide) (PLGA), was investigated. This study aimed to determine whether it was feasible to treat neuropathic pain with PLGA nanoparticles that could be precisely administered to spinal cord microglia. First, it was confirmed that enhanced green fluorescent protein (EGFP) was well-expressed when HELK293T cells treated with PLGA/EGFP nanoparticles. Second, PLGA nanoparticles conjugated with a fluorescent molecule were discovered to be selectively absorbed by microglia over other spinal cord cells when intrathecally administered. PLGA nanoparticles were synthesized to contain three types of anti-inflammatory genes (interleukin [IL]-4, IL-10, and transforming growth factor beta-1 [TGFβ1]). Finally, in a neuropathic pain mouse model, PLGA/IL-4, PLGA/IL-10, and PLGA/TGFβ1 were administered intrathecally, and pain-alleviating effects were measured by the von Frey test. All three nanoparticle-treated groups showed partial analgesic efficacy, with the PLGA/IL-4 treatment group demonstrating the greatest potency. Through this research, a new nanoparticle capable of targeting microglia was identified. It was also confirmed that neuropathic pain could be alleviated by delivering anti-inflammatory cytokine genes to microglia. Therefore, modulating microglial activity using nanoparticles promises to be an effective strategy for fundamentally addressing neuropathic pain. 통증은 위험을 감지하여 빠른 회피를 하거나 질병을 알아차리기 위한 목적의 감각이다. 하지만 어떤 위험이 없을 때에도 계속해서 통증이 느껴지는 질병 있는데 이를 신경병증성 통증이라고 한다. 암, 당뇨병, 감염증 또는 신경손상 등에 의해서 비정상적인 통증 신호가 계속 전달되는 신경병증성 통증이 발병될 수 있다. 촉각, 진동, 압력 등 통각이 아닌 자극에서도 통증 신호가 전달될 수 있는데 이것은 척수의 소교세포 활성이 원인이 된다. 신경손상 동물모델에서 척수 소교세포는 손상된 신경으로부터 온 여러 자극에 의해 과도하게 활성화 되고 신경영양성 인자를 분비하여 척수 후각 신경세포의 비정상적 활성을 유도한다. 따라서 소교세포의 활성을 조절하는 것이 신경병증성 통증의 근본적인 치료법이 될 수 있다. 나노입자는 1995년에 처음으로 약물전달체로서 FDA 승인을 받고 이후 활발히 연구되고 있다. 약물뿐만 아니라 DNA, mRNA, RNAi 등 여러 종류의 유전자 전달체로도 이용되고 있다. 나노입자의 활용은 아데노관련 바이러스를 이용한 유전자 전달 보다 염증반응을 덜 일으키고, 삽입 돌연변이 및 발암 확률이 아주 낮다는 장점이 있다. 또한, 나노입자는 재료, 크기, 표면 변형을 달리하면서 아주 다양한 특성을 가질 수 있어서 특정 세포 타겟팅 용도로 활용될 수 있다. 따라서, 본 연구에서는 척수 소교세포에 특이적으로 유전자 전달이 가능한 나노입자 전달체를 개발하고 더 나아가 항염증성 유전자 전달을 통한 신경병증성 통증의 완화를 도모하고자 한다. 본 논문의 제 1 장에서는 소교세포 특이적으로 유전자 전달을 하기 위한 새로운 나노입자를 발굴하였다. 유전자 전달을 하기 위해 대표적으로 쓰이는 폴리에틸렌이민을 활용하였다. 폴리에틸렌이민의 DNA 형질주입의 능력은 유지한 채 생분해성을 높이기 위해 트라이에틸렌 글라이콜 다이메타크릴레이트를 도입하였다. 이 폴리머는 생체 적합성이 높은 폴리에틸렌 글라이콜을 포함하고 폴리에틸렌이민과 결합 시 β-아미노 에스터 결합을 하기 때문에 생분해성이 높아질 수 있다. 이렇게 합성한 나노입자를 이용하면 일차배양 혼합교세포에 형질주입을 효과적으로 할 수 있고 세포독성이 아주 낮은 것을 확인하였다. 또한, 혼합 교세포 중 소교세포에 더 많은 나노입자가 들어가고 척수 강 주입 시 소교세포에만 특이적으로 들어간다는 사실을 확인하였다. 제 2 장에서는 FDA 승인이 된, 잘 알려진 나노입자 PLGA를 이용하였다. 형광물질을 결합시킨 PLGA 나노입자를 척수 강 주입하였을 때, 척수에 있는 세포 중 소교세포에 선택적으로 들어가는 것을 확인하였다. 또한 PLGA를 이용하여 형질주입이 가능한지 확인하기 위해 PLGA/EGFP 나노입자를 합성하여 HELK293T 세포에 처리해 보았을 때, EGFP 형광이 잘 발현되는 것을 확인하였다. 척수 소교세포 특이적으로 전달이 가능한 PLGA 나노입자를 활용하여 신경병증성 통증의 치료가 가능한지 확인하고자 하였다. 이를 위하여 PLGA 나노입자에 항염증성 사이토카인 3종류 (IL-4, IL-10, 그리고 TGFβ1)가 각각 포함되도록 합성하였다. 신경병증성 통증 생쥐 모델에 PLGA/IL-4, PLGA/IL-10, 그리고 PLGA/TGFβ1을 각각 척수 강 주입하고 통증행동실험을 통하여 통증 유발 정도를 측정하였다. 세 가지 나노입자를 처리한 그룹에서 모두 부분적인 진통 효능을 보였고 그 중 PLGA/IL-4 처리 그룹에서 가장 효과가 좋은 것을 확인하였다. 본 연구를 통하여 소교세포 표적이 가능한 새로운 나노입자를 발굴 하였고, 항염증성 사이토카인 유전자를 소교세포에 전달하여 신경병증성 통증이 완화되는 것을 확인하였다. 결과적으로 소교세포의 활성 조절이 신경병증성 통증을 근본적으로 치료하는 핵심 타겟이 될 수 있음을 증명하였다.

Multimodal approach for pain management : local analgesics, pharmacopuncture and time-restricted feeding

이정윤 서울대학교 대학원 2020 국내박사

Pain is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage by International Association for the Study of Pain (IASP). Since pain has both sensory-discriminative and affective-motivational dimensions, it is difficult to effectively modulate pain with a conventional pharmacological approach (NSAIDs and opioids) alone. In an effort to explore multimodal approaches to pain management, I have focused on the therapeutic effect of local analgesics (sinomenine), pharmacopuncture (acupoint stimulation with drug, Scolopendra subspinipes) and time-restricted feeding (fasting/refeeding) on animal pain models as below. In the first chapter, I revealed that sinomenine can produce peripheral analgesic effect by reducing cellular excitability of nociceptive neurons. The intraperitoneal injection of sinomenine suppressed formalin-induced pain behavior and c-Fos expression. Sinomenine inhibited voltage-gated sodium current in nociceptive neurons. Furthermore, the intraplantar injection of sinomenine also suppressed pain behavior. Therefore, sinomenine could be a potential pharmacological therapeutic agent for local anesthesia and analgesia. In the second chapter, I demonstrated that acupoint stimulation with Scolopendra subspinipes (pharmacopuncture with Scolopendra subspinipes, SSP) produces an analgesic effect via alpha2-adrenoreceptors in the spinal cord. SSP into Zusanli acupoint had a significant analgesic effect on oxaliplatin-induced neuropathic pain model. The intrathecal injection of yohimbine (alpha2-adrenoeceptor antagonist) reversed SSP-induced analgesic effect. Therefore, SSP produces an analgesic effect by activating descending pain inhibitory system, which suggests a possibility for pain management. In the third chapter, I focused on the analgesic effect of fasting and refeeding. Both 24h fasting and 2h refeeding produce analgesic effect but via different mechanisms, suggesting that endocannabinoid and opioid system is only involved in fasting-induced analgesia, whereas refeeding-induced analgesia is associated with eating behavior and calorie recovery effect. Therefore, the regulation of feeding (time-restricted feeding) may have abundant benefits to modulate pain and might provide a novel strategy for the management of pain. 통증은 국제 통증 연구 협회 (IASP)에 의해 실질적/잠재적 조직 손상과 관련된 불쾌한 감각 또는 정서적 경험으로 정의된다. 통증은 감각 및 정서적 요소를 모두 가지고 있기 때문에, 기존의 약리학적 접근법 (NSAID 및 오피오이드) 만으로는 통증을 효과적으로 조절하는 것이 어렵다. 따라서 본 학위 논문은 통증 관리에 대한 다각적인 접근 방식을 모색하기 위해 국소 진통제 (sinomenine), 약침 요법 (Scolopendra subspinipes, 오공 약침) 및 시간 제한 섭식 (금식 / 금식 후 재 섭식)의 통증 조절 가능성을 주요 연구 대상으로 하였다. 첫 번째 장에서는 sinomenine이 통각 수용 감각 신경의 흥분을 감소시킴으로써 말초에서 진통 효과를 유도 할 수 있음을 밝혔다. Sinomenine의 복강 내 투여는 포르말린으로 유도된 통증 행동 및 cFos 발현을 감소 시켰으며, sinomenine이 전압에 따라 개폐 되는 소디움 채널을 억제함을 통각 수용 감각신경에서 확인하였다. 또한 sinomenine을 하지 말단에 국소 투여하였을 때도 통증 행동 감소가 관찰되었다. 이러한 결과는 sinomenine이 국소 마취제 및 진통제로서 적용 가능함을 제시한다. 두 번째 장에서는 오공 약침을 이용한 경혈 자극이 척수에 존재하고 있는 alpha2 아드레날린 수용체를 통해 진통 효과를 유도한다는 것을 밝혔다. 오공약침을 족삼리 영역에 피하 투여 시 옥살리플라틴 처치로 인해 유발된 신경 병증성 통증을 감소시킴을 확인하였으며, alpha2 아드레날린 수용체의 길항제로 알려진 yohimbine을 척수 내에 주입하였을 때 오공 약침으로 유도된 진통 효과가 억제되었다. 따라서, 오공 약침에 의한 경혈 자극은 하행성 통증 억제 시스템을 활성화시켜 진통 효과를 나타내며, 이는 오공 약침을 이용한 통증 관리의 가능성을 시사한다. 세 번째 장에서는 금식과 재 섭식 시 보이는 진통 효과에 중점을 두었다. 자율 섭식과 비교하였을 때, 24 시간 금식과 2 시간 재섭식은 모두 진통 효과를 보였으나 진통 효과를 유도하는 기전이 다름을 확인하였다. 24 시간 금식 시 유도된 통증 억제 효과는 엔도카나비노이드와 오피오이드 시스템이 관여하는 반면, 2 시간 재섭식으로 유도된 진통 반응에는 섭식을 하는 행동 자체와 칼로리 회복이 관여 하였다. 따라서 섭식 조절 (시간 제한 섭식)은 통증 관리를 위한 새로운 치료 전략이 될 수 있을 것이라 기대된다.