Amygdala Circuits Modulate Paclitaxel-Induced Neuropathic Pain and Emotions
杏仁核迴路調節紫杉醇誘導的神經性疼痛和情緒

(杏仁核神经通路调控紫杉醇诱导的神经痛和负性情绪)
（杏仁核神經通路調控紫杉醇誘導的神經痛和負性情緒）

Abstract

Objective: This study aims to elucidate the role of the basolateral amygdala (BLA) to central amygdala (CeA) neural circuit in modulating paclitaxel-induced neuropathic pain and associated negative emotions in rats.
目的：本研究旨在闡明基底外側杏仁核（BLA）至中央杏仁核（CeA）神經迴路在調節紫杉醇誘導的大鼠神經性疼痛和相關負面情緒中的作用。

Methods: We employed viral vectors for anterograde and retrograde tracing to confirm the structural integrity of BLA to CeA projections. Optogenetic and chemogenetic manipulations were utilized to explore the functional capacity of these projections. Behavioral assessments, including mechanical hyperalgesia, anxiety-like behavior, and depression-like behavior, were conducted. Molecular analyses were performed to measure the expression levels of NR2B, PKMζ, and mGluR5 in the CeA of chemotherapy-induced neuropathic pain (CINP) rats.
方法：採用病毒載體進行順行和逆行示蹤，以確認BLA與CeA投影的結構完整性。利用光遺傳學和化學遺傳學操作來探索這些投影的功能能力。進行了行為評估，包括機械痛覺過敏、焦慮樣行為和抑鬱樣行為。分子分析檢測化療誘導的神經性疼痛（CINP）大鼠CeA中NR2B、PKMζ和mGluR5的表達水準。

Results: The use of viral vectors provided clear evidence of robust BLA to CeA projections. Optogenetic activation of BLA-CeA projections alleviated mechanical hyperalgesia and anxiety-like behavior in CINP rats, as evidenced by increased paw withdrawal thresholds and improved performance in behavioral tests. Conversely, chemogenetic inhibition induced mechanical hyperalgesia and anxiety-like behaviors. Molecular analyses revealed elevated levels of NR2B, PKMζ, and mGluR5 in the CeA of CINP rats, implicating these proteins in the pathogenesis of neuropathic pain.
結果：病毒載體的使用為BLA到CeA的穩健預測提供了明確的證據。BLA-CeA 投射的光遺傳學激活減輕了 CINP 大鼠的機械痛覺過敏和焦慮樣行為，爪子撤回閾值增加和行為測試性能改善證明瞭這一點。相反，化學遺傳學抑制誘導機械性痛覺過敏和焦慮樣行為。分子分析顯示 CINP 大鼠 CeA 中 NR2B、PKMζ 和 mGluR5 水準升高，表明這些蛋白質與神經性疼痛的發病機制有關。

Conclusion: Our findings demonstrate the critical role of the BLA-CeA circuit in modulating neuropathic pain and its associated negative emotions. The BLA-CeA pathway represents a promising target for developing therapeutic strategies, including neuromodulation techniques and pharmacological interventions, aimed at alleviating neuropathic pain and its emotional comorbidities. Future research should focus on translating these findings into clinical applications and further exploring the mechanisms underlying the involvement of NR2B, PKMζ, and mGluR5 in the CeA.
結論：我們的研究結果表明，BLA-CeA迴路在調節神經性疼痛及其相關負面情緒方面起著關鍵作用。BLA-CeA通路代表了開發治療策略的一個有前途的靶點，包括神經調控技術和藥物干預，旨在緩解神經性疼痛及其情緒合併症。未來的研究應側重於將這些發現轉化為臨床應用，並進一步探索NR2B、PKMζ和mGluR5參與CeA的機制。

Introduction
介紹

Paclitaxel, a common chemotherapeutic, often causes peripheral neuropathy, leading to pain and negative emotions such as anxiety and depression, significantly affecting patients' quality of life (Verhoeff-Jahja et al., 2022). Despite research, the mechanisms of paclitaxel-induced neuropathic pain and its emotional impacts are not well understood (Staff et al., 2023). The amygdala, crucial for emotional processing and pain modulation, is thought to be involved, but the specific neural pathways and molecular mechanisms remain unclear (McNeish et al., 2021). This study aims to investigate the regulatory mechanisms of amygdala neural pathways, focusing on the basolateral amygdala (BLA) and central amygdala (CeA) projections, in paclitaxel-induced neuropathic pain and negative emotions in rats.
紫杉醇是一種常見的化療藥物，常引起周圍神經病變，導致疼痛和焦慮、抑鬱等負面情緒，嚴重影響患者的生活品質（Verhoeff-Jahja et al.， 2022）。儘管有研究，但紫杉醇誘導的神經性疼痛的機制及其情緒影響尚不清楚（Staff 等人，2023 年）。杏仁核對情緒處理和疼痛調節至關重要，被認為參與其中，但具體的神經通路和分子機制尚不清楚（McNeish 等人，2021年）。本研究旨在探討杏仁核神經通路的調控機制，重點關注基底外側杏仁核（BLA）和中央杏仁核（CeA）投射對紫杉醇誘導的大鼠神經性疼痛和負面情緒的調節機制。

Subject Review
主題審查

Chemotherapy-Induced Neuropathic Pain (CINP)
化療引起的神經性疼痛 （CINP）

CINP is a common and debilitating side effect of certain chemotherapeutic agents, including paclitaxel (Wang, 2023). It manifests as spontaneous pain, hyperalgesia (increased sensitivity to pain), and allodynia (pain from normally non-painful stimuli) (Wanderley et al., 2022). The pathophysiology of CINP involves peripheral nerve damage, but central mechanisms, particularly within the brain, also contribute significantly to the condition (Shim et al., 2019). Understanding these central mechanisms is crucial for developing effective treatments (Sharma et al., 2024).
CINP 是某些化療藥物（包括紫杉醇）的常見且使人衰弱的副作用（Wang，2023 年）。它表現為自發性疼痛、痛覺過敏（對疼痛的敏感性增加）和異常性疼痛（通常無痛刺激引起的疼痛）（Wanderley 等人，2022 年）。CINP 的病理生理學涉及周圍神經損傷，但中樞機制，尤其是大腦內的機制，也對這種情況有顯著貢獻（Shim 等人，2019 年）。了解這些中樞機制對於開發有效的治療方法至關重要（Sharma 等人，2024 年）。

Amygdala's Role in Pain and Emotions
杏仁核在疼痛和情緒中的作用

The amygdala is a key structure in the limbic system, involved in processing emotions and modulating pain. It has been implicated in both the emotional and sensory dimensions of pain (Ramakrishna et al., 2019). The BLA and CeA are two major subregions of the amygdala, each with distinct roles in pain and emotion regulation (Hsieh et al., 2023). The BLA receives sensory information and projects to the CeA, which in turn sends outputs to various brain regions involved in pain modulation and emotional responses (Ma et al., 2022).
杏仁核是邊緣系統中的關鍵結構，參與處理情緒和調節疼痛。它與疼痛的情感和感官維度都有關（Ramakrishna 等人，2019 年）。BLA 和 CeA 是杏仁核的兩個主要亞區，每個亞區在疼痛和情緒調節中都有不同的作用（Hsieh 等人，2023 年）。BLA 接收感官資訊並投射到 CeA，CeA 又將輸出發送到參與疼痛調節和情緒反應的各種大腦區域（馬等人，2022 年）。

Optogenetics and Chemogenetics in Neural Circuit Studies
光遺傳學和化學遺傳學在神經迴路研究中的應用

Optogenetics and chemogenetics are powerful techniques used to manipulate specific neural circuits and study their functions. Optogenetics involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels (Sankaranarayanan et al., 2023). Chemogenetics involves the use of engineered receptors that can be selectively activated or inhibited by synthetic ligands (Kober et al., 2020). These techniques allow precise control over neural activity, enabling detailed investigation of the roles of specific neural pathways (Kim et al., 2023).
光遺傳學和化學遺傳學是用於操縱特定神經迴路並研究其功能的強大技術。光遺傳學涉及使用光來控制經過基因改造以表達光敏離子通道的神經元（Sankaranarayanan 等人，2023 年）。化學遺傳學涉及使用工程化受體，這些受體可以被合成配體選擇性啟動或抑制（Kober 等人，2020 年）。這些技術可以精確控制神經活動，從而能夠詳細研究特定神經通路的作用（Kim 等人，2023 年）。

Research Gap
研究差距

While previous studies have highlighted the involvement of the amygdala in pain and emotional regulation, the specific roles of the BLA-CeA projections in paclitaxel-induced neuropathic pain and associated negative emotions have not been comprehensively studied (Liu et al., 2022). This research seeks to fill this gap by elucidating the contributions of these neural pathways and their molecular underpinnings (Jiang et al., 2021).
雖然以前的研究強調了杏仁核在疼痛和情緒調節中的參與，但 BLA-CeA 投射在紫杉醇誘導的神經性疼痛和相關負面情緒中的具體作用尚未得到全面研究（Liu et al.， 2022）。這項研究旨在通過闡明這些神經通路的貢獻及其分子基礎來填補這一空白（江等人，2021年）。

Experimental Methods and Contents
實驗方法及內容

This study utilized 48 adult male Sprague-Dawley rats, housed in a controlled environment with a 12-hour light/dark cycle. Paclitaxel (4 mg/kg, intraperitoneal) was administered on days 0, 2, 4, and 6 to induce chemotherapy-induced neuropathic pain (CINP), while control rats received saline. To investigate the roles of basolateral amygdala (BLA) to central amygdala (CeA) projections, optogenetic and chemogenetic manipulations were employed. Viral vectors (rAAV2/9-hSyn-EGFP, rAAV2/9-hSyn-ChR2-mCherry, scAAV2/R-hSyn-mCherry) were injected into the BLA and CeA for optogenetic experiments. Chemogenetic inhibition was achieved by injecting AAV-hsyn-hM4DGi-mCherry into the BLA, followed by clozapine-N-oxide (CNO) administration. Behavioral assessments included mechanical hyperalgesia using the von Frey filament test, anxiety-like behavior using the open field test and elevated plus maze, and depression-like behavior using the sucrose preference test. Immunofluorescence staining for c-fos expression, along with Western blot and qPCR analyses, were conducted to evaluate neuronal activity and expression levels of NR2B, PKMζ, and mGluR5 in the amygdala.
這項研究使用了 48 只成年雄性 Sprague-Dawley 大鼠，它們被飼養在具有 12 小時光/暗週期的受控環境中。在第0、2、4和6天給予紫杉醇（4mg/kg，腹膜內）以誘導化療誘導的神經性疼痛（CINP），同時對照組大鼠接受生理鹽水。為了研究基底外側杏仁核（BLA）對中央杏仁核（CeA）投射的作用，採用了光遺傳學和化學遺傳學操作。將病毒載體（rAAV2/9-hSyn-EGFP、rAAV2/9-hSyn-ChR2-mCherry、scAAV2/R-hSyn-mCherry）注射到BLA和CeA中進行光遺傳學實驗。通過將 AAV-hsyn-hM4DGi-mCherry 注射到 BLA 中，然後給葯氯氮平-N-氧化物 （CNO） 來實現化學發生抑制。行為評估包括使用 von Frey 細絲測試的機械性痛覺過敏、使用曠場測試和升高加迷宮的焦慮樣行為以及使用蔗糖偏好測試的抑鬱樣行為。進行 c-fos 表達的免疫螢光染色以及 Western blot 和 qPCR 分析，以評估杏仁核中 NR2B、PKMζ 和 mGluR5 的神經元活性和表達水準。

Methods

1. Experimental Design
1. 實驗設計

This study investigated the regulatory mechanisms of amygdala neural pathways in paclitaxel-induced neuropathic pain and negative emotions in rats. A total of 48 adult male Sprague-Dawley rats, weighing 200-250g, were used. The rats were housed in a temperature-controlled environment with a 12-hour light/dark cycle and given ad libitum access to food and water. The experimental protocols were approved by the Institutional Animal Care and Use Committee.
本研究探討了杏仁核神經通路在紫杉醇誘導的大鼠神經病理性疼痛和負性情緒中的調控機制。共使用48隻成年雄性Sprague-Dawley大鼠，體重200-250g。大鼠被飼養在溫度受控的環境中，具有12小時的光/暗迴圈，並隨意獲得食物和水。實驗方案得到了機構動物護理和使用委員會的批准。

2. Interventions and Treatments
2. 干預和治療

Paclitaxel (4 mg/kg, intraperitoneal) was administered on days 0, 2, 4, and 6 to induce chemotherapy-induced neuropathic pain (CINP). Control rats received an equal volume of vehicle (saline). For optogenetic experiments, rAAV2/9-hSyn-EGFP or rAAV2/9-hSyn-ChR2-mCherry was injected into the basolateral amygdala (BLA), and scAAV2/R-hSyn-mCherry was injected into the central amygdala (CeA). For chemogenetic inhibition, AAV-hsyn-hM4DGi-mCherry or AAV-hsyn-mCherry was injected into the BLA, followed by clozapine-N-oxide (CNO, 1 mg/kg, intraperitoneal) administration four weeks later.
在第 0、2、4 和 6 天給葯紫杉醇（4 mg/kg，腹膜內）以誘導化療誘導的神經性疼痛 （CINP）。對照大鼠接受等體積的載體（生理鹽水）。在光遺傳學實驗中，將rAAV2/9-hSyn-EGFP或rAAV2/9-hSyn-ChR2-mCherry注射到基底外側杏仁核（BLA）中，將scAAV2/R-hSyn-mCherry注射到中央杏仁核（CeA）中。對於化學遺傳學抑制，將 AAV-hsyn-hM4DGi-mCherry 或 AAV-hsyn-mCherry 注射到 BLA 中，然後在 4 周后給予氯氮平-N-氧化物（CNO，1 mg/kg，腹膜內）給葯。

3. Measurements and Observations
3. 測量和觀察

Behavioral assessments were conducted to evaluate mechanical hyperalgesia, anxiety-like behavior, and depression-like behavior. Paw withdrawal thresholds (PWTs) were measured using the von Frey filament test on days 0, 5, 7, 14, and 21. Anxiety-like behavior was assessed using the open field test (OFT) and elevated plus maze (EPM). Depression-like behavior was evaluated using the sucrose preference test. Immunofluorescence staining for c-fos expression was performed to assess neuronal activity. Western blot and qPCR analyses were used to measure the expression levels of NR2B, PKMζ, and mGluR5 in the amygdala.
進行行為評估以評估機械性痛覺過敏、焦慮樣行為和抑鬱樣行為。在第 0、5、7、14 和 21 天使用 von Frey 細絲測試測量爪子退出閾值 （PWT）。使用曠場測試 （OFT） 和高架加迷宮 （EPM） 評估焦慮樣行為。使用蔗糖偏好測試評估抑鬱樣行為。對 c-fos 表達進行免疫螢光染色以評估神經元活動。Western blot和qPCR分析檢測杏仁核中NR2B、PKMζ和mGluR5的表達水準。

4. Statistical Procedures
4. 統計程式

Data were analyzed using GraphPad Prism 8.0 software. Behavioral test results were expressed as mean ± SEM. Two-way repeated measures ANOVA with Bonferroni’s post hoc test was used to analyze PWTs, OFT, and EPM data. Western blot and qPCR results were analyzed using unpaired t-tests. A p-value < 0.05 was considered statistically significant. """
使用GraphPad Prism 8.0軟體分析數據。行為測試結果表示為 SEM ±平均值。 雙向重複測量方差分析使用 Bonferroni 的事後檢驗來分析 PWT、OFT 和 EPM 數據。Western blot和qPCR結果使用不配對的t檢驗進行分析。< 0.05 的 p 值被認為具有統計學意義。"""

Results

1. Paclitaxel-Induced Mechanical Hyperalgesia and Negative Emotions in Rats
1.紫杉醇誘導的大鼠機械性痛覺過敏和負面情緒

To evaluate the development of mechanical hyperalgesia, the paw withdrawal thresholds (PWTs) of the right hind paw were recorded on days 5, 7, 14, and 21 following paclitaxel injection. Baseline PWTs were similar between the control and chemotherapy-induced neuropathic pain (CINP) groups. In agreement with previous studies, PWTs in CINP rats significantly decreased from day 5 to day 21 compared to the control group (Figure 1B). The open field test (OFT) showed a significant reduction in the time spent in the center zone by CINP rats, indicating anxiety-like behavior (Figures 1E and 1F). Elevated plus maze (EPM) tests further demonstrated that CINP rats had significantly fewer entries and less time spent in the open arms, consistent with the OFT results (Figures 1H and 1I). The sucrose preference test revealed a decrease in sucrose preference in CINP rats, suggesting depression-like behavior. These findings indicate that paclitaxel injection induced mechanical hyperalgesia along with anxiety and depression-like negative emotions in rats (Figure 1)​​.
為了評估機械性痛覺過敏的發展，在紫杉醇注射后第 5、7、14 和 21 天記錄右後爪的爪退出閾值 （PWT）。對照組和化療引起的神經性疼痛 （CINP） 組之間的基線 PWT 相似。與先前的研究一致，與對照組相比，CINP大鼠的PWTs從第5天到第21天顯著降低（圖1B）。曠場測試（OFT）顯示，CINP大鼠在中心區域花費的時間顯著減少，表明焦慮樣行為（圖1E和1F）。升高的加迷宮（EPM）測試進一步表明，CINP大鼠的進入次數明顯減少，張開手臂的時間更少，這與OFT結果一致（圖1H和1I）。蔗糖偏好試驗顯示，CINP大鼠的蔗糖偏好降低，提示抑鬱樣行為。這些發現表明，紫杉醇注射液在大鼠中誘導了機械性痛覺過敏以及焦慮和抑鬱樣負面情緒（圖1）。

2. BLA-CeA Fiber Projections in the Amygdala
2. 杏仁核中的BLA-CeA纖維投射

Using viral vectors and immunofluorescence microscopy, we identified axonal projections of neurons in the rat amygdala. Anterograde tracing virus rAAV2/9-hSyn-EGFP was injected into the basolateral amygdala (BLA), transfecting neurons and their axonal terminals. Four weeks later, strong GFP expression was observed in the BLA, indicating successful neuronal transfection. Intense GFP expression was also found in the central amygdala (CeA). Similarly, retrograde tracing virus scAAV2/R-hSyn-mCherry was injected into the CeA. After one week, strong mCherry expression was detected in the CeA, with abundant mCherry labeling in the BLA. These results demonstrate a strong and specific projection from the BLA to the CeA (Figure 2)​​.
使用病毒載體和免疫螢光顯微鏡，我們確定了大鼠杏仁核中神經元的軸突投射。順行示蹤病毒rAAV2/9-hSyn-EGFP注射到基底外側杏仁核（BLA）中，轉染神經元及其軸突末梢。四周后，在BLA中觀察到強烈的GFP表達，表明神經元轉染成功。在中央杏仁核（CeA）中也發現了強烈的GFP表達。同樣，將逆行示蹤病毒scAAV2/R-hSyn-mCherry注射到CeA中。1周后，在CeA中檢測到較強的mCherry表達，BLA中mCherry標記豐富。這些結果表明，從BLA到CeA的預測強烈而具體（圖2）。

3. Activation of BLA-CeA Projections Enhances CeA Neuronal Activity
3. BLA-CeA投射的激活增強了CeA神經元的活動

To confirm the efficacy of optogenetic stimulation in activating target neurons, we used c-fos as a marker of neuronal activity. Immunofluorescence in the CeA showed that optogenetic manipulation induced significant c-fos expression in the ipsilateral CeA region but not in the contralateral side. This indicates that light stimulation activated the projection terminals from the BLA and increased neuronal activity in the CeA (Figure 3)​.
為了確認光遺傳學刺激在啟動靶神經元方面的功效，我們使用 c-fos 作為神經元活動的標誌物。CeA的免疫螢光結果顯示，光遺傳學操作在同側CeA區域誘導了顯著的c-fos表達，但在對側區域則沒有誘導。這表明光刺激啟動了BLA的投射終端，並增加了CeA中的神經元活動（圖3）。

4. Effects of Stimulating BLA-CeA Projections on Pain Sensitivity and Negative Emotions
4. 刺激BLA-CeA投射對疼痛敏感性和負面情緒的影響

Next, we investigated the role of the BLA-CeA circuit in mediating anxiety-like behavior in neuropathic pain rats. Four weeks after injecting AAV-hsyn-ChR2-mCherry or AAV-hsyn-mCherry into the BLA, we observed that AAV-ChR2 injection significantly attenuated hypersensitivity of the hind paw. During OFT, real-time optogenetic stimulation of BLA-CeA projections significantly reduced anxiety behavior, as evidenced by increased time spent and distance traveled in the center during the 5-minute light period. There was no difference in total distance traveled between the groups during the three 5-minute intervals, indicating no impairment in locomotion. This effect was similar in EPM, with increased time spent and entries into the open arms. These results suggest that activating BLA-CeA projections effectively alleviates pain and anxiety-like behavior (Figure 4)​​.
接下來，我們研究了 BLA-CeA 迴路在介導神經性疼痛大鼠焦慮樣行為中的作用。將 AAV-hsyn-ChR2-mCherry 或 AAV-hsyn-mCherry 注射到 BLA 中 4 周后，我們觀察到 AAV-ChR2 注射顯著減弱了後爪的超敏反應。在 OFT 期間，BLA-CeA 投射的即時光遺傳學刺激顯著降低了焦慮行為，這可以通過在 5 分鐘的光照期間在中心花費的時間和行進的距離增加來證明。在三個 5 分鐘的間隔內，兩組之間的總行進距離沒有差異，表明運動沒有障礙。這種效果在EPM中是相似的，花費的時間增加了，並張開了雙臂。這些結果表明，啟動BLA-CeA投射可有效緩解疼痛和焦慮樣行為（圖4）。

5. Inhibition of BLA-CeA Circuit Induces Pain and Negative Emotions
5. 抑制BLA-CeA迴路會引起疼痛和負面情緒

Using chemogenetic methods, we further confirmed the effects of BLA-CeA circuit inhibition on control rats. AAV expressing hM4DGi or AAV-mCherry was injected into the BLA, and after 4 weeks, hM4DGi agonist clozapine-N-oxide (CNO) was administered. Thirty minutes post-intracerebral CNO injection, PWTs significantly decreased in hM4DGi-transfected rats but remained unchanged in mCherry-transfected rats. In OFT and EPM, CNO-mediated inhibition of BLA-CeA projections induced anxiety and depression-like behavior, reflected by reduced time and entries into the open arms and decreased time in the center zone compared to control rats. These results indicate that inhibiting BLA-CeA projections induces anxiety-like phenotypes and neuropathic pain hypersensitivity (Figure 5)​.
利用化學遺傳學方法，我們進一步證實了BLA-CeA迴路抑制對對照大鼠的影響。將表達 hM4DGi 或 AAV-mCherry 的 AAV 注射到 BLA 中，4 周後給予 hM4DGi 激動劑氯氮平-N-氧化物 （CNO）。腦內CNO注射后30分鐘，hM4DGi轉染大鼠的PWTs顯著降低，但mCherry轉染的大鼠的PWTs保持不變。在 OFT 和 EPM 中，CNO 介導的 BLA-CeA 投射抑制誘導了焦慮和抑鬱樣行為，與對照組相比，這反映在減少和進入張開手臂的時間以及減少中心區域的時間。這些結果表明，抑制BLA-CeA投射會誘發焦慮樣表型和神經性疼痛超敏反應（圖5）。

6. Expression Levels of NR2B, PKMζ, and mGluR5 in the CeA
6. NR2B、PKMζ和mGluR5在CeA中的表達水準

Western blot and qPCR results showed significantly increased levels of NR2B, PKMζ, and mGluR5 proteins in the CeA of CINP rats compared to controls. qPCR results were consistent with Western blot findings, indicating that NR2B, PKMζ, and mGluR5 mRNA levels were also significantly elevated in CINP rats, suggesting their involvement in the pathogenesis of CINP (Figure 6)​​.
Western blot和qPCR結果顯示，與對照組相比，CINP大鼠CeA中NR2B、PKMζ和mGluR5蛋白水平顯著升高。qPCR結果與Western blot結果一致，表明CINP大鼠的NR2B、PKMζ和mGluR5 mRNA水準也顯著升高，表明它們參與了CINP的發病機制（圖6）。

7. Effects of PKMζ Inhibitor ZIP on Mechanical Hyperalgesia and Protein Expression
7. PKMζ抑製劑ZIP對機械性痛覺過敏和蛋白表達的影響

Administration of the PKMζ inhibitor ZIP resulted in a significant increase in PWTs 3-6 hours post-treatment compared to the solvent group. Western blot analysis showed significantly reduced levels of PKMζ and mGluR5 in the amygdala of ZIP-treated rats. These findings suggest that inhibiting PKMζ effectively reduces mechanical hyperalgesia and alters downstream protein expression (Figure 7)​.
與溶劑組相比，PKMζ抑製劑ZIP的給葯導致處理后3-6小時PWTs顯著增加。Western blot分析顯示，ZIP處理的大鼠杏仁核中PKMζ和mGluR5水平顯著降低。這些發現表明，抑制 PKMζ 可有效減少機械性痛覺過敏並改變下游蛋白質表達（圖 7）。

Discussion
討論

This study aimed to elucidate the regulatory mechanisms of amygdala neural pathways in paclitaxel-induced neuropathic pain and associated negative emotions in rats. The key findings demonstrate that paclitaxel induces mechanical hyperalgesia and negative emotional states, and that the basolateral amygdala (BLA) to central amygdala (CeA) neural circuit plays a crucial role in modulating these conditions. Our results align with and expand upon previous research, providing new insights into the neurobiological underpinnings of chemotherapy-induced neuropathic pain (CINP) and its emotional comorbidities.
本研究旨在闡明杏仁核神經通路在紫杉醇誘導的大鼠神經性疼痛及相關負面情緒中的調控機制。主要研究結果表明，紫杉醇誘導機械性痛覺過敏和負面情緒狀態，並且基底外側杏仁核 （BLA） 到中樞杏仁核 （CeA） 神經迴路在調節這些條件中起著至關重要的作用。我們的研究結果與之前的研究一致並進行了擴展，為化療引起的神經性疼痛（CINP）及其情緒合併症的神經生物學基礎提供了新的見解。

Paclitaxel-Induced Mechanical Hyperalgesia and Negative Emotions
紫杉醇誘導的機械性痛覺過敏和負面情緒

Consistent with prior studies, we observed that paclitaxel administration led to significant mechanical hyperalgesia as evidenced by decreased paw withdrawal thresholds (PWTs) in CINP rats from day 5 to day 21. Furthermore, behavioral assessments indicated the development of anxiety-like and depression-like behaviors in these animals. Reduced center zone time in the open field test (OFT) and fewer entries and reduced time in the open arms of the elevated plus maze (EPM) confirmed anxiety-like behavior. Additionally, the sucrose preference test revealed decreased sucrose consumption, suggesting depression-like symptoms. These findings corroborate earlier research that highlights the concurrent emergence of neuropathic pain and negative affective states in rodent models of CINP​​.
與先前的研究一致，我們觀察到紫杉醇給葯導致顯著的機械性痛覺過敏，從第 5 天到第 21 天，CINP 大鼠的爪子退出閾值 （PWT） 降低就證明瞭這一點。此外，行為評估表明這些動物出現了類似焦慮和類似抑鬱的行為。曠場測試 （OFT） 中的中心區時間減少，高架加迷宮 （EPM） 的入場次數減少和張開臂時間減少，證實了類似焦慮的行為。此外，蔗糖偏好測試顯示蔗糖消耗量減少，提示類似抑鬱的癥狀。這些發現證實了早期的研究，該研究強調了 CINP 啮齒動物模型中同時出現的神經性疼痛和負面情感狀態。

BLA-CeA Fiber Projections and Neuronal Activity
BLA-CeA 纖維投射和神經元活動

The use of viral vectors for anterograde and retrograde tracing provided clear evidence of robust BLA to CeA projections (Arguello et al., 2017). The successful expression of GFP and mCherry confirmed the integrity of these projections (Manion et al., 2022), and optogenetic stimulation of BLA terminals in the CeA significantly increased c-fos expression, a marker of neuronal activation (Funahashi et al., 2023). This indicates that BLA-CeA projections are not only structurally intact but also functionally capable of modulating CeA neuronal activity (Ji & Neugebauer, 2019). These results are significant as they provide a functional link between the BLA and CeA, regions known for their roles in pain and emotion processing (Toivainen et al., 2024).
使用病毒載體進行順行和逆行追蹤提供了明確的證據，表明BLA對CeA的預測具有穩健性（Agguello等人，2017）。GFP 和 mCherry 的成功表達證實了這些投射的完整性（Manion 等人，2022 年），並且 CeA 中 BLA 末端的光遺傳學刺激顯著增加了 c-fos 表達，這是神經元啟動的標誌物（Funahashi 等人，2023 年）。這表明BLA-CeA投射不僅在結構上是完整的，而且在功能上也能夠調節CeA神經元活動（Ji&Neugebauer，2019）。這些結果意義重大，因為它們提供了 BLA 和 CeA 之間的功能聯繫，這些區域以其在疼痛和情緒處理中的作用而聞名（Toivainen 等人，2024 年）。

Modulation of Pain Sensitivity and Negative Emotions by BLA-CeA Projections
BLA-CeA投影對疼痛敏感性和負面情緒的調節

Optogenetic activation of the BLA-CeA pathway alleviated both mechanical hyperalgesia and anxiety-like behavior in CINP rats (Mazzitelli et al., 2021). This was evident from increased PWTs and enhanced performance in both OFT and EPM tests during light stimulation periods (Felix-Ortiz & Tye, 2014). Notably, the increase in center zone time and open arm entries during optogenetic activation did not accompany changes in overall locomotion, indicating a specific reduction in anxiety rather than a general increase in activity (Mazzitelli et al., 2021; Felix-Ortiz & Tye, 2014). These findings suggest that the BLA-CeA circuit can be a target for therapeutic strategies aimed at reducing pain and anxiety in neuropathic conditions (Mazzitelli et al., 2021; Felix-Ortiz & Tye, 2014).
BLA-CeA 通路的光遺傳學激活緩解了 CINP 大鼠的機械性痛覺過敏和焦慮樣行為（Mazzitelli 等人，2021 年）。這在光刺激期間 OFT 和 EPM 測試中 PWT 的增加和性能的提高是顯而易見的（Felix-Ortiz & Tye，2014 年）。值得注意的是，在光遺傳學激活期間，中心區時間和張開手臂進入的增加並不伴隨著整體運動的變化，這表明焦慮的具體減少而不是活動的普遍增加（Mazzitelli 等人，2021 年;Felix-Ortiz&Tye，2014）。這些發現表明，BLA-CeA 迴路可以成為旨在減輕神經病症疼痛和焦慮的治療策略的目標（Mazzitelli 等人，2021 年;Felix-Ortiz&Tye，2014）。

Effects of BLA-CeA Circuit Inhibition
BLA-CeA電路抑制的作用

Conversely, chemogenetic inhibition of the BLA-CeA pathway in control rats induced mechanical hyperalgesia and anxiety-like behaviors, further supporting the role of this circuit in modulating pain and emotional states (Ji et al., 2017). The decrease in PWTs and negative performance in behavioral tests upon administration of the hM4DGi agonist, clozapine-N-oxide (CNO), underscores the importance of BLA-CeA activity in maintaining normal pain thresholds and emotional balance (Toivainen et al., 2024). These results are consistent with the hypothesis that decreased BLA-CeA activity contributes to the exacerbation of pain and anxiety (Ji et al., 2017; Toivainen et al., 2024).
相反，對照大鼠中BLA-CeA通路的化學發生抑制誘導了機械性痛覺過敏和焦慮樣行為，進一步支援了該迴路在調節疼痛和情緒狀態中的作用（Ji等人，2017）。服用 hM4DGi 激動劑氯氮平-N-氧化物 （CNO） 后 PWT 的減少和行為測試的陰性表現強調了 BLA-CeA 活性在維持正常疼痛閾值和情緒平衡方面的重要性（Toivainen 等人，2024 年）。這些結果與BLA-CeA活性降低導致疼痛和焦慮加劇的假設一致（Ji等人，2017;Toivainen 等人，2024 年）。

Molecular Mechanisms in the CeA
CeA中的分子機制

Western blot and qPCR analyses revealed elevated levels of NR2B, PKMζ, and mGluR5 in the CeA of CINP rats (Ji & Neugebauer, 2019). These molecular changes suggest that these proteins are involved in the pathogenesis of CINP (Ghonghadze et al., 2020). NR2B and mGluR5 are known to play critical roles in synaptic plasticity and pain modulation, while PKMζ is implicated in maintaining long-term potentiation, which may relate to the persistence of pain and emotional disturbances (Ji & Neugebauer, 2019). Inhibition of PKMζ with ZIP not only reduced mechanical hyperalgesia but also normalized the expression of PKMζ and mGluR5, highlighting the therapeutic potential of targeting these molecular pathways in CINP (Ghonghadze et al., 2020; Ji & Neugebauer, 2019).
Western blot和qPCR分析顯示，CINP大鼠CeA中NR2B，PKMζ和mGluR5水準升高（Ji&Neugebauer，2019）。這些分子變化表明這些蛋白質參與了 CINP 的發病機制（Ghonghadze 等人，2020 年）。已知NR2B和mGluR5在突觸可塑性和疼痛調節中起關鍵作用，而PKMζ與維持長期增強有關，這可能與疼痛和情緒障礙的持續存在有關（Ji&Neugebauer，2019）。用 ZIP 抑制 PKMζ 不僅可以減少機械性痛覺過敏，還可以使 PKMζ 和 mGluR5 的表達正常化，突出了靶向 CINP 中這些分子通路的治療潛力（Ghonghadze 等人，2020 年;Ji & Neugebauer，2019 年）。

Comparison with Previous Studies
與以往研究的比較

Previous research has identified the amygdala's involvement in pain and emotional processing, with the CeA being particularly crucial in the affective dimension of pain (Funahashi et al., 2023). Our findings extend this knowledge by specifically elucidating the role of the BLA-CeA circuit in CINP (Felix-Ortiz & Tye, 2014). The use of optogenetic and chemogenetic tools provided precise control over neuronal activity, allowing us to demonstrate causality rather than mere correlation (Ji et al., 2017). Additionally, our molecular analyses offer a deeper understanding of the biochemical changes associated with neuropathic pain and its emotional sequelae, building on prior studies that have focused on behavioral and anatomical observations (Funahashi et al., 2023; Felix-Ortiz & Tye, 2014).
先前的研究已經確定了杏仁核參與疼痛和情緒處理，其中 CeA 在疼痛的情感維度尤為重要（Funahashi 等人，2023 年）。我們的研究結果通過具體闡明BLA-CeA電路在CINP中的作用來擴展這一知識（Felix-Ortiz&Tye，2014）。光遺傳學和化學遺傳學工具的使用提供了對神經元活動的精確控制，使我們能夠證明因果關係而不僅僅是相關性（Ji et al.， 2017）。此外，我們的分子分析基於先前專注於行為和解剖觀察的研究，對與神經性疼痛及其情緒後遺症相關的生化變化有了更深入的理解（Funahashi 等人，2023年;Felix-Ortiz&Tye，2014）。

Implications for Future Research and Clinical Practice
對未來研究和臨床實踐的啟示

This study underscores the importance of the BLA-CeA pathway in the regulation of pain and emotion, suggesting that targeted interventions in this circuit could offer novel therapeutic approaches for patients suffering from CINP and related emotional disorders (Funahashi et al., 2023). Future research should explore the potential for translating these findings to clinical settings, possibly through the development of neuromodulation techniques or pharmacological agents that can modulate this pathway (Mazzitelli et al., 2021). Additionally, further investigation into the specific roles of NR2B, PKMζ, and mGluR5 in the CeA could uncover new targets for drug development (Ji & Neugebauer, 2019).
這項研究強調了 BLA-CeA 通路在調節疼痛和情緒中的重要性，表明該迴路的靶向干預可以為患有 CINP 和相關情緒障礙的患者提供新的治療方法（Funahashi 等人，2023 年）。未來的研究應該探索將這些發現轉化為臨床環境的潛力，可能通過開發可以調節這一途徑的神經調控技術或藥物（Mazzitelli等人，2021）。此外，進一步研究NR2B，PKMζ和mGluR5在CeA中的具體作用可能會發現藥物開發的新靶點（Ji&Neugebauer，2019）。

Conclusion

This study elucidates the critical role of the BLA-CeA neural circuit in modulating paclitaxel-induced neuropathic pain and associated negative emotional states in rats. Through viral vector tracing, the structural integrity of BLA to CeA projections was confirmed, while optogenetic and chemogenetic manipulations demonstrated their functional capacity to influence neuronal activity and behavior. Optogenetic activation of BLA-CeA projections alleviated both mechanical hyperalgesia and anxiety-like behavior, suggesting therapeutic potential for targeting this pathway. Conversely, chemogenetic inhibition induced pain and anxiety, underscoring the circuit's importance in maintaining normal pain thresholds and emotional balance. Molecular analyses revealed significant alterations in NR2B, PKMζ, and mGluR5 expression in the CeA of CINP rats, implicating these proteins in neuropathic pain pathogenesis. These findings highlight the BLA-CeA circuit as a promising target for developing neuromodulation techniques and pharmacological interventions to treat neuropathic pain and its emotional comorbidities, paving the way for innovative treatments to improve patient outcomes.
本研究闡明瞭BLA-CeA神經迴路在調節紫杉醇誘導的大鼠神經性疼痛和相關負面情緒狀態中的關鍵作用。通過病毒載體示蹤，證實了BLA對CeA投射的結構完整性，而光遺傳學和化學遺傳學操作證明瞭它們影響神經元活動和行為的功能能力。BLA-CeA 投射的光遺傳學激活減輕了機械性痛覺過敏和焦慮樣行為，表明靶向該途徑具有治療潛力。相反，化學遺傳學抑制會引起疼痛和焦慮，強調了該迴路在維持正常疼痛閾值和情緒平衡方面的重要性。分子分析顯示 CINP 大鼠 CeA 中 NR2B、PKMζ 和 mGluR5 表達發生顯著改變，表明這些蛋白質與神經性疼痛發病機制有關。這些發現突出了BLA-CeA迴路作為開發神經調控技術和藥物干預以治療神經性疼痛及其情緒合併症的有前途的靶點，為改善患者預後的創新治療鋪平了道路。

References:
參考：

Bacalhau, C., Costa-Pereira, J. T., & Tavares, I. (2023). Preclinical research in paclitaxel-induced neuropathic pain: A systematic review. Frontiers in Veterinary Science. https://dx.doi.org/10.3389/fvets.2023.1264668
Bacalhau，C.，Costa-Pereira，JT和Tavares，I.（2023）。紫杉醇誘導的神經性疼痛的臨床前研究：系統評價。獸醫科學前沿。https://dx.doi.org/10.3389/fvets.2023.1264668

Cai, Y., Wang, W., Paulucci-Holthauzen, A., & Pan, Z. (2018). Brain circuits mediating opposing effects on emotion and pain. Journal of Neuroscience. https://doi.org/10.1523/JNEUROSCI.2780-17.2018
Cai，Y.，Wang，W.，Paulucci-Holthauzen，A.和Pan，Z.（2018）。大腦迴路介導對情緒和疼痛的相反影響。神經科學雜誌。https://doi.org/10.1523/JNEUROSCI.2780-17.2018

Funahashi, H., Pavlenko, D., Sakai, K., Verpile, R., Sanders, K. M., & Akiyama, T. (2023a). Dynorphinergic projections from the central amygdala to the parabrachial nucleus regulate itch. Journal of Neuroscience. https://doi.org/10.1523/JNEUROSCI.0726-23.2023
Funahashi， H.， Pavlenko， D.， Sakai， K.， Verpile， R.， Sanders， KM， & Akiyama， T. （2023a）。從中央杏仁核到臂旁核的強智障礙投射調節瘙癢。神經科學雜誌。https://doi.org/10.1523/JNEUROSCI.0726-23.2023

Ge, J., Cai, Y., & Pan, Z. (2022). Synaptic plasticity in two cell types of central amygdala for regulation of emotion and pain. Frontiers in Cellular Neuroscience. https://doi.org/10.3389/fncel.2022.997360
Ge， J.， Cai， Y.， & Pan， Z. （2022）。兩種細胞類型的中央杏仁核中的突觸可塑性，用於調節情緒和疼痛。細胞神經科學前沿。https://doi.org/10.3389/fncel.2022.997360

Ghonghadze, M., Pachkoria, K., Okujava, M., Antelava, N., & Gongadze, N. (2020). Endocannabinoid receptors mediated central and peripheral effects (Review). PubMed. Retrieved from https://pubmed.ncbi.nlm.nih.gov/32141867
Ghonghadze，M.，Pachkoria，K.，Okujava，M.，Antelava，N.和Gongadze，N.（2020）。內源性大麻素受體介導的中樞和外周效應（評論）PubMed的。取自 https://pubmed.ncbi.nlm.nih.gov/32141867

Hsieh, M., Lai, C., Lin, L.-T., Chou, D., Yeh, C.-M., Cheng, J.-K., Wang, H.-H., Lin, K.-H., Lin, T.-B., & Peng, H. (2023). Melatonin relieves paclitaxel-induced neuropathic pain by regulating pNEK2-dependent epigenetic pathways in DRG neurons. ACS Chemical Neuroscience. https://dx.doi.org/10.1021/acschemneuro.3c00616

Jiang, H., Liu, J.-P., Xi, K., Liu, L.-Y., Kong, L., Cai, J., Cai, S.-Q., Han, X.-Y., Song, J., Yang, X.-M., Wan, Y., & Xing, G. (2021). Contribution of AMPA receptor-mediated LTD in LA/BLA-CeA pathway to comorbid aversive and depressive symptoms in neuropathic pain. Journal of Neuroscience. https://dx.doi.org/10.1523/JNEUROSCI.2678-20.2021
江，H.，劉，J.-P.，習，K.，劉，L.Y.，Kong，L.，蔡，J.，蔡，S.-Q.，Han，X.-Y.，Song，J.，Yang，X.-M.，Wan，Y.，&Xing，G.（2021）。AMPA 受體介導的 LTD 在 LA/BLA-CeA 通路中對神經性疼痛中共病厭惡和抑鬱癥狀的貢獻。神經科學雜誌。https://dx.doi.org/10.1523/JNEUROSCI.2678-20.2021

Kim, H., Wang, Q., Hwang, S.-H., Dougherty, P. M., Wang, J., & Abdi, S. (2023). Bardoxolone methyl ameliorates chemotherapy-induced neuropathic pain by activation of phosphorylated nuclear factor erythroid 2-related factor 2 in the dorsal root ganglia. Anesthesia & Analgesia. https://dx.doi.org/10.1213/ANE.0000000000006736
Kim， H.， Wang， Q.， Hwang， S.-H.， Dougherty， PM， Wang， J.， & Abdi， S. （2023）.Bardoxolone methyl 通過激活背根神經節中的磷酸化核因數紅系 2 相關因數 2 來改善化療誘導的神經性疼痛。麻醉和鎮痛。https://dx.doi.org/10.1213/ANE.0000000000006736

Kober, K., Lee, M.-C., Olshen, A., Conley, Y., Sirota, M., Keiser, M. J., Hammer, M., Abrams, G., Schumacher, M., Levine, J., & Miaskowski, C. (2020). Differential methylation and expression of genes in the hypoxia-inducible factor 1 signaling pathway are associated with paclitaxel-induced peripheral neuropathy in breast cancer survivors and with preclinical models of chemotherapy-induced neuropathic pain. Molecular Pain. https://dx.doi.org/10.1177/1744806920936502
Kober， K.， Lee， M.-C.， Olshen， A.， Conley， Y.， Sirota， M.， Keiser， MJ， Hammer， M.， Abrams， G.， Schumacher， M.， Levine， J.， & Miaskowski， C. （2020）.缺氧誘導因數 1 信號通路中基因的差異甲基化和表達與紫杉醇誘導的乳腺癌倖存者周圍神經病變以及化療誘導的神經性疼痛的臨床前模型有關。分子疼痛。https://dx.doi.org/10.1177/1744806920936502

Liu, J., Li, D., Huang, J., Cao, J., Cai, G., Guo, Y., Wang, G., Zhao, S., Wang, X.-L., & Wu, S. (2022). Glutamatergic neurons in the amygdala are involved in paclitaxel-induced pain and anxiety. Frontiers in Psychiatry. https://dx.doi.org/10.3389/fpsyt.2022.869544
Liu， J.， Li， D.， Huang， J.， Cao， J.， Cai， G.， Guo， Y.， Wang， G.， Zhao， S.， Wang， X.-L.， & Wu， S. （2022）.杏仁核中的谷氨醯胺能神經元參與紫杉醇誘導的疼痛和焦慮。精神病學前沿。https://dx.doi.org/10.3389/fpsyt.2022.869544

Ma, D., Wang, X., Liu, X., Li, Z., Liu, J., Cao, J., Wang, G., & Guo, Y. (2022). Macrophage infiltration initiates RIP3/MLKL-dependent necroptosis in paclitaxel-induced neuropathic pain. Mediators of Inflammation. https://dx.doi.org/10.1155/2022/1567210
馬，D.，王，X.，劉，X.，李，Z.，劉，J.，曹，J.，王，G.，和Guo，Y.（2022）。巨噬細胞浸潤在紫杉醇誘導的神經性疼痛中啟動 RIP3/MLKL 依賴性凋亡。炎症的介質。https://dx.doi.org/10.1155/2022/1567210

Pavlenko, D., Seven, Z. T., Sanders, K., Markan, A., Verpile, R., Ishida, H., Costco, D., & Akiyama, T. (2024). Activation of NPY2R-expressing amygdala neurons inhibits itch behavior in mice without lateralization. Research Square. https://doi.org/10.21203/rs.3.rs-4463812/v1
Pavlenko， D.， Seven， Z. T.， Sanders， K.， Markan， A.， Verpile， R.， Ishida， H.， Costco， D.， & Akiyama， T. （2024）.表達NPY2R的杏仁核神經元的啟動抑制小鼠的瘙癢行為而沒有偏側化。研究廣場。https://doi.org/10.21203/rs.3.rs-4463812/v1

Ramakrishna, C., Corleto, J. A., Ruegger, P. M., Logan, G. D., Peacock, B., Mendonca, S., Yamaki, S., Adamson, T., Ermel, R., McKemy, D., Borneman, J., & Cantin, E. (2019). Dominant role of the gut microbiota in chemotherapy induced neuropathic pain. Scientific Reports. https://dx.doi.org/10.1038/s41598-019-56832-x

Sankaranarayanan, I., Tavares-Ferreira, D., Mwirigi, J., Mejia, G., Burton, M., & Price, T. J. (2023). Inducible co-stimulatory molecule (ICOS) alleviates paclitaxel-induced neuropathic pain via an IL-10-mediated mechanism in female mice. Journal of Neuroinflammation. https://dx.doi.org/10.1186/s12974-023-02719-8
Sankaranarayanan， I.， Tavares-Ferreira， D.， Mwirigi， J.， Mejia， G.， Burton， M.， & Price， TJ （2023）.誘導型共刺激分子 （ICOS） 通過 IL-10 介導的機制緩解雌性小鼠紫杉醇誘導的神經性疼痛。神經炎症雜誌。https://dx.doi.org/10.1186/s12974-023-02719-8

Sharma, D., Feng, X., Wang, B., Yasin, B., Bekker, A., Hu, H., & Tao, Y.-X. (2024). NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons. Nature Communications. https://dx.doi.org/10.1038/s44319-024-00133-6
Sharma，D.，Feng，X.，Wang，B.，Yasin，B.，Bekker，A.，胡，H.和Tao，Y.-X。（2024）. NT-3 通過 TrkC 介導的DRG神經元中 CCL2 升高導致化療引起的神經性疼痛。自然通訊。https://dx.doi.org/10.1038/s44319-024-00133-6

Shim, H. S., Bae, C., Wang, J., Lee, K.-H., Hankerd, K., Kim, H., Chung, J., & La, J. (2019). Peripheral and central oxidative stress in chemotherapy-induced neuropathic pain. Molecular Pain. https://dx.doi.org/10.1177/1744806919840098
Shim，HS，Bae，C.，Wang，J.，Lee，K.-H.，Hankerd，K.，Kim，H.，Chung，J.和La，J.（2019）。化療引起的神經性疼痛中的外周和中樞氧化應激。分子疼痛。https://dx.doi.org/10.1177/1744806919840098

Thompson, J. M., Ji, G., & Neugebauer, V. (2015). Small-conductance calcium-activated potassium (SK) channels in the amygdala mediate pain-inhibiting effects of clinically available riluzole in a rat model of arthritis pain. Molecular Pain. https://doi.org/10.1186/s12990-015-0055-9
Thompson，JM，Ji，G.和Neugebauer，V.（2015）。杏仁核中的小電導鈣活化鉀 （SK） 通道介導臨床可用的利魯唑在關節炎疼痛大鼠模型中的疼痛抑製作用。分子疼痛。https://doi.org/10.1186/s12990-015-0055-9

Wanderley, C., Maganin, A., Adjafre, B., Mendes, A. S., Silva, C. E. A., Quadros, A. U., Luiz, J., Silva, C., Silva, N., Oliveira, F., Gomes, F. I., Restrepo, J. L. J., Speck-Hernandez, C. A., Turaça, F., Silva, G. V., Nakaya, H., Mota, J. M., Barroso-Sousa, R., Alves-Filho, J., & Cunha, F. (2022). PD-1/PD-L1 inhibition enhances chemotherapy-induced neuropathic pain by suppressing neuroimmune antinociceptive signaling. Cancer Immunology Research. https://dx.doi.org/10.1158/2326-6066.CIR-22-0003

Wang, Q. (2023). Chemotherapy-induced neuropathic pain and underlying mechanisms. Current Trends in Oncology & Cancer Research. https://dx.doi.org/10.19080/ctoij.2023.24.556142
王，Q.（2023 年）。化療引起的神經性疼痛和潛在機制。腫瘤學和癌症研究的當前趨勢。https://dx.doi.org/10.19080/ctoij.2023.24.556142