Electro-acupuncture potentiates the disulphide-reducing activities of thioredoxin system by increasing thioredoxin expression in ischemia-reperfused rat brains

Reactive oxygen species can directly affect the conformation and activity of sulfhydryl-containing proteins by oxidation of their thiol moiety. During the process of ischemia-reperfusion, the thioredoxin (Trx) system (consisting of thioredoxin reductase (TR), Trx and NADPH) prevents susceptible proteins from this oxidative modification. Oxidative damage is one of the most damaging stress in ischemia. If oxidative stress could be minimized, the damage occurred will be minimized accordingly. We therefore investigated whether electroacupuncture (EA) treatment at Fengchi (GB20) or Zusanli (ST36) acupoints in post-ischemic rats could increase TR-related activities and Trx expression which would translate into maintaining the intact thiol moiety of susceptible proteins in the surrounding. Our results indicated that EA treatment at either acupoint increased the Trx expression in ischemic-reperfused brain tissues. Induced Trx expressed levels gradually increased from post-ischemia day 1 to day 4. Statistical analysis revealed that there was no observable difference in the effect of EA treatment at GB20 and ST36. Sham EA treatment did not induce any Trx expression. EA at either acupoint did not alter TR activities in both non-ischemic and ischemic-reperfused rat brains. Taken overall, our finding suggests that EA treatment at GB20 or ST36 could increase Trx expression which could minimize oxidative modifications of thiol groups of surrounding proteins.     

Photoacoustic microscopy of electronic acupuncture (EA) effect in small animals

Acupuncture has been an effective treatment for various pain in China for several thousand years. However, the mechanisms underlying this mysterious ancient healing are still largely unknown. Here we applied photoacoustic microscopy (PAM) to investigate brain hemodynamic changes in response to electronic acupuncture (EA) at ST36 (Zusanli). Due to the high optical absorption of blood at 532 nm, PAM could sensitively probe changes in hemoglobin concentration (HbT, i.e., cerebral blood volume [CBV]) of cortical regions in high resolution. Six healthy mice were stimulated at the acupoint and three healthy mice were stimulated at sham points. Remarkable CBV changes in sensorimotor and retrosplenial agranular cortex were observed. Results showed the potential of PAM as a visualization tool to study the acupuncture effect on brain hemodynamics in animal models.

( a ) Schematic showing the stimulation points. ( b ) B‐scan images overlaid with mouse atlas. ( c ) & ( d ) Statistical results of CBV changes from cortical regions.     

Photoacoustic microscopy for evaluating a lipopolysaccharide‐induced inflammation model in mice

Photoacoustic microscopy (PAM) is a noninvasive imaging technique and is excellent to study structural and functional changes in the microcirculation. In this work, a lipopolysaccharide (LPS)‐induced inflammation model in mice is noninvasively evaluated by PAM. PAM is used to image the microvascular structural changes in mice for 8 hours after the LPS with different concentrations is applied. Quantitative analysis of five vessel parameters is conducted, which shows that the rate of reduction in microvasculature is highly dependent on the applied LPS concentrations. For low‐concentration LPS, changes in the microvasculature are not obvious over the observation period, whereas for high‐concentration LPS, quick and marked reduction in the microvasculature is observed. In addition, changes in capillaries are more significant than those in relatively large vessels. The results show that PAM is able to evaluate the inflammation mouse model by studying structural (and potentially functional) changes in the microcirculation. Furthermore, PAM may have potential for early intervention and treatment plan optimization of sepsis by monitoring the microcirculation and inflammatory response.      

The real-time detection of acupuncture-induced extracellular ATP mobilization in acupoints and exploration of its role in acupuncture analgesia

Our and in vitro studies had confirmed that mechanosensitive ATP release and accumulation in acupoints was elicited by acupuncture (AP), which might be a pivotal step for triggering AP analgesia. But to date, the dynamics of extracellular ATP (eATP) in the interstitial space during AP process was poorly known, mainly due to the low temporal resolution of the current detection approach. This study attempted to capture rapid eATP signals in vivo in the process of needling, and further explored the role of this eATP mobilization in initiating AP analgesic effect. Ipsilateral 20-min needling was applied on Zusanli acupoint (ST36) of complete Freund’s adjuvant (CFA)–induced ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in the interstitial space was microdialyzed and real-time quantified by luciferin-luciferase assay at 1-min interval with the aid of the microfluid chip. We revealed in behavioral tests that modulation of eATP levels in ST36 influenced AP analgesic effect on ankle arthritis. A transient eATP accumulation was induced by needling that started to mobilize at 4 min, climbed to the peak of 11.21 nM within 3.25 min and gradually recovered. Such AP-induced eATP mobilization was significantly impacted by ankle inflammation, needling depth, needle manipulation, and the presence of local ecto-nucleotidases. This work reveals that needling elicits a transient eATP mobilization in acupoints, which contributes to initiating AP analgesia. This study will help us better understand the peripheral mechanism of AP analgesia and guide clinicians to optimize the needle manipulations to improve AP efficacy.     

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.      

Acupuncture Induces Divergent Alterations of Functional Connectivity within Conventional Frequency Bands: Evidence from MEG Recordings

As an ancient Chinese healing modality which has gained increasing popularity in modern society, acupuncture involves stimulation with fine needles inserted into acupoints. Both traditional literature and clinical data indicated that modulation effects largely depend on specific designated acupoints. However, scientific representations of acupoint specificity remain controversial. In the present study, considering the new findings on the sustained effects of acupuncture and its time-varied temporal characteristics, we employed an electrophysiological imaging modality namely magnetoencephalography with a temporal resolution on the order of milliseconds. Taken into account the differential band-limited signal modulations induced by acupuncture, we sought to explore whether or not stimulation at Stomach Meridian 36 (ST36) and a nearby non-meridian point (NAP) would evoke divergent functional connectivity alterations within delta, theta, alpha, beta and gamma bands. Whole-head scanning was performed on 28 healthy participants during an eyes-closed no-task condition both preceding and following acupuncture. Data analysis involved calculation of band-limited power (BLP) followed by pair-wise BLP correlations. Further averaging was conducted to obtain local and remote connectivity. Statistical analyses revealed the increased connection degree of the left temporal cortex within delta (0.5–4 Hz), beta (13–30 Hz) and gamma (30–48 Hz) bands following verum acupuncture. Moreover, we not only validated the closer linkage of the left temporal cortex with the prefrontal and frontal cortices, but further pinpointed that such patterns were more extensively distributed in the ST36 group in the delta and beta bands compared to the restriction only to the delta band for NAP. Psychophysical results for significant pain threshold elevation further confirmed the analgesic effect of acupuncture at ST36. In conclusion, our findings may provide a new perspective to lend support for the specificity of neural expression underlying acupuncture.     

Structure and function of the microcirculatory bed in the preserved heart]

The morphological analysis of the state of the heart during hypertermal perfusion with different conservants reveals clear dependence of the microcirculation and the activity of the heart upon the type of the conservant. Perfusion with a salt solution and hemodilution is accompanied by pronounced disorders in microcirculation and unsatisfactory parameters of the cardiac activity. Conservation with cryoprecipitated plasma is characterized by comparatively less microcirculatory disorders, but fails to give a reliable safety of the heart. When using medium 199, changes in microcirculation were found to be minimal and parameters of cardiac activity were satisfactory. In the complex of non-specific changes in microcirculatory vessels the maximum structural lability was revealed in blood capillaries and vessels of the postcapillary-venular link.     

Development of the uterine microcirculatory bed in postnatal ontogeny

Age peculiarities of common structure of the microcirculatory pathways, specific volumes and amount of microvessels, zonal parameters of pericapillary microcirculation of metabolites, glycosamine glycans and glycoproteins contents, phosphatase and ATPase activity, collagen and elastic fibers of the microcirculatory bed vessels have been studied in 190 unchanged uteri, beginning from 36-week-old fetuses up to 35 years of age. The microcirculatory bed of myometrium during the period of postnatal ontogenesis investigated possesses a polymer-homonomous structural organization; its base make myoangiomas, including the terminal arteriole with precapillaries and collecting venule, that run from it. Similar structure of the myometrium microcirculatory pathways is already observed in fetuses of late antenatal period. In endometrium formed glanduloangionomas are revealed in the prepubertal age. Increase in glycosamine glycans contents is stated in the microvessel walls and in the uterine stroma during the process of its development. Compensatory-adaptive changes in the uterine microcirculatory bed are described during the newborn period up to the puberty. Functionally mature structures of the microcirculatory pathways in the submucosal and vascular layers of myometrium, endometrium and in the cervix uteri are found to be formed earlier than in other areas of the organ.     

Isotropic high resolution optoacoustic imaging with linear detector arrays in bi‐directional scanning

Optoacoustic (photoacoustic) imaging is often performed with one‐dimensional transducer arrays, in analogy to ultrasound imaging. Optoacoustic imaging using linear arrays offers ease of implementation but comes with several performance drawbacks, in particular poor elevation resolution, i.e. the resolution along the axis perpendicular to the focal plane. Herein, we introduce and investigate a bi‐directional scanning approach using linear arrays that can improve the imaging performance to quasi‐isotropic transverse resolution. We study the approach theoretically and perform numerical simulations and phantom measurements to evaluate its performance under defined conditions. Finally, we discuss the features and the limitations of the proposed method.

The poor elevation resolution in a linear scan (left image) is overcome by the proposed bi‐directional scanning approach that yields isotropic transverse resolution (right).     

同一药物不同剂量足三里穴位注射对心气虚证的效应对比研究

目的 研究参芪扶正注射液足三里穴位注射对心气虚证的效应与用药剂量的关系.方法 通过负重游泳及灌服大剂量心得安法获得心气虚证大鼠模型,对各治疗组分别进行不同剂量参芪扶正注射液的足三里穴注治疗.连续治疗10 d后,观察并记录大鼠的一般状况和症状;通过ELISA法检测各组血清心钠素（ANP）及环磷酸腺苷（cAMP）含量;通过比色法检测各组血清超氧化物歧化酶（SOD）活性;通过HE染色法检测各组心肌组织病理改变.结果 和正常对照组相比,模型对照组出现疲软无力,舌质发紫,呼吸急促等明显的心气虚症状;血清ANP浓度升高,cAMP浓度降低,SOD活性降低,均具有极其显著性意义（P〈0.001）; 心肌组织病理示：炎细胞浸润明显,心肌细胞严重水肿,排列紊乱.和模型组相比,各治疗组症状缓解,血清ANP浓度降低,cAMP浓度升高、SOD活性增强,差异均具有显著性意义（P〈0.05或P〈0.01或P〈0.001）,其中参芪0.05 mL组变化最小（P〈0.05）,参芪0.20 mL组变化最大（P〈0.001）;心肌组织病理改变减轻,其中参芪0.20 mL组最接近正常.结论 参芪扶正注射液足三里穴位注射能有效治疗心气虚证,且其疗效与用药剂量在一定剂量范围（0.05～0.20）mL内成正相关.     

电针对小鼠肝脏血流灌注量影响的激光散斑成像显示

目的：应用激光散斑成像技术连续监测电针过程小鼠肝脏表面血流灌注图像,研究电针不同穴位对肝脏血流灌注量的影响,探讨激光散斑技术在针灸效应研究中的应用价值。方法：采用Moor．FLPI激光散斑血流成像系统分别对足三里组、曲泉组、非经非穴组正常小鼠电针30min以及不电针对照组连续观察30min过程中肝脏表面血流灌注量变化进行观察,分析电针不同穴位、各个时点肝脏血流变化的规律。结果：（1）肝脏激光散斑图显示电针后各电针组肝脏表面整体血流灌注均增加,肝门附近区域灌注量增加幅度大于肝脏边缘区域;（2）电针各时点各电针组肝脏血流灌注量均出现增加,电针0～20min灌注量增加率为足三里组〉曲泉组〉非经非穴组;电针25～30min为足三里组〉非经非穴组〉曲泉组。结论：激光散斑血流成像技术能够精确记录显示电针过程肝脏表面的微循环变化情况,电针可以增强正常小鼠肝脏血流灌注量,电针增加肝脏血流灌注的效应存在穴位特异性。     

Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways.     

Inside Front Cover: Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo (J. Biophotonics 2/2019)

A reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system has been developed. For the first time, the lateral resolution and the depth‐of‐field of the BB‐ and GB‐PAM were measured. It was demonstrated that BB‐PAM is a powerful tool for a wide range of biomedical research including RBC migration in blood vessels at various depths in vivo and observation of cell migration or cell culture. Further details can be found in the article by Byullee Park, Hoyong Lee, Seungwan Jeon, et al. ( e201800215 ).

     

Laser stimulation of the acupoint ‘Zusanli’ (ST.36) on the radiopharmaceutical biodistribution in <Emphasis Type="BoldItalic">Wistar</Emphasis> rats

Laser used to stimulate acupoints is called laser acupuncture (LA). It is generally believed that similar clinical responses to manual acupuncture can be achieved. Here we analysed the effects of the laser (904 nm) at the ‘Zusanli’ acupoint (ST.36) of the stomach meridian on the biodistribution of the radiopharmaceutical Na^99mTcO₄. Wistar rats were divided into control (CG) and experimental groups (EG). The EG were exposed daily to the laser (904 nm) at ST.36 with 1 joule/min (40 mW/cm²) for 1 min. The animals of the CG were not exposed to laser at all. On the 8th day after LA, the animals were sedated and Na^99mTcO₄ was administered. After 10 min, the animals were all sacrificed and the organs removed. The radioactivity was counted in each organ to calculate the percentage of radioactivity of the injected dose per gram (%ATI/g). Comparison of the %ATI/g in EG and CG was performed by Mann-Whitney test. The %ATI/g was significantly increased in the thyroid due to the stimulation of the ST.36 by laser. It is possible to conclude that the stimulation of ST.36 does lead to biological phenomena that interfere with the metabolism of the thyroid.     

Blood aggregation and the system of microcirculation in experimental atherosclerosis, its spontaneous regression and hemosorption

Blood aggregate state and microcirculation were studied during development and spontaneous regression of experimental atherosclerosis and following hemosorption. It has been shown that experimental atherosclerosis is not only accompanied by changes in blood lipid composition, but also by disturbances in the structure and function of microcirculatory bed. Normalization of blood lipid composition and recovery of blood aggregate state and microcirculatory bed structure and function were not observed during spontaneous regression. Repeated hemosorption enhances atherosclerosis regression, normalizes lipid composition of blood and biological membranes and promotes the recovery of microcirculation.     

Revealing metabolite biomarkers for acupuncture treatment by linear programming based feature selection

Background

Acupuncture has been practiced in China for thousands of years as part of the Traditional Chinese Medicine (TCM) and has gradually accepted in western countries as an alternative or complementary treatment. However, the underlying mechanism of acupuncture, especially whether there exists any difference between varies acupoints, remains largely unknown, which hinders its widespread use.

Results

In this study, we develop a novel Linear Programming based Feature Selection method (LPFS) to understand the mechanism of acupuncture effect, at molecular level, by revealing the metabolite biomarkers for acupuncture treatment. Specifically, we generate and investigate the high-throughput metabolic profiles of acupuncture treatment at several acupoints in human. To select the subsets of metabolites that best characterize the acupuncture effect for each meridian point, an optimization model is proposed to identify biomarkers from high-dimensional metabolic data from case and control samples. Importantly, we use nearest centroid as the prototype to simultaneously minimize the number of selected features and the leave-one-out cross validation error of classifier. We compared the performance of LPFS to several state-of-the-art methods, such as SVM recursive feature elimination (SVM-RFE) and sparse multinomial logistic regression approach (SMLR). We find that our LPFS method tends to reveal a small set of metabolites with small standard deviation and large shifts, which exactly serves our requirement for good biomarker. Biologically, several metabolite biomarkers for acupuncture treatment are revealed and serve as the candidates for further mechanism investigation. Also biomakers derived from five meridian points, Zusanli (ST36), Liangmen (ST21), Juliao (ST3), Yanglingquan (GB34), and Weizhong (BL40), are compared for their similarity and difference, which provide evidence for the specificity of acupoints.

Conclusions

Our result demonstrates that metabolic profiling might be a promising method to investigate the molecular mechanism of acupuncture. Comparing with other existing methods, LPFS shows better performance to select a small set of key molecules. In addition, LPFS is a general methodology and can be applied to other high-dimensional data analysis, for example cancer genomics.

     

Perspectives,potentials and trends of ex vivo and in vivo optical molecular pathology

It is pivotal for medical applications, such as noninvasive histopathologic characterization of tissue, to realize label‐free and molecule‐specific representation of morphologic and biochemical composition in real‐time with subcellular spatial resolution. This unmet clinical need requires new approaches for rapid and reliable real‐time assessment of pathologies to complement established diagnostic tools. Photonic imaging combined with digitalization offers the potential to provide the clinician the requested information both under in vivo and ex vivo conditions. This report summarizes photonic approaches and their use in combination with image processing, machine learning and augmented virtual reality that might solve current challenges in modern medicine. Details are given for pathology, intraoperative diagnosis in head and neck cancer and endoscopic diagnosis in gastroenterology.      

Assesment of apoptosis induced changes in scattering using optical coherence tomography

The aim of this study is to identify changes in scattering with optical coherence tomography (OCT) and relate these measurements with mitochondrial changes during the initiation of apoptosis. Human retinal pigment epithelial cells were cultured and apoptosis was induced using 10% alcohol. Using the attenuation coefficient and backscattering, changes were measured during cell death in a cell‐pellet and monolayer respectively. To confirm apoptosis, fluorescent activated cell sorting was used. Mitochondrial activity during apoptosis was assessed using an oxidative stress assay and fluorescent confocal microscopy. Pelleted apoptotic cells measured with OCT showed a clear rise while untreated cells showed a very small increase in attenuation coefficient. Monolayered apoptotic cells displayed a distinct increase, while untreated cells showed a small increase in the backscattering. Apoptosis was confirmed by FACS experiments. Mitochondrial changes during the onset of apoptosis were also measured. The results demonstrate that apoptotic cell death could be monitored in real‐time by OCT. Changes in the scattering after induction of apoptosis are likely to be related to changes in the intracellular morphology. Oxidative stress‐induced mitochondrial swelling could be responsible for the initial increase, while cell blebbing and secondary necrosis subsequently for the observed decrease in scattering.

     

Wavelength‐Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS) for noninvasive early cancer detection and tissue hypoxia monitoring

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO₂). Unlike conventional photoacoustic spectroscopy, WM‐DPAS measures simultaneously two signals induced from square‐wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy‐ and oxy‐hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two‐wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM‐DPAS to detect very small changes in total hemoglobin concentration (C_Hb) and oxygenation levels, thereby identifying pre‐malignant tumors before they are anatomically apparent. The non‐invasive nature also makes WM‐DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM‐DPAS can be tuned for different applications such as quick cancer screening and accurate StO₂ quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM‐DPAS theory has been validated with sheep blood phantom measurements.

Sensitivity comparison between conventional single‐ended signal and differential signal.