Investigation of optimizing indocyanine green solution for in vivo lymphatic research using near-infrared fluorescence indocyanine green lymphangiography.

Despite the tireless efforts of many researchers in lymphatic research, indocyanine green (ICG) solution conditions suitable for lymphatic circulation tests have not been perfectly established yet. We aimed to investigate the optimal in vivo conditions of ICG solution to avoid photobleaching and quenching effects, which may affect the accuracy of lymphatic circulation evaluation. After ICG fluorescence intensity (or ICG intensity) was assessed under different in vitro conditions, the image quality of brachial lymph nodes (LNs) and collecting lymphatic vessels (LVs) in eight rats was investigated. The in vitro results showed that ICG intensity depends on concentration and time in various solvents; however, the brightest intensity was observed at a concentration of 8-30 µg/mL in all solvents. ICG concentration in the albumin (bovine serum albumin; BSA) solution and rat's plasma showed more than two times higher fluorescence intensity than in distilled water (DW) in the same range. However, saline reduced the intensity by almost half compared to DW. In the in vivo experiment, we obtained relatively high-quality images of the LNs and LVs using ICG in the BSA solution. Even at low concentrations, the result in the BSA solution was comparable to those obtained from high-concentration solutions commonly used in conventional circulation tests. This study provides valuable information about the conditions for optimal ICG intensity in near infrared fluorescence indocyanine green (NIRF-ICG) lymphangiography, which may be useful not only for the diagnosis of lymphatic circulation diseases such as lymphedema but also for preclinical research for the lymphatic system.

In Vivo Dynamic and Static Analysis of Lymphatic Dysfunction in Lymphedema Using Near-Infrared Fluorescence Indocyanine Green Lymphangiography.

BACKGROUND: Near-infrared fluorescence indocyanine green lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatics and reveals not only the spatial distribution of drainage (static analysis) but also information on the lymphatic contraction (dynamic analysis). METHODS: We have produced total lymphatic obstruction in the upper limbs of 18 Sprague-Dawley rats through the dissection of proximal (brachial and axillary) lymph nodes and 20-Gy radiation (dissection limbs). After the model formation for 1 week, 9 animal models were observed for 6 weeks using near-infrared fluorescence indocyanine green lymphangiography by injecting 6-µL ICG-BSA (indocyanine green-bovine serum albumin) solution of 20-µg/mL concentration. The drainage pattern and leakage of lymph fluid were evaluated and time-domain signals of lymphatic contraction were observed in the distal lymphatic vessels. The obtained signals were converted to frequency-domain spectrums using signal processing. RESULTS: The results of both static and dynamic analyses proved to be effective in accurately identifying the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and increased leakage of lymph fluid to the periphery of the vessels compared with the control (normal) limbs. Meanwhile, the waveforms were changed and the contractile signal frequency increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 to 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored for the follow-up period, indicating a persistent lymphatic obstruction. CONCLUSIONS: The dynamic analysis could detect the abnormalities of lymphatic circulation by observing the characteristics of signals, and it provided additional evaluation indicators that cannot be provided by the static analysis. Our findings may be useful for the early detection of the circulation problem as a functional evaluation indicator of the lymphatic system.

The effects of postoperative treadmill exercise on rats with secondary lymphedema.

Cancer-related lymphedema (LE) is often caused by radiotherapy and surgery such as lymph node dissection (LND). Previous studies have reported that exercise is beneficial to relieve LE, but the changes in the lymphatic system following exercise are still unclear. This study aimed to examine the changes in lymphatic drainage pathways over the exercise period and beneficial effects of exercise in rats with LE. Twelve rats were randomly allocated into exercise and control groups (EG and CG; n = 6 each). To obtain LE, inguinal and popliteal LND followed by 20 Gy irradiation was performed. Treadmill exercise was 30 minutes/day, 5 days/week over the four-week period. Consecutive indocyanine green (ICG) lymphography images were collected and classified into five patterns: i) linear; ii) splash; iii) stardust; iv) diffuse, and v) none. Ankle thickness was measured weekly. Histopathological evaluation was performed to examine the skin thickness, collagen area fraction (%) and lymphatic vessel density in harvested tissue. ICG lymphography exhibited more linear and splash patterns in the EG at week 3. The difference of swelling between both groups was significantly different at week 4 (p = 0.016). Histopathologic data revealed a thinner epidermis (p = 0.041) and dermis (p = 0.002), lower collagen area fraction (%, p = 0.002), and higher lymph vessel density (p = 0.002) in the EG than the CG. In conclusion, we found that postoperative exercise can facilitate improvement in lymphatic fluid retention in the lymphedema rat model, resulting in improvement of pathological conditions in the lymphatic system.

The effects of inspiratory muscle training with pulmonary rehabilitation on NSCLC patients during radiation therapy: A pilot clinical study.

BACKGROUND: The effects of inspiratory muscle training (IMT) with pulmonary rehabilitation (PR) on patients with non-small cell lung cancer (NSCLC) receiving radiotherapy (RT) have not previously been reported. This pilot study aimed to determine the effectiveness of IMT with PR on respiratory muscles and exercise capacity of NSCLC patients receiving RT. METHODS: We retrospectively analyzed 20 patients who underwent RT for NSCLC. The rehabilitation included IMT, stretching, strengthening, and aerobic exercises three times a week for 4 weeks with concurrent RT. IMT training lasted 10 min, consisting of one cycle of 30 breaths using the Powerbreathe KH1 device in the hospital by a physical therapist. Patients underwent two IMT sessions at home daily at an intensity of approximately 30%-50% of the participant's maximum inspiratory muscle pressure (MIP) using the threshold IMT tool. We analyzed the results from the respiratory muscle strength test, pulmonary function test, 6-min walk test (6MWT), cardiopulmonary function test, cycle endurance test (CET), Inbody test, grip measurement, knee extensor/flexor strength measurement, Cancer Core Quality of Life Questionnaire (EORTCQ-C30), and NSCLC 13 (EORTC-LC13). RESULTS: There were no adverse events during evaluation and IMT with PR. MIP (60.1 ± 25.1 vs. 72.5 ± 31.9, p = 0.005), 6MWT (439.2 ± 97.1 vs. 60.7 ± 97.8, p = 0.002), CET (181.39 ± 193.12 vs. 123.6 ± 87.6, p = 0.001), knee extensor (14.4 ± 5.3 vs. 17.4 ± 5, p = 0.012), and knee flexor (14.0 ± 5.2 vs. 16.9 ± 5.5, p = 0.004) significantly improved after IMT with PR. CONCLUSION: IMT with PR appears effective on respiratory muscles and exercise capacity without adverse events in NSCLC patients who underwent RT.

Epigenetic modification of gene expression in cancer cells by terahertz demethylation.

Terahertz (THz) radiation can affect the degree of DNA methylation, the spectral characteristics of which exist in the terahertz region. DNA methylation is an epigenetic modification in which a methyl (CH3) group is attached to cytosine, a nucleobase in human DNA. Appropriately controlled DNA methylation leads to proper regulation of gene expression. However, abnormal gene expression that departs from controlled genetic transcription through aberrant DNA methylation may occur in cancer or other diseases. In this study, we demonstrate the modification of gene expression in cells by THz demethylation using resonant THz radiation. Using an enzyme-linked immunosorbent assay, we observed changes in the degree of global DNA methylation in the SK-MEL-3 melanoma cell line under irradiation with 1.6-THz radiation with limited spectral bandwidth. Resonant THz radiation demethylated living melanoma cells by 19%, with no significant occurrence of apurinic/apyrimidinic sites, and the demethylation ratio was linearly proportional to the power of THz radiation. THz demethylation downregulates FOS, JUN, and CXCL8 genes, which are involved in cancer and apoptosis pathways. Our results show that THz demethylation has the potential to be a gene expression modifier with promising applications in cancer treatment.

Lymphatic channel sheet of polydimethylsiloxane for preventing secondary lymphedema in the rat upper limb model.

Secondary lymphedema is a severe complication of cancer treatment, but there is no effective curative method yet. Lymph node dissection and radiation therapy for cancer treatment may lead to secondary lymphedema, which is a chronic disease induced by malfunction of lymphatic flow. The lymphatic channel sheet (LCS) is an artificial micro-fluidic structure that was fabricated with polydimethylsiloxane to maintain lymphatic flow and induce lymphangiogenesis. The structure has two-dimensional multichannels that increase the probability of lymphangiogenesis and allow for relatively easy application. We verified the efficacy of the lymphatic channel sheet through macroscopic and microscopic observation in small animal models, which underwent brachial lymph node dissection and irradiation. The lymphatic channel sheet enabled the successful transport of lymphatic fluid from the distal to the proximal area in place of the removed brachial lymph nodes. It prevented swelling and abnormal lymphatic drainage during the follow-up period. Lymphangiogenesis was also identified inside the channel by histological analysis after 8 weeks. According to these experimental results, we attest to the roles of the lymphatic channel sheet as a lymphatic pathway and scaffold in the rat upper limb model of secondary lymphedema. The lymphatic channel sheet maintained lymphatic flow after lymph node dissection and irradiation in an environment where lymph flow is cut off. It also relieved symptoms of secondary lymphedema by providing a lymph-friendly space and inducing lymphangiogenesis.

Clinical Utility of Bioelectrical Impedance Analysis Parameters for Evaluating Patients with Lower Limb Lymphedema after Lymphovenous Anastomosis.

BACKGROUND: In lymphedema, lymphatic fluid accumulates in the interstitial space, and localized swelling appears. Lymphovenous anastomosis (LVA) is the most widely used surgery to rebuild a damaged lymphatic system; however, assessing outcome of LVA involves performing volume measurements, which provides limited information on body composition changes. Therefore, we analyzed the bioelectrical impedance analysis (BIA) parameters that can reflect the status of lymphedema patients who underwent LVA. METHODS: We retrospectively reviewed records of 42 patients with unilateral lower extremity lymphedema who had LVA. We measured the perioperative BIA parameters such as extracellular water (ECW) ratio and volume as defined by the percentage of excess volume (PEV). We evaluated the relationship between the amount of change in PEV and in BIA parameters before and after surgery. We confirmed the correlation between ΔPEV and BIA parameters using Spearman's correlation. RESULTS: Most patients included had secondary lymphedema due to cancer. Average age was 51.76 years and average body mass index was 23.27. PEV and all BIA parameters after surgery showed a significant difference (p < 0.01) compared with preoperative measurements. The ECW ratio aff/unaff showed the strongest correlation with PEV with a correlation coefficient of 0.473 (p < 0.01). CONCLUSION: Our findings suggest that BIA parameters, especially ECW ratio aff/unaff could reflect the status of patients with lower limb lymphedema after LVA. Appropriate use of BIA parameters may be useful in the postoperative surveillance of patients.

Numerical Model for Cerebrovascular Hemodynamics with Indocyanine Green Fluorescence Videoangiography.

OBJECTIVE: The use of indocyanine green videoangiography (ICG-VA) to assess blood flow in the brain during cerebrovascular surgery has been increasing. Clinical studies on ICG-VA have predominantly focused on qualitative analysis. However, quantitative analysis numerical modelling for time profiling enables a more accurate evaluation of blood flow kinetics. In this study, we established a multiple exponential modified Gaussian (multi-EMG) model for quantitative ICG-VA to understand accurately the status of cerebral hemodynamics. METHODS: We obtained clinical data of cerebral blood flow acquired the quantitative analysis ICG-VA during cerebrovascular surgery. Varied asymmetric peak functions were compared to find the most matching function form with clinical data by using a nonlinear regression algorithm. To verify the result of the nonlinear regression, the mode function was applied to various types of data. RESULTS: The proposed multi-EMG model is well fitted to the clinical data. Because the primary parameters-growth and decay rates, and peak center and heights-of the model are characteristics of model function, they provide accurate reference values for assessing cerebral hemodynamics in various conditions. In addition, the primary parameters can be estimated on the curves with partially missed data. The accuracy of the model estimation was verified by a repeated curve fitting method using manipulation of missing data. CONCLUSION: The multi-EMG model can possibly serve as a universal model for cerebral hemodynamics in a comparison with other asymmetric peak functions. According to the results, the model can be helpful for clinical research assessment of cerebrovascular hemodynamics in a clinical setting.

Anatomy and relationships of forelimb lymph nodes in Sprague-Dawley rats: A detailed dissecting approach.

Background: Constructing a reliable animal model for preclinical treatment of secondary lymphedema is challenging because the anatomical characteristics near the lymph nodes are understudied. Therefore, this study examined the detailed anatomical relationship between the axillary lymph node flaps (ALNFs) and brachial lymph node flaps (BLNFs) in the forelimb of Sprague-Dawley (SD) rats. Materials and methods: Ten male rats, weighing 250-300 g, were used. The ALNFs and BLNFs on either side of the rat forelimbs were dissected. The two lymph node flaps (LNFs) were immediately harvested to analyze their physical characteristics (via imaging process software) and microscopic structure (via histology examinations). Results: A total of 20 ALNFs and BLNFs from 10 rats were harvested and analyzed. ALNF dissection was simpler and lasted a shorter time than BLNF dissection (p < 0.0001). The left LNFs were more difficult to dissect than the right LNFs (p < 0.0001). In physical characteristics of LNFs, the area (p < 0.001) of LNFs and the number of lymph nodes (p < 0.0001) associated with ALNFs were greater than those associated with BLNFs, but the pedicle lengths of ALNFs were shorter than that of BLNFs (p < 0.0001). No significant difference in the diameter of the venous and arterial pedicles was noted between the two LNFs (p > 0.05). Conclusion: This study reported detailed physical characteristics of ALNFs and BLNFs in SD rat forelimbs, assessing the respective area of LNFs, number of lymph nodes, and lengths and diameters of vascular pedicles. Moreover, this study suggested an efficient method to perform a study of LNFs by describing the operation process and repeatedly measuring the operation time.

Transformation of terahertz vibrational modes of cytosine under hydration.

Cytosine and cytosine monohydrate are representative biomolecules for investigating the effect of hydrogen bonds in deoxyribonucleic acid. To better understand intermolecular interactions, such as hydrogen bonds, between nucleobases it is necessary to identify the low-frequency vibrational modes associated with intermolecular interactions and crystalline structures. In this study, we investigated the characteristic low-frequency vibrational modes of cytosine and cytosine monohydrate using terahertz time-domain spectroscopy (THz-TDS). The crystal geometry was obtained by the powder X-ray diffraction technique. The optimized atomic positions and the normal modes in the terahertz region were calculated using density functional theory (DFT), which agreed well with the experimental results. We found that overall terahertz absorption peaks of cytosine and cytosine monohydrate consist of collective vibrations mixed with intermolecular and intramolecular vibrations in mode character analysis, and that the most intense peaks of both samples involve remarkable intermolecular translational vibration. These results indicate that THz-TDS combined with DFT calculations including mode character analysis can be an effective method for understanding how water molecules contribute to the characteristics of the low-frequency vibrational modes by intermolecular vibrations with hydrogen bonding in biological and biomedical applications.

Determining terahertz resonant peaks of biomolecules in aqueous environment.

The resonant peaks of biomolecules provide information on the molecules' physical and chemical properties. Although many biomolecules have resonant peaks in the terahertz region, it is difficult to observe their specific signals in aqueous environments. Hence, this paper proposes a method for determining these peaks. We found the specific resonant peaks of a modified nucleoside, 5-methlycytidine and modified HEK293T DNA in an aqueous solution through baseline correction. We evaluated the consistency of various fitting functions used for determining the peaks with various parameters. We separated two resonance peaks of 5-methlycytidine at 1.59 and 1.97 THz and for artificially methylated HEK293T DNA at 1.64 and 2.0 THz.

Effective demethylation of melanoma cells using terahertz radiation.

Terahertz (THz) demethylation is a photomedical technique applied to dissociate methyl-DNA bonds and reduce global DNA methylation using resonant THz radiation. We evaluated the performance of THz demethylation and investigated the DNA damage caused by THz irradiation. The demethylation rate in M-293T DNA increased linearly with the irradiation power up to 48%. The degree of demethylation increased with exposure to THz radiation, saturating after 10 min. Although THz demethylation occurred globally, most of the demethylation occurred within the partial genes in the CpG islands. Subsequently, we performed THz demethylation of melanoma cells. The degree of methylation in the melanoma cell pellets decreased by approximately 10-15%, inducing ∼5-8 abasic sites per 105 bp; this was considerably less than the damaged DNA irradiated by the high-power infrared laser beam used for generating THz pulses. These results provide initial data for THz demethylation and demonstrate the applicability of this technique in advanced cancer cell research. THz demethylation has the potential to develop into a therapeutic procedure for cancer, similar to that involving chemical demethylating agents.

Detection and manipulation of methylation in blood cancer DNA using terahertz radiation.

DNA methylation is a pivotal epigenetic modification of DNA that regulates gene expression. Abnormal regulation of gene expression is closely related to carcinogenesis, which is why the assessment of DNA methylation is a key factor in cancer research. Terahertz radiation may play an important role in active demethylation for cancer therapy because the characteristic frequency of the methylated DNA exists in the terahertz region. Here, we present a novel technique for the detection and manipulation of DNA methylation using terahertz radiation in blood cancer cell lines. We observed the degree of DNA methylation in blood cancer at the characteristic resonance of approximately 1.7 THz using terahertz time-domain spectroscopy. The terahertz results were cross-checked with global DNA methylation quantification using an enzyme-linked immunosorbent assay. We also achieved the demethylation of cancer DNA using high-power terahertz radiation at the 1.7-THz resonance. The demethylation degrees ranged from 10% to 70%, depending on the type of cancer cell line. Our results show the detection of DNA methylation based on the terahertz molecular resonance and the manipulation of global DNA methylation using high-power terahertz radiation. Terahertz radiation may have potential applications as an epigenetic inhibitor in cancer treatment, by virtue of its ability to induce DNA demethylation, similarly to decitabine.

Terahertz imaging of metastatic lymph nodes using spectroscopic integration technique.

Terahertz (THz) imaging was used to differentiate the metastatic states of frozen lymph nodes (LNs) by using spectroscopic integration technique (SIT). The metastatic states were classified into three groups: healthy LNs, completely metastatic LNs, and partially metastatic LNs, which were obtained from three mice without infection and six mice infected with murine melanoma cells for 30 days and 15 days, respectively. Under histological examination, the healthy LNs and completely metastatic LNs were found to have a homogeneous cellular structure but the partially metastatic LNs had interfaces of the melanoma and healthy tissue. THz signals between the experimental groups were not distinguished at room temperature due to high attenuation by water in the tissues. However, a signal gap between the healthy and completely metastatic LNs was detected at freezing temperature. The signal gap could be enhanced by using SIT that is a signal processing method dichotomizing the signal difference between the healthy cells and melanoma cells with their normalized spectral integration. This technique clearly imaged the interfaces in the partially metastatic LNs, which could not be achieved by existing methods using a peak point or spectral value. The image resolution was high enough to recognize a metastatic area of about 0.7 mm size in the partially metastatic LNs. Therefore, this pilot study demonstrated that THz imaging of the frozen specimen using SIT can be used to diagnose the metastatic state of LNs for clinical application.

Terahertz molecular resonance of cancer DNA.

Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.