Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

Significance: We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. Aim: In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). Approach: A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. Results: The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. Conclusions: The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Optimal blue light irradiation conditions for the treatment of acne vulgaris in a mouse model.

BACKGROUND: Although blue light is one of the therapeutic approaches used to treat acne vulgaris (AV), there is no consensus on its effectiveness. As a result, it is not recommended in the major acne vulgaris treatment guidelines. OBJECTIVE: The goal of this study was to look into the mechanism, safety, and efficacy of blue light therapy. We achieved this by examining the pathological response, inflammation, and depth of light penetration in a mouse model of cystic AV. METHODS: The aims of the study were addressed by exposing the mice to light with a wavelength of 415 nm under four different irradiation conditions. The exposure was done for five consecutive days followed by a no irradiation period of 72 h. RESULTS: Blue light treatment was most effective when irradiation was performed at 100 mW/cm2 for 20 min for five consecutive days. Inflammatory responses emerged 72 h after the final irradiation dose was administered. These responses were not associated with apoptosis as cleaved caspase-3 staining revealed no significant increases in apoptosis in the skin under any of the tested conditions. Blue light reached the superficial layer of the acne cyst at 5% of the total irradiation power and was attenuated by half for every 50 µm of progress through the cyst. CONCLUSION: In conclusion, blue light could control severe dermatologic inflammatory responses; therefore, it can be used to irradiate AV with high inflammation levels on a daily basis until improvement is observed. In addition, porphyrin, a metabolite of Cutibacterium acnes, and reactive oxygen species generated by the surrounding skin tissue may have essential roles in AV treatment.

Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

1270 nm near-infrared light as a novel vaccine adjuvant acts on mitochondrial photoreception in intradermal vaccines.

With the development of laser technology in the 1960s, a technique was developed to inject intradermal vaccines immediately after irradiating the skin with laser light to elicit an adjuvant effect, referred to as "laser adjuvant." We have been investigating the mechanism of laser adjuvant in influenza mouse models using noninvasive continuous-wave (CW) near-infrared (NIR) light mainly at a wavelength of 1064 nm, and have shown that the production of reactive-oxygen-species (ROS) in the skin and mast cells in the skin tissue plays an important role in the laser adjuvant effect. The new wavelength of 1270 nm NIR light is characterized by its ability to elicit the same vaccine adjuvant effect as other wavelengths at a lower energy, and may be suitable for clinical applications. In this study, we investigated the physiological activity of CW1270 nm NIR light in mast cells, its biological activity on mouse skin, and the durability of the vaccine adjuvant effect in influenza vaccine mouse models. We show that irradiation of mast cells with 1270 nm NIR light produced ROS and ATP, and irradiation of isolated mitochondria also produced ATP. In mouse skin, the relative expression levels of chemokine mRNAs, such as Ccl2 and Ccl20, were increased by irradiation with 1270 and 1064 nm NIR light at minimum safe irradiance. However, the relative expression of Nfkb1 was increased at 1064 nm, but not at 1270 nm. Serum anti-influenza IgG antibody titers increased early after immunization with 1064 nm, whereas with 1270 nm, there was not only an early response of antibody production but also persistence of antibody titers over the medium- to long-term. Thus, to our knowledge, we show for the first time that 1270 nm NIR light induces ROS and ATP production in mitochondria as photoreceptors, initiating a cascade of laser adjuvant effects for intradermal vaccines. Additionally, we demonstrate that there are wavelength-specific variations in the mechanisms and effects of laser adjuvants. In conclusion, CW1270 nm NIR light is expected to be clinically applicable as a novel laser adjuvant that is equivalent or superior to 1064 nm NIR light, because it can be operated at low energy and has a wavelength-specific adjuvant effect with medium- to long-lasting antibody titer.

Photoacoustic imaging of fresh human surgically and endoscopically resected gastrointestinal specimens.

Objective: Photoacoustic (PA) imaging is a novel noninvasive technique that offers high-contrast tomographic imaging with ultrasound-like resolution at depths of centimeters, enabling visualization of deep small vessels. The aim of this pilot study was to survey the characteristics of deep vessel networks in the mucosa of neoplastic gastrointestinal (GI) lesions using PA imaging. Methods: Specimens of patients who had undergone surgical and endoscopic resection for GI lesions were included in this study. The PA/ultrasound imaging system for clinical research is characterized by a technology that can superimpose a PA image over an ultrasound image. Three-dimensional PA images were acquired for the resected specimen before fixation. The stomach and colon of live pigs were incised, and the walls were scanned from the mucosa. Results: A total of 32 specimens (nine esophageal, 12 gastric, 11 colorectal) were scanned. The pathological diagnoses were adenomas (n = 2), intramucosal cancers (n = 14), and invasive cancers (n = 16). The deep vessel networks of all lesions could be visualized. In the intramucosal lesions, the deep vessel network was similar to that of a normal tissue. In invasive cancers, the thick and prominent vessel network was visible in the surface layer of esophageal cancers, infiltrated area of gastric cancers, and surface layer and infiltrated area of colorectal cancers. In the images of living pigs, visualizing the vascular network deeper than the submucosa in both the stomach and large intestine was possible. Conclusion: Our study confirmed that the deep vessel networks of neoplastic GI lesions were visible by PA imaging.

Chromatic-aberration-free multispectral optical-resolution photoacoustic microscopy using reflective optics and a supercontinuum light source.

A supercontinuum (SC) light source enables multispectral photoacoustic imaging at excitation wavelengths in the visible-to-near-infrared range. However, for such a broad optical wavelength range, chromatic aberration is non-negligible. We developed a multispectral optical-resolution photoacoustic microscopy (MS-OR-PAM) setup with a nanosecond pulsed SC light source and a reflective objective lens to avoid chromatic aberration. Chromatic aberrations generated by reflective and conventional objective lenses were compared, and the images acquired using the reflective objective were not affected by chromatic aberration. Hence, MS-OR-PAM with the reflective objective was used to distinguish red blood cells from melanoma cells via spectral subtraction processing.

Photocrosslinked gelatin hydrogel improves wound healing and skin flap survival by the sustained release of basic fibroblast growth factor.

Biomaterials traditionally used for wound healing can act as a temporary barrier to halt bleeding, prevent infection, and enhance regeneration. Hydrogels are among the best candidates for wound healing owing to their moisture retention and drug-releasing properties. Photo-polymerization using visible light irradiation is a promising method for hydrogel preparation since it can easily control spatiotemporal reaction kinetics and rapidly induce a single-step reaction under mild conditions. In this study, photocrosslinked gelatin hydrogels were imparted with properties namely fast wound adherence, strong wet tissue surface adhesion, greater biocompatibility, long-term bFGF release, and importantly, ease of use through the modification and combination of natural bio-macromolecules. The production of a gelatin hydrogel made of natural gelatin (which is superior to chemically modified gelatin), crosslinked by visible light, which is more desirable than UV light irradiation, will enable its prolonged application to uneven wound surfaces. This is due to its flexible shape, along with the administration of cell growth factors, such as bFGF, for tissue regeneration. Further, the sustained release of bFGF enhances wound healing and skin flap survival. The photocrosslinking gelatin hydrogel designed in this study is a potential candidate to enhance wound healing and better skin flap survival.

Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis.

Current cartilage regenerative therapies are not fully effective in treating osteoarthritis of the knee (OAK). We have developed chondrocyte sheets for autologous transplantation and tested these in in vitro and in vivo preclinical studies, and have reported that the transplantation of chondrocyte sheets promoted hyaline cartilage repair in rat, rabbit, and minipig models. However, autologous transplantation of chondrocyte sheets has yet to be reported in humans. Here, we report our combination therapy in which conventional surgical treatment for OAK, is followed by autologous chondrocyte sheet transplantation for cartilage repair. Eight patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV receive the therapy. Patients are thoroughly assessed by preoperative and postoperative X-rays, magnetic resonance imaging (MRI), arthroscopy, Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm Knee Score (LKS), and a laser-induced photoacoustic method to assess cartilage viscoelasticity. Arthroscopic biopsies of all patients are performed 12 months after transplantation for histological evaluation. The properties of the chondrocyte sheets are evaluated using gene expression analysis to investigate the ability to predict the clinical and structural outcomes of the therapy. For this small initial longitudinal series, combination therapy is effective, as assessed by MRI, arthroscopy, viscoelasticity, histology, and the clinical outcomes of KOOS and LKS. Gene marker sets identified in autologous chondrocyte sheets may be predictive of the overall KOOS, LKS, and histological scores after therapy. These predictive gene sets may be potential alternative markers for evaluating OAK treatment.

Spectral-differential-based unmixing for multispectral photoacoustic imaging.

We propose the use of a spectral differential method (SDM) to emphasize the spectral peaks of multispectral photoacoustic images. Because contrast agent signals have spectral peaks at the contrast agent absorption peak, the SDM can selectively emphasize contrast agent signals. Unlike the conventional spectral fitting method (SFM), the SDM does not require reference background spectra and, consequently, does not suffer from separation error caused by the deviation of reference spectra from the measured spectra. We performed multispectral photoacoustic imaging of tissue-mimicking phantoms and subcutaneous tumors of mice injected with small organic molecule-based contrast agents. Contrast agent images obtained by the SDM were clearer than those obtained by SFM.

Pilot Study of Prostate Cancer Angiogenesis Imaging Using a Photoacoustic Imaging System.

OBJECTIVES: To investigate a link between the appearance of photoacoustic imaging (PAI) and microvasculature in prostate cancer and to assess the feasibility of PAI for angiogenesis imaging in prostate cancer. METHODS: We have developed a PAI system equipped with a transrectal ultrasound (TRUS)-type probe. Three patients who underwent PAI just before prostate biopsy and subsequently underwent radical prostatectomy were included. The PAI appearance was retrospectively reviewed, and in each patient, 4 representative areas were selected: 1 with high PAI signal intensity, 1 with low PAI signal intensity, 1 peripheral to the index tumor, and 1 inside the index tumor. The correlation of PAI intensity with 3 microvascular parameters-microvascular density, total vascular area (TVA), and total vascular length (TVL)-assessed by CD34-immunostaining in resected specimens was analyzed. RESULTS: In all 3 patients the PAI intensity, TVA, and TVL in areas with high-intensity PAI signals were significantly higher than those in areas with low-intensity PAI signals, suggesting that PAI appearance describes the distribution of microvasculature in prostatic tissue correctly. All index tumors showed a ring-like PAI appearance consisting of a peripheral area of high signal intensity completely or partially surrounding an area with low signal intensity. The PAI intensity, TVA, and TVL in the periphery of the index tumors were significantly higher than those inside of the index tumors. CONCLUSION: The intensity of PAI signals might reflect the microvascularity in normal prostatic tissues and index tumors. PAI could be a novel modality for imaging prostate cancer angiogenesis.

Appropriate timing of blood sampling for blood gas analysis in the ventilated rabbit.

BACKGROUND: Arterial and venous blood gas analyses (BGAs) are essential to evaluate devices that measure biological oxygenation. The appropriate timing of blood sampling for BGA after respiratory rate (RR) change in animal experiments has not been reported. This study investigated the appropriate timing of blood sampling for BGA in ventilated rabbits and whether venous samples are an alternative to arterial samples. MATERIALS AND METHODS: Under general anesthesia, 14 rabbits (body weight, 3.02 ± 0.09 kg) were ventilated and their RR was changed (40/min, 30/min, and 20/min). Blood was sampled through cervical arterial and venous catheters. Experiment 1: in seven rabbits, arterial BGA was measured at 0, 0.5, 1, 2, 3, 5, 10, 15, and 20 min after the RR change. Experiment 2: in seven different rabbits, simultaneous arterial and venous BGA were measured at 0, 2, 5, 10, 15, and 20 min after the RR change. RESULTS: Oxygen partial pressure (PO2) and saturation (SO2) of the arterial blood stabilized 0.5 min after the RR changed. In venous BGA, no index stabilized during observation. The arterial and venous values of the carbon dioxide partial pressure (PCO2) and pH had significant correlations (arterial PCO2 = 0.9316 × venous PCO2-4.4425 [r = 0.9178]; arterial pH = 1.0835 × venous pH-0.5795 [r = 0.9453]). CONCLUSIONS: In ventilated rabbits, arterial PO2 and SO2 stabilized in 0.5 min. No venous value stabilized after the RR change. Only the PCO2 and pH of venous samples may be an alternative to arterial samples under the defined formula.

A pilot study of photoacoustic imaging system for improved real-time visualization of neurovascular bundle during radical prostatectomy.

BACKGROUND: Photoacoustic imaging, a noninvasive imaging based on optical excitation and ultrasonic detection, enables one to visualize the distribution of hemoglobin and acquire a map of microvessels without using contrast agents. We examined whether it helps visualize periprostatic microvessels and improves visualization of the neurovascular bundle. METHODS: We developed a photoacoustic imaging (PAI) system with a hand-held probe combining optical illumination and a conventional linear array ultrasound probe. In experiments with a phantom model, it was able to visualize vessels with diameters as small as 300 µm within a depth of 10 mm. We also developed a TRUS type probe for our photoacoustic imaging system and used it to intraoperatively monitor periprostatic tissues in seven patients with clinically organ-confined prostate cancer who were undergoing non-nerve-sparing retropubic radical prostatectomy. Images of periprostatic tissues from resected prostatectomy specimens were also obtained using the linear photoacoustic probe, and the consistency of the microvessel distribution and co-existence of nerve fibers was examined by double immunostaining of paraffin-embedded sections with anti-CD31 and anti-S-100 antibodies. RESULTS: Intraoperative monitoring of periprostatic tissues with the TRUS photoacoustic probe showed substantial signals on the posterolateral surface of the prostate and clearly demonstrated the location and extent of the neurovascular bundle better than does TRUS alone. Photoacoustic images of the periprostatic tissues in resected specimens also showed substantial signals that were especially strong on the posterolateral surface of the prostate. Nerve fibers were closely co-localized with periprostatic microvessels and the pattern of their distribution was consistent with that of PAI signals. CONCLUSIONS: The intraoperative photoacoustic imaging located the microvascular complex in the neurovascular bundle. Moreover, the neurovascular bundle was easier to identify by PAI than by TRUS alone, suggesting that PAI could be helpful in nerve-sparing radical prostatectomy.

Improved angiogenesis and healing in crush syndrome by fibroblast growth factor-2-containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles.

BACKGROUND: We produced fibroblast growth factor (FGF)-2-containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles (FGF-2 + F/P NPs). The purpose of this study was to evaluate the effectiveness of the local administration of FGF-2 + F/P NPs on repairing crush syndrome (CS)-injured lesions after compression release using a nonlethal and reproducible CS injury rat model. MATERIALS AND METHODS: The hind limbs of the anesthetized rats were compressed for 6 h using 3.6 kg blocks, as previously described. The effects of administering FGF-2 + F/P NPs (group A), F/P NPs alone (group B), FGF-2 alone (group C), and saline (control; group D) were examined. Motor function, surface blood flow in the hind limbs, and the wet/dry weight ratio in the tibialis anterior muscle were examined for 1-28 d after the compression release. Histologic analyses were also performed. RESULTS: At the middle and late stages (3-28 d after the compression release), group A had higher scores in the motor function, improved blood flow, increased number of blood vessels, and faster recovered muscle tissue, compared with the other groups. There was no significant difference in enhanced edema in the tibialis anterior muscle among all groups. CONCLUSIONS: The local administration of FGF-2 + F/P NPs to a CS-injured lesion was effective in repairing damaged muscle tissue after compression release.

Image reconstruction of the absorption coefficients with l 1-norm minimization from photoacoustic measurements.

BACKGROUND: The photoacoustic (PA) imaging by considering light propagation into image reconstruction process can provide quantitative information of photon absorbers, such as hemoglobin and exogenous dyes, and to improve their imaging contrasts. METHODS: A 2D image reconstruction of the distribution of the light absorption coefficient from the PA measurements with light source and ultrasound transducer placed at the identical position was tested. The PA pressures were formulated with the PA wave equation and the photon diffusion equation. The relation between the PA pressure and the absorption coefficient was linearized. The image reconstruction was carried out by minimizing the squared error between the measured and calculated PA signals. The l 1-norm of the reconstructed image was simultaneously minimized to improve the localization of the reconstructed target in the image. The image reconstruction with the l 1-norm minimization was compared to that with the Tikhonov regularization by numerical simulation and phantom experiment. In phantom experiment, an aqueous solution of the intralipid and the indocyanine green was used as the measured object. The PA probe had optical fiber for illumination and piezoelectric film for detection placed at the identical position. RESULTS: The l 1-norm minimization reconstructed more localized target than the Tikhonov regularization. CONCLUSIONS: The l 1-norm minimization is useful for the sparse PA image reconstruction.

Uncooled infrared camera for the noninvasive visualization of the vascular flow in an anastomotic vessel during neurological surgery: technical note.

We herein present our experience to assess intraoperative confirmation of vascular patency with an uncooled infrared camera in extracranial-intracranial (EC-IC) bypass surgery. This camera had distinguishing characteristics, including its small size, light weight, and adequate temperature resolution (< 0.022 degrees). We used a simplified zoom germanium lens as a preliminary study to verify the potential of using this camera to assess the vascular flow of the end-to-side anastomosis model in rats. In addition, we evaluated the vascular flow in continuous clinical series using this infrared camera during EC-IC bypass in 14 patients (17 sides). This infrared camera offers real-time information on the vascular patency of end-to-side anastomosis vessels of all relevant diameters. The spatial resolution and image quality are satisfactory, and the procedure can be safely repeatable. We have shown that the infrared camera could be a new and feasible technology for intraoperative imaging of the vascular flow and is considered to be clinically useful during cerebrovascular surgery.

Responses of cancer cells induced by photodynamic therapy.

Photodynamic therapy (PDT) involves the administration of a photosensitizer, followed by local irradiation of tumor tissues using a laser of an appropriate wavelength to activate the photosensitizer. Since multiple cellular signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic effect, understanding the responses of cancer cells to PDT will aid in the development of new interventions. This review describes the possible cell-death signaling pathways initiated by PDT. In addition, we describe our latest findings regarding the induction of expression of miRNAs specific to apoptosis in cancer cells and the induction of antitumor immunity following PDT against cancer cells. A more detailed understanding of the molecular mechanisms related to PDT will potentially improve long-term survival of PDT treated patients.

Blue laser irradiation generates intracellular reactive oxygen species in various types of cells.

UNLABELLED: Abstract Objective: The purpose of this study was to measure intracellular reactive oxygen species (ROS) production after laser irradiation in various types of cells. BACKGROUND DATA: ROS are considered to be the key secondary messengers produced by low-level laser therapy (LLLT). Although various mechanisms for the effects of LLLT have been proposed, and intracellular ROS were indicated as the one of the key factors, direct measurement of intracellular ROS of several types of cells after different wavelength lasers irradiation has not been reported. MATERIALS AND METHODS: Various types of cells were used in this study: mouse preadipocytes (3T3-L1), prechondrocytes (ATDC5), myoblasts (C2C12), mesenchymal stromal cells (KUSA-A1), lung cancer cells (LLC), insulinoma cells (MIN6), fibroblasts (NIH-3T3), human cervix adenocarcinoma cells (HeLa), macrophages differentiated from lymphocytes (THP-1) after treatment with phorbol ester, and rat basophilic leukemia cells (RBL-2H3). Cells were irradiated with a blue laser (wavelength: 405 nm), a red laser (wavelength: 664 nm) or a near infrared laser (wavelength: 808 nm) at 100 mW/cm(2) for 60 or 120 sec. Intracellular ROS levels were measured by fluorometric assay using the intracellular ROS probe, CM-H2DCFDA in a flow cytometer. RESULTS: After a blue laser irradiation, intracellular ROS levels were increased in all types of cells. In contrast, intracellular ROS generation was not observed after irradiation with a red laser or near-infrared laser. CONCLUSIONS: Potential sources of intracellular ROS were excited by blue laser irradiation, resulting in ROS production within cells. Although the low-level intracellular ROS should be generated after a red or a near-infrared laser irradiation, the only high level intracellular ROS were detected by the ROS probe used in this study. As ROS are considered to be key secondary messengers, the specific functional regulation of cells by laser irradiation will be studied in a future study.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential.

BACKGROUND: Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span. METHODS: We transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo. RESULTS: Our results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components. CONCLUSIONS: Focal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Novel TNF-α receptor 1 antagonist treatment attenuates arterial inflammation and intimal hyperplasia in mice.

AIM: Tumor necrosis factor receptor 1 (TNFR1) participates importantly in arterial inflammation in genetically altered mice; however it remains undetermined whether a selective TNFR1 antagonist inhibits arterial inflammation and intimal hyperplasia. This study aimed to determine the effect and mechanism of a novel TNFR1 antagonist in the suppression of arterial inflammation. METHODS: We investigated intimal hyperplasia in IL-1 receptor antagonist-deficient mice two weeks after inducing femoral artery injury in an external vascular cuff model. All mice received intraperitoneal injections of TNFR1 antagonist (PEG-R1antTNF) or normal saline twice daily for 14 days. RESULTS: PEG-R1antTNF treatment yielded no adverse systemic effects, and we observed no significant differences in serum cholesterol or blood pressure in either group; however, selective PEG-R1antTNF treatment significantly reduced intimal hyperplasia (19,671±4,274 vs. 11,440±3,292 µm(2); p=0.001) and the intima/media ratio (1.86±0.43 vs. 1.34±0.36; p=0.029), compared with saline injection. Immunostaining revealed that PEG-R1antTNF inhibits Nuclear factor-κB (NF-κB), suppressing smooth muscle cell (SMC) proliferation and decreasing chemokine and adhesion molecule expression, and thus decreasing intimal hyperplasia and inflammation. CONCLUSIONS: Our data suggest that PEG-R1antTNF suppresses SMC proliferation and inflammation by inhibiting NF-κB. This study highlights the potential therapeutic benefit of selective TNFR1 antagonist therapy in preventing intimal hyperplasia and arterial inflammation.

The influence of Ho:YAG laser irradiation on intervertebral disc cells.

BACKGROUND AND OBJECTIVE: Various types of laser have been reported for percutaneous laser disc decompression (PLDD). The aim of this study was to understand the effects on intervertebral disc cells following Ho:YAG laser irradiation, using a three-dimensional culture model, and consider appropriate irradiation conditions. STUDY DESIGN/MATERIALS AND METHODS: Intervertebral discs from the lumbar spine were obtained from 36 female Japanese white rabbits and processed to obtain isolated cells in three-dimensional cultures. Photoacoustic and photothermal effects were investigated by irradiating three-dimensional cultures with Ho:YAG laser at 27 or 54 J. Residual cell counts after irradiation were estimated based on DNA content according to fluorometric assay. Lactate dehydrogenase levels were also investigated as a marker of damage to cell plasma membranes. Finally, proteoglycan synthesis was measured by rapid filtration assay of (35) S incorporation, as an index of matrix synthesis. RESULTS: Residual cell count tended to be higher in the 27-J group. Plasma membrane damage was higher and remained high longer after irradiation in the 54-J group. Proteoglycan synthesis was higher in the 27-J group than in the 54-J group, with some conditions (e.g., 90 mJ/pulse condition) showing marked activation of proteoglycan synthesis maintained for a long time after irradiation. CONCLUSIONS: Three-dimensional culture models of intervertebral disc cells are useful for clarifying relationships between cell reactions and photoacoustic and photothermal effects after laser irradiation. Total energy is closely related to optimization of irradiation conditions, which may allow optimization of cytoprotection and promotion of matrix synthesis in clinical practice.