Mechanical Interatomic Bond Formation in Ethanol and Methanol-Ethanol Mixture by Laser-Driven Shock Waves.

Molecules, which were predicted to be produced by C-C or C-O bond formation between ethanol molecules induced by a laser-driven shock wave, were identified by gas chromatography-mass spectrometry. Moreover, the laser irradiation of a methanol-ethanol mixture revealed the formation of C-C and C-O bonds between both components. Particularly, four hemiacetals (methoxymethanol, 1-methoxyethanol, ethoxymethanol, and 1-ethoxyethanol) were identified in the Ar-saturated alcohol samples, whereas acetalization dominated sufficiently in the CO2-saturated samples, significantly reducing the hemiacetals. It was verified that some molecules were produced by the dropout of an ethanol part during the C-C or C-O bond formation, supporting the contribution of laser-driven shock waves.

Meningeal Damage and Interface Astroglial Scarring in the Rat Brain Exposed to a Laser-Induced Shock Wave(s).

In the past decade, signature clinical neuropathology of blast-induced traumatic brain injury has been under intense debate, but interface astroglial scarring (IAS) seems to be convincing. In this study, we examined whether IAS could be replicated in the rat brain exposed to a laser-induced shock wave(s) (LISW[s]), a tool that can produce a pure shock wave (primary mechanism) without dynamic pressure (tertiary mechanism). Under certain conditions, we observed astroglial scarring in the subpial glial plate (SGP), gray-white matter junctions (GM-WM), ventricular wall (VW), and regions surrounding cortical blood vessels, accurately reproducing clinical IAS. We also observed shock wave impulse-dependent meningeal damage (dural microhemorrhage) in vivo by transcranial near-infrared (NIR) reflectance imaging. Importantly, there were significant correlations between the degree of dural microhemorrhage and the extent of astroglial scarring more than 7 days post-exposure, suggesting an association of meningeal damage with astroglial scarring. The results demonstrated that the primary mechanism alone caused the IAS and meningeal damage, both of which are attributable to acoustic impedance mismatching at multi-layered tissue boundaries. The time course of glial fibrillary acidic protein (GFAP) immunoreactivity depended not only on the LISW conditions but also on the regions. In the SGP, significant increases in GFAP immunoreactivity were observed at 3 days post-exposure, whereas in the GM-WM and VW, GFAP immunoreactivity was not significantly increased before 28 days post-exposure, suggesting different pathological mechanisms. With the high-impulse single exposure or the multiple exposure (low impulse), fibrotic reaction or fibrotic scar formation was observed, in addition to astroglial scarring, in the cortical surface region. Although there are some limitations, this seems to be the first report on the shock-wave-induced IAS rodent model. The model may be useful to explore potential therapeutic approaches for IAS.

Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

Research Application of Laser-induced Shock Wave for Studying Blast-induced Cochlear Injury.

The ear is the organ most susceptible to explosion overpressure, and cochlear injuries frequently occur after blast exposure. Blast exposure can lead to sensorineural hearing loss (SNHL), which is an irreversible hearing loss that negatively affects the quality of life. Detailed blast-induced cochlear pathologies, such as the loss of hair cells, spiral ganglion neurons, cochlear synapses, and disruption of stereocilia, have been previously documented. However, determining cochlear sensorineural deterioration after a blast injury is challenging because animals exposed to blast overpressure usually experience tympanic membrane perforation (TMP), which causes concurrent conductive hearing loss. To evaluate pure sensorineural cochlear dysfunction, we developed an experimental animal model of blast-induced cochlear injury using a laser-induced shock wave. This method avoids TMP and concomitant systemic injuries and reproduces the functional decline in the SNHL component in an energy-dependent manner after LISW exposure. This animal model could be a platform for elucidating the pathological mechanisms and exploring potential treatments for blast-induced cochlear dysfunction.

Laser nanoprocessing via an enhanced longitudinal electric field of a radially polarized beam.

Single-shot laser ablation is performed on the surface of a transparent glass material using a radially polarized femtosecond beam. Theoretical and experimental investigation revealed the significant role of the material interface under high-numerical-aperture conditions. The longitudinal electric field at the focus was remarkably enhanced due to the total reflection on the interface when a radially polarized beam was focused on the back surface of the glass from the inside using an immersion lens. This focusing condition enabled the fabrication of a small ablation hole sized 67 nm. This study offers a novel, to the best of our knowledge, approach to realize laser nanoprocessing with radially polarized beams.

Multibeam continuous axial scanning two-photon microscopy for in vivo volumetric imaging in mouse brain.

This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450 µm from the surface. In vivo volumetric Ca2+ imaging at a volume rate of 1.5 Hz within a depth range of 130-200 µm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.

In vivo visualization of burn depth in skin tissue of rats using hemoglobin parameters estimated by diffuse reflectance spectral imaging.

Significance: Burn injuries represent a global public health problem that kills an estimated 180,000 people annually. Non-fatal burns result in prolonged hospitalization, disfigurement, and disability. The most common, convenient, and widely used method for assessing burn depth is physical or visual examination, but the accuracy of this method is reportedly poor (60% to 75%). Rapid, correct assessment of burn depth is very important for the optimal management and treatment of burn patients. New methods of burn depth assessment that are inexpensive, simple, rapid, non-contact, and non-invasive are therefore needed. Aim: The aim of this study was to propose an approach to visualize the spatial distribution of burn depth using hemoglobin parameters estimated from spectral diffuse reflectance imaging and to demonstrate the feasibility of the proposed approach for differentiating burn depth in a rat model of scald burn injury. Approach: The new approach to creating a spatial map of burn depth was based on canonical discriminant analysis (CDA) of total hemoglobin concentration, tissue oxygen saturation, and methemoglobin saturation as estimated from spectral diffuse reflectance images. Burns of three different degrees of severity were created in rat dorsal skin by 10-s exposure to water maintained at 70°C, 78°C, and 98°C, respectively. Spectral images for dorsal regions were acquired under anesthesia immediately after burn injury and at 24 h, 48 h, and 72 h after injury. Results: Most areas of images in the group with skin exposed to 70°C water and 98°C water were classified as 70°C burn and 98°C burn, respectively. In contrast, no significant difference between areas classified as 78°C burn and 98°C burn from 24 h to 72 h was evident in the group with skin exposed to 78°C water, suggesting that burn depth was heterogeneous. Conclusions: The proposed approach combining diffuse reflectance spectral imaging and CDA appears promising for differentiating 70°C burns from 78°C burns and 98°C burns, and 98°C burns from 70°C burns and 78°C burns at 24 to 72 h after burn injury in a rat model of scald burn injury.

In vivo monitoring of hemoglobin derivatives in a rat thermal injury model using spectral diffuse reflectance imaging.

INTRODUCTION: To demonstrate the feasibility of our previously proposed Diffuse reflectance spectral imaging (DRSI) method for in vivo monitoring of oxygenated hemoglobin, deoxygenated hemoglobin, methemoglobin, tissue oxygen saturation, and methemoglobin saturation in a rat scald burn wound model and assess whether the method could be used for differentiating the burn depth groups in rats based on the hemoglobin parameters. METHODOLOGY: Superficial dermal burns (SDBs), deep dermal burns (DDBs), and deep burns (DBs) were induced in rat dorsal skin using a Walker-Mason method. An approach based on multiple regression analysis for spectral diffuse reflectance images aided by Monte Carlo simulations for light transport was used to quantify the hemoglobin parameters. Canonical discriminant analysis (CDA) was performed to discriminate SDB, DDB, and DB. RESULTS: CDA using the total hemoglobin concentration, tissue oxygen saturation, and methemoglobin saturation as the independent variables showed good performance for discriminating the SDB, DDB, and DB groups immediately after burn injury and the SDB group from the DDB and DB groups 24-72 h after burn injury. CONCLUSIONS: The DRSI method with multiple regression analysis for quantification of oxygenated hemoglobin, deoxygenated hemoglobin, and methemoglobin proved to be reliable for monitoring these hemoglobin derivatives in the rat experimental burn injury model. The parameters of tissue oxygen saturation, methemoglobin saturation, and total hemoglobin concentration are promising for the differentiating the degree of burn injury using CDA.

Trigeminal Sensitization in a Closed Head Model for Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) is often accompanied by neurological and ocular symptoms that involve trigeminal nerve pathways. Laser-induced shock wave (LISW) was applied to the skull of male rats as a model for mTBI, while behavioral and neural recording methods were used to assess trigeminal function. The LISW caused greater eye wiping behavior to ocular instillation of hypertonic saline (Sham = 4.83 ± 0.65 wipes/5 min, LISW = 12.71 ± 1.89 wipes/5 min, p < 0.01) and a marked reduction in the time spent in bright light consistent with enhanced periocular and intraocular hypersensitivity, respectively (Sham = 16.3 ± 5.6 s, LISW = 115.5 ± 27.3 s, p < 0.01). To address the early neural mechanisms of mTBI, single trigeminal brainstem neurons, identified by activation to corneal or dural mechanical stimulation, were recorded in trigeminal subnucleus interpolaris/caudalis (Vi/Vc) and trigeminal subnucleus caudalis/upper cervical cord (Vc/C1) regions. The LISW caused marked sensitization to hypertonic saline and to exposure to bright light in neurons of both regions (p < 0.05). Laser speckle imaging revealed an increase in meningeal arterial blood flow to bright light after LISW (Sham = 4.7 ± 2.0 s, LISW = 469.0 ± 37.9 s, p < 0.001). Local inhibition of synaptic activity at Vi/Vc, but not at Vc/C1, by microinjection of CoCl2, prevented light-evoked increases in meningeal blood flow in LISW-treated rats. By contrast, topical meningeal application of phenylephrine significantly reduced light-evoked responses of Vi/Vc and Vc/C1 neurons. These data suggested that neurons in both regions became sensitized after LISW and were responsive to changes in meningeal blood flow. Neurons at the Vi/Vc transition and at Vc/C1, however, likely serve different roles in mediating the neurovascular and sensory aspects of mTBI.

Transvascular delivery of talaporfin sodium to subcutaneous tumors in mice by nanosecond pulsed laser-induced photomechanical waves.

BACKGROUND: We previously developed a site-specific transvascular drug delivery system (DDS) based on photomechanical waves (PMWs) or laser-induced stress/shock waves (LISWs). In this study, we investigated the validity of this method to deliver a clinical photosensitizer, talaporfin sodium (TS), to subcutaneous tumors in mice and to enhance the efficacy of photodynamic therapy (PDT). METHODS: TS solution (2.5 mg/kg) was intravenously injected into mice. Immediately thereafter, PMWs were applied to the tumor by irradiating a laser target with a Q-switched ruby laser pulse (0.8 J/cm2). Five hours after TS administration, some tumors were excised to evaluate the depth distribution of the delivered TS under a fluorescence microscope. Other tumors were subjected to PDT by irradiating the tissues with a 665 nm continuous-wave laser diode (75 mW/cm2, 667 s) at this timepoint. The effects of PDT were evaluated on the basis of the two primary therapeutic mechanisms of TS-mediated PDT: i) damage to tumor cells and ii) damage to endothelial cells of tumor vessels, i.e., the vascular shutdown effect on tumors. RESULTS: PMW application significantly increased the accumulation of TS in the tumor parenchyma but not in the tumor vessel walls; the endothelial cell junctions of tumor vessels should be the route of TS delivery enhanced by PMWs. Thus, as a result of PMW application followed by PDT, while the vascular shutdown effect on the tumors was not enhanced, direct damage to the tumor cells was increased, resulting in significant tumor growth retardation without body weight loss for 7 days after treatment.

Enhanced Therapeutic Effects of an Antitumor Agent on Subcutaneous Tumors in Mice by Photomechanical Wave-based Transvascular Drug Delivery.

Purpose: We previously developed a site-selective transvascular drug delivery system based on nanosecond pulsed laser-induced photomechanical waves (PMWs). In this study, we applied this method to the delivery of cisplatin (cis-diamminedichloroplatinum, CDDP) to a subcutaneous tumor in a mouse and examined its antitumor effects. Methods: A mouse tumor model with subcutaneous inoculation of human head and neck cancer cells (FaDu cells) was used. The mice were divided into four groups: control without any treatment (control), CDDP application only (CDDP only), PMW application only (PMW only) and combined application of PMWs and CDDP (PMW+CDDP). A PMW was generated by irradiating a laser target, which was placed on the skin over the tumor, with a ruby laser pulse (fluence, 1.6 J/cm2). A CDDP solution was intraperitoneally injected into the mice (2.5 mg/kg). Results: Until 7 days posttreatment, the tumor volume in the control group monotonically increased, while the tumor volumes in the CDDP-only group and PMW-only group did not change greatly and that in the PMW+CDDP group slightly decreased. Afterward, the tumors started to regrow in all treatment groups, but the tumor growth rate was considerably low in the PMW+CDDP group. There was a significant difference in the time courses of tumor volume between the PMW+CDDP group and the control group for up to 14 days posttreatment. The ratio of the Ki-67-positive (proliferative) areas to the whole tumor regions in the PMW+CDDP group was significantly smaller than that in the control group at 7 days posttreatment. These results are attributable to the synergistic effects of enhanced extravasation of CDDP and mechanical tumoricidal effect by PMWs. Conclusion: The combined application of CDDP and PMWs significantly improved the antitumor effects on mouse subcutaneous tumors.

Visceral hypersensitivity induced by mild traumatic brain injury via the corticotropin-releasing hormone receptor: An animal model.

BACKGROUND: Mild blast-induced traumatic brain injury (bTBI) induces various gut symptoms resembling human irritable bowel syndrome (IBS) as one of mental and behavioral disorders. However, the underlying mechanisms remain unclear. We investigated whether the extremely localized brain impact extracranially induced by laser-induced shock wave (LISW) evoked IBS-like phenomenon including visceral hypersensitivity and intestinal hyperpermeability in rats. METHODS: The rats were subjected to LISW on the scalp to shock the entire brain. Visceral hypersensitivity was evaluated by the threshold pressure of abdominal withdrawal reflex (AWR) using a colorectal distension test. Permeability was evaluated by the concentration of penetrating FITC-dextran from intestine and the mRNA expression levels of tight junction family proteins. Involvement of corticotropin-releasing factor receptor (CRFR) 1 and 2 was examined by evaluating mRNA expression and modulating CRFR function with agonist, recombinant CRF (10 µg/kg), and antagonist, astressin (33 µg/kg). High-throughput sequencing of the gut microbiota was performed by MiSeqIII instrument and QIIME tool. KEY RESULTS: The thresholds of the AWR were significantly lowered after LISW. Permeability was increased in small intestine by LISW along with decreased expression of tight junction ZO-1. LISW significantly increased CRFR1 expression and decreased CRFR2 expression. Visceral hypersensitivity was significantly aggravated by CRFR agonist and suppressed by CRFR antagonist. The α- and ß-diversity of the fecal microbiota was altered after LISW. CONCLUSIONS AND INFERENCES: LISW provoked visceral hypersensitivity, small intestinal hyperpermeability, altered expression of CRFRs and changes in the microbiota, suggesting that genuine bTBI caused by LISW can induce a pathophysiology comparable to that of human IBS.

Nanoprocessing of Self-Suspended Monolayer Graphene and Defect Formation by Femtosecond-Laser Irradiation.

We demonstrate the femtosecond-laser processing of self-suspended monolayer graphene grown by chemical vapor deposition, resulting in multipoint drilling with holes having a diameter of <100 nm. Scanning transmission electron microscopy revealed the formation of many nanopores on the laser-irradiated graphene. Furthermore, atomic-level defects as well as nanopores were found in the graphene membrane by high-resolution transmission electron microscopy, while the overall crystal structure remained intact. Raman spectroscopy showed an increase in the defect density with an increase in the number of laser shots, suggesting that the nanopore formation triggered the creation of the <100 nm holes. The approach presented herein can offer an experimental insight into the simulation of atomic dynamics in graphene under femtosecond-laser irradiation. The thorough examination of the atomic defect formation and secondary effect of surface cleaning observed in this study would help develop engineering methods for graphene and other two-dimensional materials in the future.

Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes.

For the treatment of irreversible, extensive skin damage, artificial skins or cultured skins are useful when allogeneic skins are unavailable. However, most of them lack vasculature, causing delayed perfusion and hence delay or failure in engraftment of the tissues. We previously developed a prevascularized three-dimensional (3D) cultured skin based on the layer-by-layer cell coating technique (LbL-3D skin), in which cells are seeded and laminated on a porous polymer membrane for medium supply to the thick cultured tissue. Recent animal studies have demonstrated that LbL-3D skin can achieve rapid perfusion and high graft survival after transplantation. However, there were practical issues with separating LbL-3D skins from the membranes before transplantation and the handling separated LbL-3D skins for transplantation. To address these problems, in this study, we examined the use of biodegradable porous polymer membranes that enabled the transplantation of LbL-3D skins together with the membranes, which could be decomposed after transplantation. Thin films made from poly (lactic-co-glycolic acid) (PLGA) were irradiated with femtosecond laser pulses to create micro through-holes, producing porous membranes. We designed and fabricated culture inserts with the PLGA membranes and cultivated LbL-3D skins with 2 × 106 neonatal normal human dermal fibroblasts and 1 × 104 human umbilical vein endothelial cells in the dermis of 20 cell layers and 1 × 105 neonatal human epidermal keratinocytes in the epidermis. Histological analyses revealed that the skins cultured on the PLGA membranes had thickness of about 400 µm and that there were no defects in the quality of the skins cultured on the PLGA membranes when compared with those cultured on the conventional (nonbiodegradable) commercial membranes. The cultured LbL-3D skins were then transplanted together with the PLGA membranes onto full-thickness excisional wounds in mice. At 7 days posttransplantation onto a mouse, the tissues above and below the membrane were connected through the holes with collagen-positive fibers that appeared to migrate from both the host and donor sides, and favorable reepithelization was observed throughout the transplanted skin region. However, insufficient engraftment was observed in some cases. Thus, further optimization of the membrane conditions would be needed to improve the transplantation outcome.

In vivo imaging of nitric oxide in the male rat brain exposed to a shock wave.

While numerous studies have suggested the involvement of cerebrovascular dysfunction in the pathobiology of blast-induced traumatic brain injury (bTBI), its exact mechanisms and how they affect the outcome of bTBI are not fully understood. Our previous study showed the occurrence of cortical spreading depolarization (CSD) and subsequent long-lasting oligemia/hypoxemia in the rat brain exposed to a laser-induced shock wave (LISW). We hypothesized that this hemodynamic abnormality is associated with shock wave-induced generation of nitric oxide (NO). In this study, to verify this hypothesis, we used an NO-sensitive fluorescence probe, diaminofluorescein-2 diacetate (DAF-2 DA), for real-time in vivo imaging of male Sprague-Dawley rats' brain exposed to a mild-impulse LISW. We observed the most intense fluorescence, indicative of NO production, along the pial arteriolar walls during the period of 10-30 min post-exposure, parallel with CSD occurrence. This post-exposure period also coincided with the early phase of hemodynamic abnormalities. While the changes in arteriolar wall fluorescence measured in rats receiving pharmacological NO synthase inhibition by nitro-L-arginine methyl ester (L-NAME) 24 h before exposure showed a temporal profile similar to that of changes observed in LISW-exposed rats with CSD, their intensity level was considerably lower; this suggests partial involvement of NOS in shock wave-induced NO production. To the best of our knowledge, this is the first real-time in vivo imaging of NO in rat brain, confirming the involvement of NO in shock-wave-induced hemodynamic impairments. Finally, we have outlined the limitations of this study and our future research directions.

Burn depth assessment by dual-wavelength light emitting diodes-excited photoacoustic imaging in rats.

Accurate burn depth assessment is crucial to determine treatment plans for burn patients. We have previously proposed a method for performing burn depth assessments based on photoacoustic (PA) imaging, and we have demonstrated the validity of this method, which allows the successful detection of PA signals originating from the blood under the bloodless burned tissue, using rat burn models. Based on these findings, we started a clinical study in which we faced two technical issues: (1) When the burn depth was shallow, PA signals due to skin contamination and/or melanin in the epidermis (surface signals) could not be distinguished from PA signals originating from the blood in the dermis; (2) the size of the system was too large. To solve these issues, we propose a burn depth diagnosis based on dual-wavelength light emitting diodes (LEDs)-excited PA imaging. The use of LEDs rendered the system compact compared to the previous one that used a conventional solid-state laser. We replicated human burned skin by applying a titrated synthetic melanin solution onto the wound surface in albino rat burn models and measured their burn depths by PA excitation at 690 and 850 nm, where melanin and haemoglobin show greatly different absorption coefficients. As a result, the surface signals were eliminated by subtracting the PA signals at 690 nm from those at 850 nm. The resultant estimated burn depths were strongly correlated with the histological assessment results. The validity of the proposed method was also examined using a burn model of rats with real melanin.

The cause of acute lethality of mice exposed to a laser-induced shock wave to the brainstem.

Air embolism is generally considered the most common cause of death within 1 h of a blast injury. Shock lung, respiratory arrest, and circulatory failure caused by vagal reflexes contribute to fatal injuries that lead to immediate death; however, informative mechanistic data are insufficient. Here we used a laser-induced shock wave (LISW) to determine the mechanism of acute fatalities associated with blast injuries. We applied the LISW to the forehead, upper neck, and thoracic dorsum of mice and examined their vital signs. Moreover, the LISW method is well suited for creating site-specific damage. Here we show that only mice with upper neck exposure, without damage elsewhere, died more frequently compared with the other injured groups. The peripheral oxygen saturation (SpO2) of the former mice significantly decreased for < 1 min [p < 0.05] but improved within 3 min. The LISW exposure to the upper neck region was the most lethal factor, affecting the respiratory function. Protecting the upper neck region may reduce fatalities that are related to blast injuries.

Effects of Isolated and Combined Exposure of the Brain and Lungs to a Laser-Induced Shock Wave(s) on Physiological and Neurological Responses in Rats.

Blast-induced traumatic brain injury (bTBI) has been suggested to be caused by direct head exposure and by torso exposure to a shock wave (thoracic hypotheses). It is unclear, however, how torso exposure affects the brain in real time. This study applied a mild-impulse laser-induced shock wave(s) (LISW[s]) only to the brain (Group 1), lungs (Group 2), or to the brain and lungs (Group 3) in rats. Because LISWs are unaccompanied by a dynamic pressure in principle, the effects of acceleration can be excluded, allowing analysis of the pure primary mechanism (the effects of a shock wave). For all rat groups, real-time monitoring of the brain and systemic responses were conducted for up to 1 h post-exposure and motor function assessments for up to seven days post-exposure. As reported previously, brain exposure alone caused cortical spreading depolarization (CSD), followed by long-lasting hypoxemia and oligemia in the cortices (Group 1). It was found that even LISW application only to the lungs caused prolonged hypoxemia and mitochondrial dysfunction in the cortices (Group 2). Importantly, features of CSD and mitochondrial dysfunction were significantly exacerbated by combined exposure (Group 3) compared with those caused by brain exposure alone (Group 1). Motor dysfunction was observed in all exposure groups, but their time courses differed depending on the groups. Rats with brain exposure alone exhibited the most evident motor dysfunction at one day post-exposure, and after that, it did not change much for up to seven days post-exposure. Alternatively, two groups of rats with lung exposure (Group 2 and Group 3) exhibited continuously aggravated motor functions for up to seven days post-exposure, suggesting different mechanisms for motor dysfunction caused by brain exposure and that caused by lung exposure. As for the reported thoracic hypotheses, our observations seem to support the volumetric blood surge and vagovagal reflex. Overall, the results of this study indicate the importance of the torso guard to protect the brain and its function.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device.

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 µm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 µm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Viability Improvement of Three-Dimensional Human Skin Substitutes by Photobiomodulation during Cultivation.

Three-dimensional (3D) cultured skin containing vascular networks is a useful skin substitute that enables rapid reperfusion after transplantation. During its cultivation, however, insufficient nutrient delivery to the thick cultured tissue from the surrounding culture medium decreases the tissue viability. To solve this problem, in this study, we applied photobiomodulation (PBM), which can optically activate the electron transport chain of mitochondria, to human 3D skin cultures constructed using the layer-by-layer cell coating technique. PBM was applied once 5 days after the start of epidermal differentiation using a light-emitting diode array with a center wavelength of 440, 523, 658 or 823 nm at a constant light intensity of 15 mW cm-2 for 50 or 600 s. Two days after PBM, we assessed the viability of the tissues by a water-soluble tetrazolium-8 assay, adenosine triphosphate measurements and live/dead cell imaging, and the results showed that the PBM at 823 nm for 50 s (0.75 J cm-2 ) significantly improved the viability of the 3D-cultured skin.