Deciphering perivascular macrophages and microglia in the retinal ganglion cell layers.

Introduction: The classically defined two retinal microglia layers are distributed in inner and outer plexiform layers. Although there are some reports that retinal microglia are also superficially located around the ganglion cell layer (GCL) in contact with the vitreous, there has been a lack of detailed descriptions and not fully understood yet. Methods: We visualized the microglial layers by using CX3CR1-GFP (C57BL6) transgenic mice with both healthy and disease conditions including NaIO3-induced retinal degeneration models and IRBP-induced auto-immune uveitis models. Result: We found the GCL microglia has two subsets; peripheral (pph) microglia located on the retinal parenchyma and BAM (CNS Border Associated Macrophage) which have a special stretched phenotype only located on the surface of large retinal veins. First, in the pph microglia subset, but not in BAM, Galectin-3 and LYVE1 are focally expressed. However, LYVE1 is specifically expressed in the amoeboid or transition forms, except the typical dendritic morphology in the pph microglia. Second, BAM is tightly attached to the surface of the retinal veins and has similar morphology patterns in both the healthy and disease conditions. CD86+ BAM has a longer process which vertically passes the proximal retinal veins. Our data helps decipher the basic anatomy and pathophysiology of the retinal microglia in the GCL. Discussion: Our data helps decipher the basic anatomy and pathophysiology of the retinal microglia in the GCL.

A Distinct Microglial Cell Population Expressing Both CD86 and CD206 Constitutes a Dominant Type and Executes Phagocytosis in Two Mouse Models of Retinal Degeneration.

Microglial cells are the key regulators of inflammation during retinal degeneration (RD) and are conventionally classified as M1 or M2. However, whether the M1/M2 classification exactly reflects the functional classification of microglial cells in the retina remains debatable. We examined the spatiotemporal changes of microglial cells in the blue-LED and NaIO3-induced RD mice models using M1/M2 markers and functional genes. TUNEL assay was performed to detect photoreceptor cell death, and microglial cells were labeled with anti-IBA1, P2RY12, CD86, and CD206 antibodies. FACS was used to isolate microglial cells with anti-CD206 and CD86 antibodies, and qRT-PCR was performed to evaluate Il-10, Il-6, Trem-2, Apoe, and Lyz2 expression. TUNEL-positive cells were detected in the outer nuclear layer (ONL) from 24 h to 72 h post-RD induction. At 24 h, P2RY12 was decreased and CD86 was increased, and CD86/CD206 double-labeled cells occupied the dominant population at 72 h. And CD86/CD206 double-labeled cells showed a significant increase in Apoe, Trem2, and Lyz2 levels but not in those of Il-6 and Il-10. Our results demonstrate that microglial cells in active RD cannot be classified as M1 or M2, and the majority of microglia express both CD86 and CD206, which are involved in phagocytosis rather than inflammation.

Morphologic variations of the superior intercostal vein: An anatomical study with clinical applications.

This study aimed to validate and compare the anatomical variations of the superior intercostal veins, focusing on their origin, course, anastomoses, and destination. In addition, the results were compared with findings from other relevant studies. Fifty Korean and 16 Chinese adult cadavers were dissected for this study. The superior intercostal veins were dissected and measured. In our study of 66 specimens, the right superior intercostal vein was observed in 92.3% of cases, while the left superior intercostal vein was observed in 50%. The right superior intercostal vein was subdivided into six types based on its composition, which mainly drained the second and third right posterior intercostal veins. Similarly, the left superior intercostal vein was subdivided into eight types, primarily involving the second to fourth left posterior intercostal veins. This detailed anatomical study successfully identified and classified the various morphologic types of the superior intercostal vein and reviewed the clinical significance of this vein. The findings of this study can offer valuable anatomical evidence to physicians, aiding in their understanding and utilization of the superior intercostal vein.

Intravitreal Administration of Retinal Organoids-Derived Exosomes Alleviates Photoreceptor Degeneration in Royal College of Surgeons Rats by Targeting the Mitogen-Activated Protein Kinase Pathway.

Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.

Optimal needle electromyography approach to the serratus anterior muscle.

INTRODUCTION/AIMS: There are two conventional needle electromyography (EMG) approaches to the serratus anterior (SA), both of which can result in erroneous insertion into adjacent structures such as the latissimus dorsi (LD), teres major, or external oblique abdominis muscles and pose a risk of long thoracic nerve (LTN) injury. Therefore, we identified a novel needle insertion point for the SA in cadavers that avoids other muscles and LTN injury. METHODS: This study included 17 cadavers: 12 to devise the new method and 5 to verify its accuracy. Novel landmarks were the inferior angle of the scapula (I), sternal notch (S), and xiphoid process (X). The relationships of the LD, pectoralis major (PM), SA, and LTN were determined relative to these landmarks. RESULTS: When inserting a needle into the proximal one third along the line connecting points I and X, there were adequate safety margins around the LD, PM, and LTN, and the new method had excellent accuracy. DISCUSSION: Compared to the conventional midaxillary method, our novel method improved the accuracy of needle EMG of the SA. Follow-up studies using clinical imaging techniques are needed to verify whether above findings are equally applicable in living subjects.

Avoiding Pretarsal Denervation in Lower Blepharoplasty Incisions: Refined Pretarsal Motor Nerve Anatomy.

BACKGROUND: Pretarsal atrophy is not uncommonly found in patients who have undergone a transcutaneous or transconjunctival lower blepharoplasty due to intraoperative denervation of the pretarsal orbicularis oculi. The motor supplying concept to the lower eyelid was recently updated, however, there have not yet been any guidelines to preserve motor nerves in lower blepharoplasty incisions based on the refined knowledge. METHODS: 46 fresh cadaveric hemifaces were examined to find a safe zone for a lower blepharoplasty muscle incision and a danger zone for an infraorbital incision in the transblepharoplasty midface approach. Additionally, practical anatomy about the pretarsal motor supply was also investigated in detail. RESULTS: The medial, lateral, superior, and inferior borders of the safe zone for a lower blepharoplasty muscle incision were 9.4 mm from the medial canthus line, 3 mm from the lateral canthal crease, and 6.0 and 6.5 mm from the eyelid margin, respectively. The danger zone for an infraorbital incision ranged from 9.4 mm medial to the midpupillary line to 9.7 mm lateral to the midpupillary line. The motor nerve in the danger zone abutted the distal roof of the preseptal pocket making it vulnerable to electrocautery heat. Motor nerve distribution of the lower pretarsal orbicularis oculi was fully identified. CONCLUSION: There is a safe zone for the lower blepharoplasty muscle incision which if adhered to will preserve the pretarsal motor supply and prevent muscle atrophy. There is an infraorbital danger zone, where surgeons should pay special attention to avoid electrocautery heat injury.

Photobiomodulation therapy activates YAP and triggers proliferation and dedifferentiation of Müller glia in mammalian retina.

Photobiomodulation therapy has been proposed as a promising therapeutic approach for retinal degenerative diseases. However, its effect on the regenerative capacity in mammalian retina and its intracellular signalling mechanisms remain unknown. Here, we show that photobiomodulation with 670 nm light stimulates Müller glia cell cycle re-entry and dedifferentiation into a progenitor-like state in both the uninjured and injured retina. We also find that 670 nm light treatment inhibits the Hippo pathway, which is activated in Müller glia following NaIO3-induced retinal injury. YAP, a major downstream effector of the Hippo signalling pathway was translocated into the nucleus of Müller glia along with YAP dephosphorylation in retina treated with 670 nm light. Deficiency of YAP attenuated Müller glia cell cycle re-entry and dedifferentiation. Our data reveal that the Hippo-YAP signalling pathway is associated with the photostimulatory effect on regenerative response in mammalian retina, and suggest a potential therapeutic strategy for retinal degenerative diseases. [BMB Reports 2023; 56(9): 502-507].

How the Lifting Amount of Endoscopic Brow Lifts Is Influenced by Supraorbital Nerve Tension and Brow Gliding-Layer Mobility.

BACKGROUND: Sensory nerve tension and gliding-layer mobility in the brow may be significant factors affecting postoperative brow level in an endoscopic brow lift, yet they have rarely been studied. METHODS: To investigate the effects of sensory nerve tension and gliding-layer mobility, the following measurements were performed alongside the endoscopic brow lift in 50 fresh cadaveric hemifaces: amount of brow elevation, critical lifting amount (as sensory nerves became tense), laxity of sensory nerve courses, and mobility of brow-gliding layers. The sensory nerve situations in the subperiosteal and subgaleal dissections were also observed. RESULTS: Supraorbital nerve tension limited the cephalic advancement of the forehead flap. The mean elevation of the brow was 5.8 ± 1 mm (range, 3.5 to 8.6 mm). The mean critical lifting amount was 5.3 ± 1.1 mm (range, 4.0 to 7.3 mm). The mean amount of laxity in the supraorbital nerve (the permissible amount of lift) was 4.1 ± 0.9 mm (range, 2.5 to 5.5 mm). The galeal fat pad was responsible for 60% of brow mobility. The sensory nerve was more protected by a subgaleal dissection in the brow and inferior forehead and by a subperiosteal dissection in the middle and upper forehead. CONCLUSIONS: Cephalic movement of the forehead flap is limited by supraorbital nerve tension. The permitted lifting amount varies from 2.5 to 5.5 mm. Gliding-layer mobility in the brow offsets the postoperative amount of cephalic advancement of the forehead flap. Consideration of supraorbital nerve tension and gliding-layer mobility is recommended to obtain an optimal brow level in endoscopic brow lifts.

Orthodenticle homeobox 2 is transported to lysosomes by nuclear budding vesicles.

Transcription factors (TFs) are transported from the cytoplasm to the nucleus and disappear from the nucleus after they regulate gene expression. Here, we discover an unconventional nuclear export of the TF, orthodenticle homeobox 2 (OTX2), in nuclear budding vesicles, which transport OTX2 to the lysosome. We further find that torsin1a (Tor1a) is responsible for scission of the inner nuclear vesicle, which captures OTX2 using the LINC complex. Consistent with this, in cells expressing an ATPase-inactive Tor1aΔE mutant and the LINC (linker of nucleoskeleton and cytoskeleton) breaker KASH2, OTX2 accumulated and formed aggregates in the nucleus. Consequently, in the mice expressing Tor1aΔE and KASH2, OTX2 could not be secreted from the choroid plexus for transfer to the visual cortex, leading to failed development of parvalbumin neurons and reduced visual acuity. Together, our results suggest that unconventional nuclear egress and secretion of OTX2 are necessary not only to induce functional changes in recipient cells but also to prevent aggregation in donor cells.

Amplification of olfactory signals by Anoctamin 9 is important for mammalian olfaction.

Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9-deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Thus, we concluded that the signal amplification by ANO9 is important for mammalian olfactory transduction.

Single-stranded RNA adjuvant enhances the efficacy of 10-valent human papilloma virus-like particle vaccine.

Following the development of various types of vaccines, the use of adjuvants to boost vaccine efficacy has become a focus of research. Aluminum hydroxide (alum), the most commonly used adjuvant, induces a certain immune response and ensures safety in human trials. However, alum mainly induces only a Th2 response; its Th1 response is weak. Thus, we previously developed a single-stranded ribose nucleic acid (ssRNA) adjuvant that induces a Th1 response through toll-like receptors. Here, we explored whether 10-valent human papilloma virus (HPV)-like particle (VLP) vaccine formulated with ssRNA adjuvant and alum helped to enhance immune response and maintained memory response. The mice were immunized intramuscularly twice at 2 week intervals and were inoculated 4 days after the second boost (after about 1 year). The antibody response and T cell activation were measured by Elispot, ELISA using harvested serum and splenocytes. The 10-valent HPV VLP vaccine formulated with ssRNA adjuvant and alum increased the antigen-specific immune response more than alum used alone. It increased each type-specific IgG1/IgG2a titer, and antigen-specific IFN-γ cells. Furthermore, the ssRNA adjuvant with alum induced memory response. In memory response, each type-specific IgG1/IgG2c, IFN-γ, and IL-6 cytokine, and neutralizing antibodies were increased by the ssRNA adjuvant with alum. Overall, the ssRNA adjuvant with alum induced memory responses and balanced Th1/Th2 responses. The ssRNA adjuvant and alum may help to enhance prophylactic vaccine efficacy.

Refined Concept of Motor Supply to the Medial Periorbital Area Relevant to Periorbital Surgery.

BACKGROUND: Mimetic muscles in the medial periorbital area have been thought to be innervated solely by the angular nerve. Recently, however, the upper medial palpebral branch and lower palpebral branch were reported as additional motor suppliers in this area. This study aimed to define all the motor nerve systems passing through the medial canthal area. METHODS: Motor nerve branches that passed through the medial canthal region were identified and traced thoroughly from the parotid gland to their destinations under a surgical microscopic field in 74 hemifaces. The courses, anatomical positions of, and anatomical relationships between the angular nerve and the upper medial palpebral branch were observed. RESULTS: The upper medial palpebral branch and the angular nerve were found in all samples within a 3-mm to 6-mm area lateral to the intersecting point of the medial orbital rim and medial canthal ligament. The upper medial palpebral branch supplied the upper eyelid, whereas the angular nerve supplied the extraorbicularis muscles in the medial periorbital area. The medial pretarsal area of the upper eyelid was supplied solely by the pretarsal branches of the upper medial palpebral branch, which was formed by uniting three or four minor branches that traveled throughout the anterior cheek. CONCLUSIONS: Two separate motor nerve systems, the upper medial palpebral branch and the angular nerve, exist in the medial canthal area. The upper medial palpebral branch course along the medial orbital rim is considered as a facial nerve danger zone.

Correction: Kim et al. Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy. Int. J. Mol. Sci. 2021, 22, 4273.

The authors wish to make a correction to this paper [...].

Chorion-derived perinatal mesenchymal stem cells improve cardiac function and vascular regeneration: Preferential treatment for ischemic heart disease.

BACKGROUND: Stem cell therapy has emerged as a novel treatment for heart failure after myocardial infarction (Ml). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are commonly considered because of their accessibility and usability. However, their therapeutic potential remains controversial. In our previous in vitro study, chorion-derived mesenchymal stem cells (C-MSCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) demonstrated an ability to differentiate into cardiomyocytes and neural cells, respectively. Thus, we examined whether C-MSCs had a better differentiation potential in an MI animal model. METHODS: MI was induced by ligation of the left anterior descending artery, and DiI-labeled MSCs were injected into the border of the infarcted myocardium. The left ventricular ejection fraction (LVEF) and fractional shortening (FS) were measured using echocardiograms. Masson's Trichrome staining was performed to evaluate the viable myocardium. Alpha-sarcomeric actin (α-SA), cardiac troponin-T (cTnT), and isolectin were immunolabeled to evaluate differentiation and capillary formation. RESULTS: After 8 weeks, the LVEF and FS significantly increased to a greater extent in the C-MSC-injected group with maintenance of viable myocardium, as compared to in the control, UC-MSC-, and BM-MSC-injected groups (p < 0.05). Compared to UC-MSCs and BM-MSCs, C-MSCs significantly increased the capillary density (p < 0.05) and demonstrated higher expressions of cTnT and α-SA. CONCLUSIONS: In conclusion, compared to UC-MSCs and BM-MSCs, C-MSCs showed a better therapeutic efficacy in a rat MI model.

Morphological symmetry of the radius and ulna-Can contralateral forearm bones utilize as a reliable template for the opposite side?

The most important precondition for correction of the affected forearm using data from the contralateral side is that the left and right bone features must be similar, in order to develop patient-specific instruments (PSIs) and/or utilize computer-assisted orthopedic surgery (CAOS). The forearm has complex anatomical structure, and most people use their dominant hand more than their less dominant hand, sometimes resulting in asymmetry of the upper limbs. The aim of this study is to investigate differences of the bilateral forearm bones through a quantitative comparison of whole bone parameters including length, volume, bowing, and twisting parameters, and regional shape differences of the forearm bones. In total, 132 bilateral 3D radii and ulnae 3D models were obtained from CT images, whole bone parameters and regional shape were analyzed. Statistically significant differences in whole bone parameters were not shown. Regionally, the radius shows asymmetry in the upper section of the central part to the upper section of the distal part. The ulna shows asymmetry in the lower section of the proximal part to the lower section of the central part. Utilizing contralateral side forearm bones to correct the affected side may be feasible despite regional differences in the forearm bones of around 0.5 mm.

Differential Response of Müller Cells and Microglia in a Mouse Retinal Detachment Model and Its Implications in Detached and Non-Detached Regions.

Retinal detachment (RD) is a sight-threatening condition, leading to photoreceptor cell death; however, only a few studies provide insight into its effects on the entire retinal region. We examined the spatiotemporal changes in glial responses in a mouse RD model. In electroretinography, a- and b-waves were reduced in a time-dependent manner. Hematoxylin and eosin staining revealed a gradual decrease in the outer nuclear layer throughout the retinal region. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay showed that TUNEL-positive photoreceptors increased 5 days after RD and decreased by 14 days. Glial response was evaluated by immunohistochemistry using antibodies against glial fibrillary acidic protein (GFAP, Müller glial marker) and Iba-1 (microglial marker) and osteopontin (OPN, activated microglial marker). GFAP immunoreactivity increased after 7 days in complete RD, and was retained for 14 days. OPN expression increased in microglial cells 3-7 days after RD, and decreased by 14 days in the detached and border regions. Although OPN was not expressed in the intact region, morphologically activated microglial cells were observed. These retinal glial cell responses and photoreceptor degeneration in the border and intact regions suggest that the effects of RD in the border and intact retinal regions need to be understood further.

Complement System and Potential Therapeutics in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a complex multifactorial disease characterized in its late form by neovascularization (wet type) or geographic atrophy of the retinal pigment epithelium cell layer (dry type). The complement system is an intrinsic component of innate immunity. There has been growing evidence that the complement system plays an integral role in maintaining immune surveillance and homeostasis in AMD. Based on the association between the genotypes of complement variants and AMD occurrence and the presence of complement in drusen from AMD patients, the complement system has become a therapeutic target for AMD. However, the mechanism of complement disease propagation in AMD has not been fully understood. This concise review focuses on an overall understanding of the role of the complement system in AMD and its ongoing clinical trials. It provides further insights into a strategy for the treatment of AMD targeting the complement system.

Paradoxical paramagnetic calcifications in the globus pallidus: An ex vivo MR investigation and histological validation study.

MR images based on phase contrast images have gained clinical interest as an in vivo tool for assessing anatomical and histological findings. The globus pallidus is an area of major iron metabolism and storage in the brain tissue. Calcium, another important metal in the body, is frequently deposited in the globus pallidus as well. Recently, we observed dense paramagnetic deposition with paradoxical calcifications in the globus pallidus and putamen. In this work, we explore detailed MR findings on these structures, and the histological source of the related findings using ex vivo CT and MR images. Ex vivo MR was obtained with a maximum 100 µm3 isotropic resolution using a 15.2 T MR system. 3D gradient echo images and quantitative susceptibility mapping were used because of their good sensitivity to metallic deposition, high signal-to-noise ratio, and excellent contrast to iron and calcium. We found dense paramagnetic deposition along the perforating arteries in the globus pallidus. This paramagnetic deposition was hyperdense on ex vivo CT scans. Histological studies confirmed this finding, and simultaneous deposition of iron and calcium, although more iron dominant, was observed along the vessel walls of the globus pallidus. This was an exclusive finding for the penetrating arteries of the globus pallidus. Thus, our results suggest that several strong and paradoxical paramagnetic sources at the globus pallidus can be associated with vascular degeneration.

Expression of the Endoplasmic Reticulum Stress Marker GRP78 in the Normal Retina and Retinal Degeneration Induced by Blue LED Stimuli in Mice.

Retinal degeneration is a leading cause of blindness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress response that affects cell survival and death and GRP78 forms a representative protective response. We aimed to determine the exact localization of GRP78 in an animal model of light-induced retinal degeneration. Dark-adapted mice were exposed to blue light-emitting diodes and retinas were obtained at 24 h and 72 h after exposure. In the normal retina, we found that GRP78 was rarely detected in the photoreceptor cells while it was expressed in the perinuclear space of the cell bodies in the inner nuclear and ganglion cell layers. After injury, the expression of GRP78 in the outer nuclear and inner plexiform layers increased in a time-dependent manner. However, an increased GRP78 expression was not observed in damaged photoreceptor cells in the outer nuclear layer. GRP78 was located in the perinuclear space and ER lumen of glial cells and the ER developed in glial cells during retinal degeneration. These findings suggest that GRP78 and the ER response are important for glial cell activation in the retina during photoreceptor degeneration.

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy.

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu's method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.