Ultrastructural changes in the choriocapillaris of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL/6 mice.

N-methyl-N-nitrosourea (MNU) is known to cause apoptosis of photoreceptor cells and changes in retinal pigment epithelium (RPE). However, the changes in choriocapillaris, which nourishes photoreceptor cells by diffusing tissue fluid through RPE, have not been reported in detail. Therefore, we studied the ultrastructural transformation in and around the choriocapillaris to characterize the interdependence between choriocapillaris and surrounding tissue components in a mouse model. Seven-week-old male C57BL/6 mice were given a single intraperitoneal injection of MNU (60 mg/kg of body weight). Perfusion-fixed eyeballs were examined chronologically using immunohistochemistry and electron microscopy until the photoreceptor cells were lost. Sequential ultrastructural changes were observed in photoreceptor cells, RPE, Bruch's membrane, choriocapillaris, and choroidal melanocytes after an MNU injection. The lumens of the choriocapillaris narrowed following dilation, and the vascular endothelium showed structural alterations. When the photoreceptor cells were completely lost, the choriocapillaris appeared to be in a recovery process. Our results suggest that transport abnormality through Bruch's membrane and structural changes in the choroid might have influenced the morphology of choriocapillaris. The thin wall of the choriocapillaris appears to be the cause of the vulnerability with its altered morphology.

Pericytes in Cutaneous Wound Healing.

Most of the studies on cutaneous wound healing are focused on epidermal closure. This is obviously important, as the epidermis constitutes the main barrier that separates the inner organism from the environment. However, dermal remodeling is key to achieve long-lasting healing of the area that was originally wounded. In this chapter, we summarize what is known on the stromal components that strongly influence the outcome of healing and postulate that dedifferentiation of stably differentiated cells plays a major role in the initial response to wounding, as well as in long-term wound remodeling. Specifically, we explore the available evidence implicating skin pericytes, endothelial cells, Schwann cells, and macrophages as major players in a complex symphony of cellular plasticity and signaling events whose balance will promote healing (by tissue regeneration or repair) or fibrosis.

Pathological Process of Prompt Connection between Host and Donor Tissue Vasculature Causing Rapid Perfusion of the Engineered Donor Tissue after Transplantation.

The shortage of donors for transplantation therapy is a serious issue worldwide. Tissue engineering is considered a potential solution to this problem. Connection and perfusion in engineered tissues after transplantation is vital for the survival of the transplanted tissue, especially for tissues requiring blood perfusion to receive nutrients, such as the heart. A myocardial cell sheet containing an endothelial cell network structure was fabricated in vitro using cell sheet technology. Transplantation of the three-dimensional (3D) tissue by layering myocardial sheets could ameliorate ischemic heart disease in a rat model. The endothelial cell network in the 3D tissue was able to rapidly connect to host vasculature and begin perfusion within 24 h after transplantation. In this review, we compare and discuss the engineered tissue⁻host vasculature connection process between tissue engineered constructs with hydrogels and cell sheets by histological analysis. This review provides information that may be useful for further improvements of in vivo engineered tissue vascularization techniques.

Morphological characterization of pulmonary microvascular disease in bronchopulmonary dysplasia caused by hyperoxia in newborn mice.

PURPOSE: Pulmonary microvascular injury is associated with the pathogenesis of bronchopulmonary dysplasia (BPD). To characterize the mechanisms of pulmonary vascular disease resulting from BPD, we studied the ultrastructural changes affecting pulmonary microvasculature. METHODS: Newborn ICR mice were exposed to 85% hyperoxia or normoxia for 14 days, and then normal air replacement conditions for the following 7 days. At postnatal day (P)14 and P21, lungs were harvested for ultrastructural examination and assessment of pulmonary hypertension. RESULTS: The ultrastructure of pulmonary microvasculature in the hyperoxia-exposed lungs revealed a collapsed capillary lumen. This was due to the abnormal morphology of endothelial cells (ECs) characterized by heterogeneously thick cytoplasm. Compared to normal air controls, the specimens displayed also remarkably thick blood-air barriers (BABs), most of which were occupied by EC layer components. Structural changes were accompanied by increased pulmonary artery medial thickness and right ventricular hypertrophy (RVH). Moreover, abnormalities in ECs persisted even after exposure to 7 days of normal air replacement conditions. Results were confirmed by morphometric quantification. CONCLUSION: Our results suggest that the abnormal morphology of capillary ECs and thick BABs correlates with pulmonary artery remodeling and RVH. These ultrastructural changes might represent possible mechanisms of secondary pulmonary hypertension in BPD.

Foxc2 influences alveolar epithelial cell differentiation during lung development.

FOXC2, a forkhead transcriptional factor, is a candidate gene for congenital heart diseases and lymphedema-distichiasis syndrome and yellow nail syndrome; however, there are no reports on Foxc2 and the development of the lung. We have identified lung abnormalities in Foxc2-knockout embryos during investigation of cardiac development. The aim of this study was to clarify the morphological characteristics during lung development using ICR-Foxc2 knockout lungs. Mutant fetuses at embryonic days 10.5-18.5 were obtained from mating of Foxc2+/- mice and then analyzed. Notably, Foxc2-knockout lungs appeared parenchymatous and much smaller than those of the wild-type littermates. In the Foxc2 knockout lungs, the capillary beds remained distant from the alveolar epithelium until the late stages, the number of type2 alveolar cells per alveolar progenitor cell was lower and the type1 alveolar cells were thicker in Foxc2 knockout mice. In contrast, Foxc2 expression was only detected in the mesenchyme of the lung buds at E10.5, and it disappeared at E11.5 in Foxc2-LacZ knockin mice. Furthermore, the expression of Lef1 was significantly inhibited in E11.5 lungs. All of these results suggest that the abnormalities in Foxc2 knockout mice may involve maldifferentiation of alveolar epithelial cells and capillary vessel endothelial-alveolar epithelial approach as well as lymph vessel malformation. This is the first report about relationship between Foxc2 and lung development. This animal model might provide an important clue for elucidating the mechanism of lung development and the cause of respiratory diseases.

Invariant NKT cells induce plasmacytoid dendritic cell (DC) cross-talk with conventional DCs for efficient memory CD8+ T cell induction.

A key goal of vaccine immunotherapy is the generation of long-term memory CD8(+) T cells capable of mediating immune surveillance. We discovered a novel intercellular pathway governing the development of potent memory CD8(+) T cell responses against cell-associated Ags that is mediated through cross-presentation by XCR1(+) dendritic cells (DCs). Generation of CD8(+) memory T cells against tumor cells pulsed with an invariant NKT cell ligand depended on cross-talk between XCR1(+) and plasmacytoid DCs that was regulated by IFN-α/IFN-αR signals. IFN-α production by plasmacytoid DCs was stimulated by an OX40 signal from the invariant NKT cells, as well as an HMGB1 signal from the dying tumor cells. These findings reveal a previously unknown pathway of intercellular collaboration for the generation of tumor-specific CD8(+) memory T cells that can be exploited for strategic vaccination in the setting of tumor immunotherapy.

Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis.

BACKGROUND: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of ApcMin/+ mice and ApcMin/+/Vash2-/- mice. METHODS: ApcMin/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. ApcMin/+ mice were mated with Vash2-/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2-/- gene by PCR. Intestinal tumors from ApcMin/+ mice and ApcMin/+/Vash2-/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. RESULTS: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in ApcMin/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. CONCLUSION: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.

Role of uterine contraction in regeneration of the murine postpartum endometrium.

The endometrium undergoes continuous repair and regeneration without scarring throughout the reproductive life of women. However, the mechanisms responsible for this complete restoration remain mostly unexplored. We hypothesized that the ischemic state and local hypoxia present after parturition may create a special microenvironment for endometrial healing, and that this ischemia might be caused by reduction in organ volume via postpartum uterine contraction. Here, we developed a mouse model using a combination of cesarean section and the administration of a beta 2 adrenergic receptor agonist (ritodrine hydrochloride) in postpartum mice that had been ovariectomized to exclude the effect of ovarian hormones. Our results revealed that transient hypoxia indeed occurred in postpartum uteri. Furthermore, we found that the number of M2 macrophages, which play a central role in wound healing, peaked on Postpartum Day 3 and gradually decreased thereafter in hypoxic injury sites. Almost concurrently, significant upregulation of vascular endothelial growth factor and transforming growth factor beta (TGFbeta) was observed. In particular, the antifibrotic factor TGFbeta3 was released during the endometrial healing process. These changes were significantly suppressed by inhibition of uterine contraction. Taken together, these results suggest that uterine contraction is essential, not only for hemostasis, but also for endometrial regeneration, leading to a process that involves the activation of macrophages, increased endometrial cell proliferation, and upregulation of nonfibrotic growth factors. This study paves the way to a novel approach for investigating the process of scarless wound healing.

Intravitreal injection of erythropoietin protects against retinal vascular regression at the early stage of diabetic retinopathy in streptozotocin-induced diabetic rats.

A single intravitreal injection of erythropoietin (EPO) (50 ng/eye) or phosphate-buffered saline was administered to 5-week-old Sprague-Dawley rats at the onset of diabetes mellitus (DM) to determine and evaluate the protective effect of EPO on retinal microvessels. DM was induced by an intraperitoneal injection of streptozotocin (STZ; 60 mg/kg body weight). Morphological changes in microvessels in flat retinal preparations were evaluated during the subsequent 4 weeks by three-dimensional imaging of all blood vessels stained with fluorescein isothiocyanate-conjugated tomato lectin, following immunofluorescence techniques. No marked differences were observed in the shape or density of retinal vessels and the number of retinal capillary branches of the four groups [control, EPO, DM, and DM/EPO] up to 4 weeks after STZ administration. We also observed unique type IV collagen-positive filamentous structures that lacked both cellular elements and blood circulation (lectin-/type IV+ acellular strands), suggesting regressed vessel remnants. The lectin-/type IV+ acellular strands were detected soon after the onset of DM in the diabetic rats, and the number of these structures increased in the DM group (P < 0.01). A single intravitreal injection of EPO caused a significant reduction in the number of lectin-/type IV+ acellular strands to levels observed in the control group. However, the lectin-/type IV+ acellular strands were observed in the central area of the retina near the optic disc in all four groups. Intravitreal injection of EPO resulted in downregulation of the EPO receptor, vascular endothelial growth factor (VEGF), and VEGF receptor at 4 weeks. We conclude that EPO may play a primary role against the progression of diabetic retinopathy by reducing blood vessel degeneration at a very early disease stage.

Recruited peripheral blood monocytes participate in the liver extramedullary hematopoietic milieu.

The hematopoietic microenvironment has been investigated and well defined in the bone marrow. However, there is a lack of studies on the extramedullary hematopoietic milieu such as in the liver, to which hematopoietic stem cells migrate and there commence hematopoiesis under pathological conditions such as bone marrow failure. We induced extramedullary hematopoiesis by phenylhydrazine in the adult mouse liver and investigated the immunohistochemical, ultrastructural, and molecular changes within this organ. Using an intravital lectin injection technique, we found numerous monocytes attached to the central vein prior to hematopoietic foci formation. These cells were later incorporated into the hematopoietic foci. An increase in the mRNA expressions of the monocyte attracting chemokine CCL-2 (MCP-1) was noted in the central vein region as well as in cells within the hematopoietic foci. Together with local liver components, we regard these monocytes as components of the extramedullary hematopoietic milieu. We conclude that the recruitment of extra-hepatic monocytes is an important event during extramedullary hematopoiesis in the liver and that these monocytes participate in the liver hematopoietic microenvironment.

Fine structural study of the innervation of muscle spindles in the internal oblique muscle of the abdominal wall in the adult mouse.

We examined by electron microscopy the innervation of muscle spindles in the internal oblique muscle of the mouse abdominal wall. In the equatorial region, in addition to the sensory innervation on individual intrafusal muscle fibers, sensory cross terminals were often observed between nuclear chain fibers. In the area from the juxtaequatorial region to the polar region, nuclear bag fibers were supplied by trail and plate-type motor endings, while nuclear chain fibers were innervated by sensory endings, being probably secondary sensory endings. From these findings, it is clear that the innervation patterns differ between two types of intrafusal muscle fibers.

Alteration of angiogenic patterns on B16BL6 melanoma development promoted in Matrigel.

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.

Distribution and morphological characteristics of the interstitial cells of Cajal in the ileocaecal junction of the guinea-pig.

The guinea-pig ileocaecal junction including the valve was studied by immunohistochemistry to clarify the organization of the muscle bundles, the enteric nerves and the interstitial cells of Cajal (ICC). This region clearly exhibited characteristic features in the distribution patterns of ICC in a proximal to distal direction: (1) the thickened portion of the terminal ileum immediately adjacent to the ileocecal junction contained many ICC throughout the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers, but ICC were few or absent in the rest of the ileum; (2) the ileal side of the valve contained ICC associated with the deep muscular plexus (ICC-DMP) as in the small intestine, whereas ICC-DMP were absent in the caecal side as in the caecum; (3) the valve contained many ICC-CM and ICC-LM in both the ileal and caecal sides; (4) many ICC associated with the myenteric plexus were observed in both the ileal and caecal sides of the valve, whereas they were only sparsely found in the caecum; (5) ICC were also observed around the submucosal plexus in a confined area of the terminal ileum and the ileocaecal valve. These observations provide morphological evidence that the terminal ileum and ileocaecal valve are specially equipped for their active involvement in the movement of the junctional area.

Local lymphogenic migration pathway in normal mouse spleen.

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP(+)) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP(+) cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.

Systemic transmigration of allosensitizing donor dendritic cells to host secondary lymphoid organs after rat liver transplantation.

UNLABELLED: Donor dendritic cell (DC) migration and allosensitization in host secondary lymphoid organs after liver transplantation are ill defined. We used rat models to investigate graft-derived cells and intrahost allosensitization. Liver transplantation induced diffuse blood-borne migration of donor major histocompatibility class II antigen-positive (MHCII(+)) cells and MHCI(+) cells from the graft to host secondary lymphoid organs, not only the spleen, but also lymph nodes and Peyer's patches. The migrated MHCII(+) cells included DCs and some T cells and B cells. The DCs formed clusters with host BrdU(+) cells where they up-regulated CD86(+), and a CD8(+) T cell proliferative response originated within 24 hours after liver transplantation, demonstrating that these DCs can quickly mature and trigger direct allosensitization in host lymphoid organs. Transfer of allogeneic bone marrow cells also induced DC transmigration and a similar host response. In contrast, allogeneic thoracic duct lymph cells contained many fewer transmigrating DCs, and their transfer induced a comparable T cell response but significantly weaker CD8(+) T cell proliferation. Thus, there is a different outcome via the indirect pathway by host DCs that have captured donor alloantigens. CONCLUSION: The rat liver as well as bone marrow contains an immature DC population that can systemically transmigrate through blood vessel walls of the host secondary lymphoid organs, quickly mature, and induce diffuse intrahost CD8(+) T cell responses, which may promote graft rejection.

Characterization of the sugar chain expression of normal term human placental villi using lectin histochemistry combined with immunohistochemistry.

The general sugar expression pattern was studied in 9 normal full-term human placentas by the use of 21 individual lectins in combination with immunohistochemistry for various markers to understand the function of the placenta as the site of feto-maternal interactions. In mature intermediate and terminal villi, the brush border of the syncytiotrophoblast layer strongly expressed GlcNAc (as stained by WGA, S-WGA, DSL lectins) but weakly expressed sialic acid (Mal II, SNA). The cytoplasm of the syncytiotrophoblast layer showed weak expressions of GlcNAc and Gal/GalNAc with granular patterns. The cytotrophoblast layer, as also recognized by PCNA and HAI-1, typically expressed GlcNAc (LEL etc.) and Gal/GalNAc (MAL I). We found that the cytotrophoblast layer became very thin but largely maintained its continuity in the mature villi. The basement membranes of both the trophoblast layer and the endothelial layer strongly and continuously expressed mannose (Con A, LCA) and galactose (ECL, RCA I). Although endothelial cells almost exclusively expressed sialic acid and fucose, UEA I showed a heterogeneous reactivity with endothelial cells within the same vessels. No uniform expression pattern of any sugar was seen in stromal components except for Hofbauer cells, which usually expressed GlcNAc (LEL and DSL etc.). Thus, the sugar expression analysis by lectin histochemistry combined with immunohistochemistry proved helpful to understand the sugar chain related functions of the placenta under both normal and pathological conditions.

Proliferating mesodermal cells in murine embryos exhibiting macrophage and lymphendothelial characteristics.

BACKGROUND: The data on the embryonic origin of lymphatic endothelial cells (LECs) from either deep embryonic veins or mesenchymal (or circulating) lymphangioblasts presently available remain inconsistent. In various vertebrates, markers for LECs are first expressed in specific segments of embryonic veins arguing for a venous origin of lymph vessels. Very recently, studies on the mouse have strongly supported this view. However, in the chick, we have observed a dual origin of LECs from veins and from mesodermal lymphangioblasts. Additionally, in murine embryos we have detected mesenchymal cells that co-express LEC markers and the pan-leukocyte marker CD45. Here, we have characterized the mesoderm of murine embryos with LEC markers Prox1, Lyve-1 and LA102 in combination with macrophage markers CD11b and F4/80. RESULTS: We observed cells co-expressing both types of markers (e.g. Prox1 - Lyve-1 - F4/80 triple-positive) located in the mesoderm, immediately adjacent to, and within lymph vessels. Our proliferation studies with Ki-67 antibodies showed high proliferative capacities of both the Lyve-1-positive LECs of lymph sacs/lymphatic sprouts and the Lyve-1-positive mesenchymal cells. CONCLUSION: Our data argue for a dual origin of LECs in the mouse, although the primary source of embryonic LECs may reside in specific embryonic veins and mesenchymal lymphangioblasts integrated secondarily into lymph vessels. The impact of a dual source of LECs for ontogenetic, phylogenetic and pathological lymphangiogenesis is discussed.

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method.

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.

Survival of skin allografts is prolonged in mice with a dominant-negative H-Ras.

Ras is a guanine nucleotide-binding protein that plays a major role in regulating the proliferation of T cells. To investigate the mechanism of the Ras/mitogen-activated protein kinase pathway, one of the downstream signal-transduction pathways of T-cell receptors, in the response to alloantigen, we performed full-thickness skin grafting in the major histocompatibility complex (MHC) incompatible strain BALB/c (H-2Kd) (donor) and T-cell-specific H-Ras dominant-negative (dnRas) transgenic (tg) C57BL/6 (H-2Kb) (recipient) male mice. In vitro and in vivo dnRas tg mouse T-cell proliferation and cytotoxic T lymphocyte (CTL) activity assay were also performed. The median graft survival time in control B6/wild type (wt) mouse allografts was seven days. Conversely, the dnRas tg mouse group exhibited a significant (p<0.01) prolongation of graft survival to 15 days. However, all grafts were eventually rejected after one month. Mixed lymphocyte reaction and popliteal lymph node assay revealed that T-cell proliferation was decreased in response to alloantigen, but CTL activity was not changed in the dnRas tg mice. These results suggested that Ras is essential for peripheral T lymphocytes to respond to allo-MHC antigens, and Ras may be a molecular target for controlling transplant rejection.

[Cancer stromal cells influence on the tumor angiogenic pattern].

Since the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize the tumor angiogenesis three-dimensionally in a highly metastatic mouse melanoma model, B16BL6 melanoma injected with Matrigel into syngeneic C57BL/6 mice by using confocal laser scanning microscopy and transmission electron microscopy. We found that B16 melanoma within Matrigel grew significantly quicker than B16 melanoma alone and was accompanied by altered tumor angiogenesis and avascular area. We characterized the unique patterns of avascular area by using confocal laser-scanning microscopy and transmission electron microscopy.