Three-Dimensional Ultrastructural and Volume Analysis of the Redundant Nuclear Envelope of Developing and Matured Sperm in Mice.

The ultrastructure of the nuclear envelope (NE) and redundant NE (RNE) of the spermatozoon cannot be observed in detail using conventional electron microscopy. Thus, this study aimed to employ transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography to fill this research gap. Male mice aged 13 weeks were deeply anesthetized, and the testes and vas deferens were extracted and processed for electron microscopy. In round spermatids, the acrosomal vesicle compressed the nucleus, and the acrosomal center was depressed. The nucleoli concentrated on the contralateral side of the acrosome formation site. In mature spermatozoa, the RNE accumulated in the neck with the residual bodies. The NE pores exhibited a hexagonal pattern. The body surface area and volume of the nuclei of spermatids and spermatozoa in each maturation phase were analyzed using FIB/SEM tomography. The body surface area and volume of the nuclei decreased during spermatid maturation into spermatozoa. The RNE converged at the sperm neck and possessed a honeycomb structure. The method used revealed that the nuclei of spermatids gradually condense as they mature into spermatozoa. This method may be used to analyze small tissues, such as RNE, and detect morphological abnormalities in microtissues, such as spermatozoa.

Three-Dimensional Ultrastructural Analysis of the Head-Most Mitochondrial Roots of Mice Spermatozoa Using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Tomography.

This study aimed to clarify the three-dimensional ultrastructure of head-side mice spermatozoa mitochondria. Six 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with 2% paraformaldehyde, and placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for electron microscopy. After perfusion, the vas deferens was removed, and the specimens were cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on five mitochondria on the spermatozoa head using conventional transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. Conventional TEM analysis showed that head-side mitochondria were not spiral in morphology but clearly horizontal to the sperm axis. However, this was difficult to evaluate further using conventional TEM. In the FIB/SEM analysis, the first and second head-most mitochondria were flat and straight, with no helix, and shaped as an attachment plug with two electrodes, and their tail side contacted the third mitochondrion. The third mitochondrion was shorter than the fourth and fifth and had a semicircular arching structure. The fourth and fifth mitochondria were spiral-shaped and intertwined. The redundant nuclear envelope encircled the head-most mitochondria. This ultrastructural analysis clarified that the head-most mitochondria have a unique morphology.

Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM.

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

Correlative imaging of collagen fibers and fibroblasts using CLEM optimized for picrosirius red staining and FIB/SEM tomography.

Conventional imaging for three-dimensional (3D) ultra-architectural analysis of collagen fibers and fibroblasts is time-consuming and requires numerous ultrathin sections to search the target area. Currently, no method allows 3D ultra-architectural analysis of predetermined areas including spatial relationships between collagen fibers and fibroblasts in vitro. Herein, we developed a new method for in vitro analysis of the 3D ultrastructure of fibroblasts and collagen fibers using CLEM optimized for picrosirius red staining and FIB/SEM tomography. Collagen fibers were observed between, rather than on top of, stacked cells. This method offers the advantage of mesoscopic and ultrastructural analysis, thus minimizing bias and ensuring accurate observation.

Three-dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. MATERIAL AND METHODS: The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. RESULTS: Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. CONCLUSION: These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

The Usefulness of Piezoelectric Surgery in Sagittal Split Ramus Osteotomy.

Mandibular osteotomy carries with it a risk of damaging blood vessels and nerves when using traditional surgical techniques. Piezosurgery®, is a new technique that uses ultrasonic vibration to enable bone-selective sectioning without damage to the surrounding soft tissues. However, paralysis may not be completely eliminated using Piezosurgery® for osteotomy. We investigated how piezoelectric surgery in bilateral sagittal splitting ramus osteotomy (BSSRO) affected the surrounding soft tissue. Forty-four patients with skeletal mandibular prognathism underwent mandibular setback with BSSRO. Patients were divided into two groups, those treated by the conventional chisel technique and those treated by Piezosurgery®. Osteotomy time, blood loss, and incidence of paresthesia were compared retrospectively. Osteotomy time and blood loss in the piezo group were significantly reduced compared to the chisel group. Interestingly, whereas paresthesia incidence immediately after the operation did not differ between the groups, paresthesia in the piezo group 3 months postoperatively was significantly less than in the chisel group. However, a few cases of paralysis did not recover even in the piezo group. Blood loss and osteotomy time did not correlate with the paralysis. This study demonstrates that while piezoelectric surgery does impact the nerve tissue, the use of piezoelectric surgery in BSSRO leads to significantly less long term paralysis compared to surgery done by chisel.

Three-dimensional ultrastructural analysis of the interface between an implanted demineralised dentin matrix and the surrounding newly formed bone.

Previous investigators have reported that transplanted demineralised dentin matrix (DDM) influences bone formation in vivo. However, the specific mechanism of how dentinal tubules contribute to bone formation has not been determined with regard to DDM transplantation therapy. In this study, we ultrastructurally investigated how DDM contacted the surrounding newly formed bone using a scanning electron microscopy (SEM) three-dimensional reconstruction method that is based on focused ion beam slicing and SEM (FIB/SEM). A pulverised and processed DDM derived from human teeth was implanted into rat calvarial bone defects, and a series of X-ray computed tomographic images were obtained over 12 weeks. Implants with surrounding new bone were removed and histologically examined using FIB/SEM. After obtaining objective block-face images, the target boundary face was reconstructed three-dimensionally. The osteocytes of the new bone tissue surrounding the DDM formed a network connected by their cellular processes and formed bone tissue. It is also interesting that the cellular processes of the osteocytes extended into the dentinal tubules, and that bone tissue with canaliculi had formed and filled the DDM surface.

Uncoupled mitochondria quickly shorten along their long axis to form indented spheroids, instead of rings, in a fission-independent manner.

Loss of mitochondrial membrane potential (ΔΨm) triggers dramatic structural changes in mitochondria from a tubular to globular shape, referred to as mitochondrial fragmentation; the resulting globular mitochondria are called swelled or ring/doughnut mitochondria. We evaluated the early period of structural changes during the ΔΨm loss-induced transformation after carbonyl cyanide m-chlorophenyl hydrazine (CCCP) administration using a newly developed correlative microscopic method combined with fluorescence microscopic live imaging and volume electron microscopy. We found that most mitochondria changed from a tubular shape to a globular shape without fusion or fission and typically showed ring shapes within 10 min after CCCP exposure. In contrast, most ring mitochondria did not have a true through hole; rather, they had various indents, and 47% showed stomatocyte shapes with vase-shaped cavities, which is the most stable physical structure without any structural support if the long tubular shape shortens into a sphere. Our results suggested that loss of ΔΨm triggered collapse of mitochondrial structural support mechanisms.

AGEs-RAGE overexpression in a patient with smoking-related idiopathic nodular glomerulosclerosis.

We report a case of smoking-related idiopathic nodular glomerulosclerosis (ING) with overexpression of glomerular advanced glycation end products (AGEs) and their receptor (RAGE). A 59-year-old Japanese man with nephrotic syndrome, who had a smoking history of one pack of cigarettes per day for approximately 40 years, presented with a 3-year history of urinalysis abnormalities without clinical evidence of diabetic mellitus. The patient's leg edema progressively worsened over the previous 2 years, and he was admitted to our hospital. Renal biopsy showed mesangial expansion with diabetic Kimmelstiel-Wilson-like nodular lesions, glomerular basement thickening, and arteriosclerosis. No electron-dense deposits, fibrils, or microtubule deposits were seen in the glomeruli on electron microscopy. Skin AGE level measured using AGE reader was higher in this case than the average level in age-matched Caucasians. In addition, immunohistochemical analysis revealed that N-carboxymethyl lysine, one of the major AGEs, and RAGE were overexpressed and podocin expression was decreased in the peripheral area of the glomerular nodular lesions. These observations suggest that AGEs-RAGE system may be activated in smoking-related ING, possibly leading to the progression of renal dysfunction.

An unusual finding of the auriculotemporal nerve: possible risk factor during preauricular skin incisions.

The auriculotemporal nerve (ATN) is a branch of the mandibular nerve and has been implicated for some migraines and its role in Frey's syndrome is well known. An adult cadaver was found to have a duplicated ATN. The anterior trunk ascended as the superficial temporal artery and gave off the branches to the temporomandibular joint, parotid gland, external acoustic meatus and temporal region and communicated with a posterior trunk of the ATN. The posterior trunk ascended via the subcutaneous tissues 1 mm anterior to the auricle and gave off the branches to the anterior auricular region, temporal region and communicated with the anterior trunk. Such a duplicated ATN might be injured with preauricular skin incisions. Knowledge of such an anatomical variation might assist surgeons in iatrogenic injury of the ATN.