Takao Inoué

Three‐dimensional structural changes in cerebral microvessels after transient focal cerebral ischemia in rats: Scanning electron microscopic study of corrosion casts

Pathological changes of cerebral microvessels in transient ischemia were investigated by scanning electron microscopy of vascular corrosion casts. Wistar rats were treated with middle cerebral artery (MCA) occlusion for 30 min, 1 h,  3 h,  4 h,  5 h  or  7 h  and  subsequent  reperfusion  for 2 h. The ultrastructures of the cast were observed and computer‐aided montage micrographs were obtained for visualization of the whole microvasculature in the ischemic brain hemisphere. Avascular areas representing ischemic areas were detected in the frontotemporal cortex and caudate putamen in the groups from 30 min to 5 h occlusion. Extravasation of the resin, which probably corresponded to the leakage of plasma or hemorrhage, was seen as spheroidal, conglomerative, large massive and worm‐like types. The spheroidal type, which probably indicated a small leakage or minor hemorrhage, began to appear in the 30‐min occlusion group. The conglomerative type, which probably indicated a larger leakage or moderate hemorrhage, appeared in the 3‐ to 5‐h occlusion groups. The large massive and worm‐like types, which probably indicated a significant hemorrhage, appeared in the 4‐ and 5‐h occlusion groups. The number of these extravasations increased significantly in the 4‐h occlusion group. Arterioles near the avascular area frequently showed vasospastic appearances, such as corrugations, fusiform indentations of endothelial nuclei, continuous circulatory constrictions and severe narrowing with interrupted branches. Arteriolar vasospasm possibly caused prolonged hypoperfusion even if reperfusion was achieved. The capillaries had a thin stringy appearance in the 4‐ and 5‐h occlusion groups. These changes seemed to relate closely with increased intracranial pressure by brain edema or hemorrhage. The present study suggested that the risk of brain edema or hemorrhagic infarction increased beyond 3 h of MCA occlusion, and vasospasm of the arterioles might participate in stroke pathophysiology.

Scanning electron microscopy of the basal surface of the epidermis of human digits.

The epidermal-dermal junction of the volar skin of human digits was examined by scanning electron microscopy (SEM). Fixed specimens obtained from cadavers were treated with 6 N NaOH to exfoliate the epithelium. The three-dimensional features of the basal surfaces were clearly visualized by SEM. Epithelial prolongations (intermediate and limiting ridges) protruded towards the dermis, and transverse ridges were visible between them. Nerve fibers and Schwann cells were aggregated around the sweat ducts on the intermediate ridges. Discoid structures were visible among the nerve fibers and Schwann cells. Examination by transmission electron microscopy of ultrathin sections of re-embedded specimens confirmed that the discoid structures were nerve endings that covered the Merkel cells. Minute projections were studded on the basal surfaces of basal keratinocytes. The projections in the intermediate ridges appeared to be villous, whereas the profiles of those in the limiting ridges resembled cobblestones. This morphological difference might be a reflection of function.

N-cadherin expression in palisade nerve endings of rat vellus hairs.

Palisade nerve endings (PNs) are mechanoreceptors around vellus hairs of mammals. Each lanceolate nerve ending (LN) of the PN is characterized by a sensory nerve ending symmetrically sandwiched by two processes of type II terminal Schwann cells (tSCIIs). However, the molecular mechanisms underlying the structural organization of the PN are poorly understood. Electron microscopy showed that adherens junctions appeared to adhere to the sensory nerve ending and tSCII processes, so we examined the location of the N‐cadherin adhesion system in PNs of rat vellus hairs by using immunoelectron microscopy. N‐cadherin localized near both ends of the cell boundary between sensory nerve ending and tSCII processes, which corresponded to the sites of adherens junctions. We further found cadherin‐associated proteins, α‐ and β‐catenins, at the linings of adherens junctions. Three‐dimensional reconstruction of immunoelectron microscopic serial thin sections showed four linear arrays of N‐cadherin arranged longitudinally along the LN beneath the four longitudinal borders of two tSCII processes. In contrast, sensory nerve fibers just proximal to the LNs formed common unmyelinated nerve fibers, in which N‐cadherin was located mainly at the mesaxon of type I terminal Schwann cells (tSCIs). These results suggest that the four linear arrays of N‐cadherin‐mediated junctions adhere the sensory nerve ending and tSCII processes side by side to form the characteristic structure of the LN, and the structural differences between the LNs and the proximal unmyelinated nerve fibers possibly are due to the difference in the pattern of N‐cadherin expression between sensory nerve endings and tSCII or tSCI processes. J. Comp. Neurol. 506:525–534, 2008.

Intercellular junctions between palisade nerve endings and outer root sheath cells of rat vellus hairs.

Hair follicles have a longitudinal set of sensory nerve endings called palisade nerve endings (PN). We examined the junctional structures between the PN and outer root sheath (ORS) cells of hair follicles in the rat external ear. Transmission electron microscopy of serial thin sections showed that the processes of the ORS cells penetrated the basal lamina of the hair follicle, forming intercellular junctions with the PN (PN‐ORS junctions). Two types of junctions were found: junctions between nerve endings and ORS cells (N‐ORS junctions) and those between Schwann cell processes and ORS cells (S‐ORS junctions). The N‐ORS junctions had two subtypes: 1) a short process or small eminence of the ORS cell was attached to the nerve ending (type I); or 2) a process of the ORS cell was invaginated into the nerve ending (type II). The S‐ORS junctions also had two subtypes: 1) a short process or small eminence of the ORS cell was abutted on the Schwann cell process (type I); or 2) a process of the ORS cell was invaginated into the Schwann cell process (type II). Vesicles, coated pits, coated vesicles, and endosomes were sometimes seen in nerve endings, Schwann cells, and ORS cells near the junctions. Computer‐aided reconstruction of the serial thin sections displayed the three‐dimensional structure of these junctions. These results suggested that the PN‐ORS junctions provided direct relationships between the PN and ORS in at least four different patterns. The discovery of these junctions shows the PN‐ORS relationship to be closer than previously realized. We speculate that these junctions may have roles in attachment of the PN to the ORS, contributing to increases in the sensitivity of the PN, and in chemical signaling between the PN and ORS. J. Comp. Neurol. 420:419–427, 2000.

The three-dimensional ultrastructure of intracellular organization of smooth muscle cells by scanning electron microscopy

The membranous organization of smooth muscle cells is characterized by surface caveolae, sarcoplasmic reticulum, and mitochondria. Their structures are quite different from those recognized in skeletal or cardiac muscle cells. In smooth muscle cells, the surface caveolae and the sarcoplasmic reticulum are of great interest from the viewpoint of excitation—contraction coupling.

High-resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens

Intracellular structures of rapidly frozen biological tissues were observed in 3‐D under a low‐temperature scanning electron microscope using a newly developed side‐entry type cryo‐holder. The present low‐temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo‐holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo‐holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3‐D configuration of lipid droplets which had been difficult to preserve by chemical fixation.

A candidate of organum vasculosum of the lamina terminalis with neuronal connections to neurosecretory preoptic nucleus in eels

Systemic angiotensin II (Ang II) is a dipsogen in terrestrial vertebrates and seawater teleosts. In eels, Ang II acts on the area postrema, a sensory circumventricular organ (CVO) and elicits water intake but other sensory CVOs have not yet been found in the eel forebrain. To identify sensory CVOs in the forebrain, eels were peripherally injected with Evans blue, which immediately binds to albumin, or a rabbit IgG protein. Extravasation of these proteins, which cannot cross the blood–brain barrier (BBB), was observed in the brain parenchyma of the anteroventral preoptic recess (PR) walls. Fenestrated capillaries were observed in the parenchymal margin of the ventral wall of the PR, confirming a deficit of the BBB in the eel forebrain. Immunostaining for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) detected neurons in the lateral region of the anterior parvocellular preoptic nucleus (PPa), which were strongly stained by BBB-impermeable N-hydroxysulfosuccinimide. In the periventricular region of the PPa, many neurons incorporated biotinylated dextran amine conjugated to fluorescein, a retrograde axonal tracer, injected into the magnocellular preoptic nucleus (PM), indicating neuronal connections from the PPa to the PM. The mammalian paraventricular and supraoptic nuclei, homologous to the teleost PM, receive principal neuronal projections from the organum vasculosum of the lamina terminalis (OVLT). These results strongly suggest that the periventricular subpopulation of the PPa, which is most likely to be a component of the OVLT, serves as a functional window of access for systemic signal molecules such as Ang II.

A stone-like ovarian dermoid cyst in the Douglas' Pouch of an elderly woman

The dermoid cyst or mature cystic teratoma is the most common ovarian tumor type, accounting for approximately 20% of all adult ovarian tumors. Usually, an ovarian dermoid cyst is detected as a soft mass with an adhesive pedicle in a female of reproductive age, and it is characterized by the presence of well-differentiated derivatives of the three germ layers, including fat and hair. We report on a rare case of autoamputated ovarian dermoid cyst with a thick calcified shell, which was discovered post-mortem in the Douglas’ Pouch of an 83-year-old woman. During a demonstration of an anatomical dissection for medical students at Tottori University, a large stone-like structure was discovered in the Douglas’ Pouch of an 83-year-old female who had died of pneumonia (Fig. 1a). Her medical history, including any pregnancies, was unavailable. The stone-like structure was easily removed from the Douglas’ Pouch, since it lacked pedicles or adhesions (Fig. 1b). The removed tissue was a fist-sized, yellowish-brown, oval mass (81 ¥ 74 ¥ 61 mm) with a volume of 197.4 cm and mass of 205.5 g. The rectum was compressed leftward and narrowed by the structure, whereas the sigmoid colon was dilated. The uterus, right oviduct, and right ovary were intact. However, the left ovary was absent and the distal part of the left oviduct was tapered without fimbriae. Sectioning the stone-like structure, it consisted of a hard shell of approximately 5 mm in thickness and a core containing a sebaceous muddy substance, several small dark-brown structures (2 to 5 mm in diameter), and a large amount of hair (Fig. 1c). The small dark-brown structures were confirmed by histology to be clusters of degenerated red blood cells, erythrocyte ghosts. In addition, scanning electron microscopy revealed that the hairs had cuticles (Fig. 2a), similar to human scalp hair. The sebaceous muddy substance and large amount of hair detected in the core were considered to correspond to dermoid cyst derivatives. However, X-ray microanalysis showed that calcium and phosphorus were present in the shell, suggesting that the shell contained calcium phosphate. Histological analysis showed that the shell consisted of layers of calcified and organized fibrous tissue (Fig. 2b), although it contained neither ovarian nor epithelial tissue. No malignant formation was observed in either the core or shell. Based on these results, we identified the stone-like structure as an ovarian dermoid cyst with a thick calcified shell, derived from the left ovary. The ovarian dermoid cyst reported here is extremely unusual in three respects. First, it was found in an 83-yearold woman. Second, it was covered with a calcified shell and had a stone-like structure. Third, it was found in the Douglas’ Pouch without a pedicle or adhesion to the surrounding structures. Ovarian dermoid cysts are usually found in females of reproductive age but rarely found in persons of >80 years of age. Actually, only 12 cases (0.01% of females >80 years of age) were reported in the Annual of Pathological Autopsy Cases in Japan during 2003 and 2009. It probably took a long time for this stone-like dermoid cyst to develop in the abdominal cavity of the subject. This dermoid cyst originated from the left ovary and grew to the size of a fist. Eventually, torsion of the large cyst would occur and this led to blood congestion, ischemic necrosis, wall calcification, organization, and autoamputation. The clusters of degenerated red blood cells in the core probably reflect the induction of congestion and clot formation by the torsion. Torsion and autoamputation of ovarian dermoid cysts may cause severe abdominal pain that requires surgical treatment. However if the pain is endurable, it may self-resolve. In the present case, it appears that the process of autoamputation progressed without severe symptoms. After the autoamputation event, it is likely that the ovarian dermoid cyst was isolated and incubated for a long time in the Douglas’ Pouch, until its wall was calcified to a thickness of about 5 mm. If the ovarian dermoid cyst had developed and autoamputated sometime during the reproductive years of the subject, it would have incubated for decades in the Douglas’ Pouch. The wall of an ovarian dermoid cyst is usually composed of two layers: ovarian stroma (outer layer) and epithelium (inner layer), which is typically keratinized squamous epithelium. Although partial calcification of the cyst wall has been reported frequently, total calcification of the wall is rare. Although the frequency of calcified dermoid cysts remains uncertain, it is likely to be low, given that a clinico-statistical study conducted in 1955 found only one case of completely calcified benign cystic teratoma of the ovary among 1007 subjects. The rate at which ovarian dermoid cysts are discovered after total calcification of the wall has been much lower in recent years than previously because medical imaging techniques and/or laparoscopic surgery are used to identify and remove the ovarian dermoid cyst at an earlier stage. Dermoid cysts of the Douglas’ Pouch are rare; only nine cases have been reported to date (e.g.). Of these, four cases were ovarian dermoid cysts and three cases were autoamputated. When an autoamputated dermoid cyst Pathology International 2012; 62: 771–773 doi:10.1111/pin.12002 bs_bs_banner

Simultaneous occurrence of an aberrant right subclavian artery and accessory lobe of the liver

We herein report a case showing the simultaneous occurrence of an aberrant right subclavian artery (ARSA) and accessory lobe of the liver in a 75-year-old female cadaver. In the thorax, the left aortic arch branched into the right common carotid artery, left common carotid artery, left subclavian artery, and ARSA, in that order. The ARSA was dilated at its origin to form Kommerell’s diverticulum and coursed behind the esophagus. This diverticulum seemed to press the esophagus. A right-sided thoracic duct was identified that emptied into the angulus venosus. In the right-sided neck, a nonrecurrent laryngeal nerve was found. In the abdominal cavity, an accessory lobe protruded from the anterior margin of the left liver lobe. The accessory lobe was separated from the left lobe by a transverse furrow on the anterior side. We discuss possible common causes of these anomalies during development.

Mitochondrial changes in motor neurons of homozygotes of leucine 126 TT deletion SOD1 transgenic mice

We investigated the time course of ultrastructural changes of mitochondria in the spinal cord of homozygotes of Leu126TTdel SOD1 (superoxide dismutase 1) with FLAG (signal sequence at the C‐terminal protein) transgenic mice (DF‐homo). Non‐Tg mice and wild‐type human SOD1 with FLAG epitope transgenic mice (WF) were investigated as controls for non‐onset Tg mice. Expansion and vacuolation of the mitochondrial matrix was exhibited in motor neurons in the anterior horns of DF‐homo Tg mice at the presymptomatic stage. Such mitochondrial degeneration became severe at the postsymptomatic stage. In contrast, expansion of the mitochondrial inner‐membrane space was not evident even at the terminal stage. Microvacuoles of cytoplasm and fibrillar inclusions were rarely shown from the early symptomatic stage. WF mice showed expansion and vacuolation of the mitochondrial inner membrane space at old age. Non‐Tgs showed no obvious change in mitochondria. Gold‐labeled human SOD1 immunoreactivity showed small amount of gold deposits in the vacuolated mitochondria. These results suggest that the expansion and vacuolation of mitochondrial matrix in the spinal cord of DF‐homo transgenic mice is the first pathological change, but that it is not directly caused by the aggregation of an abnormal human SOD1 protein in intermembrane space of mitochondria.