Hyaluronan dynamics during retinal development.

We have already shown the regulated expressions of hyaluronan binding molecules including versican, SPACR, and SPACRCAN, during retinal development. Here we analyzed the profiles of hyaluronan during these periods. Hyaluronan and hyaluronan synthase (HAS) expressions in chicken retinas at different ages were assessed by slot blot and real-time PCR, respectively. The molecular size of hyaluronan was determined using a Superose-6 column, and histochemistry was used to localize hyaluronan expression. Hyaluronan degradation activity was measured after adding hyaluronan to retinal extracts. In the embryonic retina, hyaluronan was mainly present in the inner plexiform and photoreceptor layers. Expression in the inner plexiform layer decreased with development and aging while expression in the photoreceptor layer remained constant. The molecular size of hyaluronan decreased from embryonic day 12 (E12) to postnatal day 1 (P1) with increasing retinal hyaluronan degradation activity. In adult retinas, hyaluronan was only present in the photoreceptor layer, and its molecular size was the highest. HAS-2 and HAS-3 expressions increased from E15 to P1, but both were reduced in adulthood. Thus hyaluronan was specifically detected where versican, SPACR, and SPACRCAN were previously demonstrated, and the synthesis and degradation of hyaluronan are regulated. Hyaluronan together with versican, SPACR, and SPACRCAN are closely related during retinal development, and may be important for retinal physiology.

Occlusal tooth wear in male F344/N rats with aging.

With the aim of clarifying the aging properties of an animal model, the progress of occlusal tooth wear (OTW) of molars in male F344/N rats was monitored. Dried maxilla and mandible specimens from 61 male F344/N rats, aged 7 to >30 months, were used. The levels of OTW of all molars were monitored with aging. The cuspis dentis of molar teeth were worn out by 7 months (M) of age, and the occlusal surface became flat. As for each molar tooth (M(1-3), numbered in accordance with its position), OTW of M(1) was more severe in the lower than in the upper jaw, whereas M(3) was more severe in the upper than the lower jaw. OTW of M(2) in both the upper and the lower jaws progressed rapidly after 27M. OTW in male F344/N rats progressed faster than in females. However, when compensated for life span, both genders had similar profiles in OTW progress with aging. This study suggested that male rats were more convenient than females as a model for gerodontological research because of the earlier course of OTW progress.

Characterization of a motif for specific binding to hyaluronan in chicken SPACR.

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body.

PURPOSE: In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan. METHODS: The new polyclonal antibodies against the amino and carboxyl termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, and their protein levels were evaluated by Western blot analysis and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot-blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation. RESULTS: The newly prepared polyclonal antibodies recognized amino and carboxyl termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-terminal end of versican bound to fibrillin microfibrils and the amino terminal end of versican bound to hyaluronan in the vitreous body. CONCLUSIONS: Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary nonpigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous.

Medial horn supporting ligament in Asian upper eyelids.

The purpose of this study was to describe the medial horn supporting ligament (MHSL) attached to the medial horn of the levator aponeurosis. The authors examined 3 different groups of specimens: gross cadaveric samples, microscopic cadaveric samples and intraoperative samples from levator resections. In all eyelids in the gross cadaveric samples, the MHSL was attached from the superior to the medial surfaces of the medial horn, and ran deeper in the direction of the trochlea in the superior region. Furthermore, the MHSL continued to the Whitnall ligament and was partially attached to the trochlea. In the microscopic samples, the MHSL was located adjacent to the levator aponeurosis in all specimens. The MHSL was constituted by comparatively thick fibers with few smooth muscle fibers. Under the MHSL, another fibrous structure existed with minute fibers including much smooth muscle. In the intraoperative samples from levator resections, the MHSL was also observed in all specimens. The MHSL was released from the medial horn in the cases of lateral tarsal shifts, but not in cases without. The MHSL, an indicator of the medial margin of the levator aponeurosis, may provide support, tension and suspension to the medial margin of the medial horn, strengthening its fragility.

Direct insertion of the medial rectus capsulopalpebral fascia to the tarsus.

PURPOSE: To clarify the insertion of the medial rectus capsulopalpebral fascia to the tarsus in Asians. METHODS: Specimens from 19 (11 right, 8 left) postmortem medial eyelids and orbits of 11 Asians (aged 45-96 years at death) were used. Samples had been fixed in 10% buffered formalin before their removal and microscopic examination. The tarsi were incised at 2 different heights in the upper and lower eyelids, as it was not disclosed which parts had the insertion of the medial rectus capsulopalpebral fascia. The first and second sections, parallel to the eyelid margin, were obtained, respectively, at 1 mm and 5 mm from the upper eyelid margin, and at 1 mm and 3 mm from the lower eyelid margin. Sections were stained with Masson trichrome. RESULTS: Both upper and lower eyelids demonstrated similar findings. The first sections, which showed the medial rectus capsulopalpebral fascia and included many smooth muscle fibers, did not insert in the tarsi. However, the deep part of Horner muscle directly inserted, whereas the superficial part went in the dense fibrous tissue closely attaching on the tarsi. Then, some of the muscle branched out in the tarsi. The second sections showed that the medial rectus capsulopalpebral fascia had a direct insertion to the tarsi. CONCLUSIONS: The tarsi are supported medially by the medial rectus capsulopalpebral fascia and Horner muscle. The "medial eyelid retractors, " comprising the medial rectus capsulopalpebral fascia and smooth muscles, were clearly defined, highlighting the relationship of the eyelid to the medial rectus muscle and offering a new pathogenesis and treatment for lateral tarsal shifts and lower medial ectropion.

Microscopic findings of lateral tarsal fixation in Asians.

PURPOSE: To identify microscopically lateral tarsal fixation in Asians. METHODS: Specimens from 19 postmortem lateral eyelids and orbits of 11 Asians (11 right, 8 left; aged 45-96 years at death) were used. Samples damaged on sectioning and samples without tarsal plates were excluded. The samples were fixed in 10% buffered formalin and examined under a microscope. Two levels of tarsus were observed in the upper and lower eyelids, suggesting the possibility of different means of fixation. The first and second sections, which were incised parallel to the eyelid margin, were obtained at 1 mm and 5 mm from the upper eyelid margin, and at 1 mm and 3 mm from the lower eyelid margin. The sections were stained with Masson trichrome. RESULTS: The first sections of all upper eyelids and those of the lower eyelids except one showed tarsal fixation by both the lateral rectus capsulopalpebral fascia (lr-CPF) and the tendon-ligament complex of the lateral canthal tendon (LCT), which in several cases received the muscle of Riolan. The second sections of all upper eyelids showed fixation by the lr-CPF and the ligamentous part of the LCT. The second sections of the lower eyelids were mostly similar to the second sections of upper eyelids, though some showed only ligamentous fixation. The lr-CPF in all cases included a small amount of smooth muscle fibers. CONCLUSIONS: The lateral aspect of the tarsus is supported by the lr-CPF and the LCT, which in some cases includes the muscle of Riolan.

Overview of the lacrimal canaliculus in microscopic cross-section.

Although many illustrations have been published seeking to explain the anatomy of the lacrimal canaliculus and its surroundings, to our knowledge, no micrograph showing the total length of the lacrimal canaliculus has ever been displayed. Here, we present a clear microscopic cross-section of the total length of an upper lacrimal canaliculus, except for the vertical part. A winding path was found in part of the canaliculus. The common canaliculus opened into the lacrimal sac almost perpendicularly and was covered by solid fibrous tissue. The transition between squamous cell epithelium and columnar epithelium did not always take place at the opening. There were superficial goblet cells, mucous secretory glands and intraluminal debris in the distal part of the canaliculus, including the common canaliculus, where the lining consisted of columnar epithelium. The clear overview of the structures presented here should be helpful in the treatment of the lacrimal canaliculus.

Development of IgA nephropathy-like disease with high serum IgA levels and increased proportion of polymeric IgA in Beta-1,4-galactosyltransferase-deficient mice.

The glycosylation of glycoproteins is important for their biological activity, conformation and stability. Recent studies indicate that aberrant glycosylation causes various human disorders. Here we report that mice lacking beta-1,4-galactosyltransferase-I (beta4GalT-I), which transfers galactose from UDP-Gal to terminal GlcNAc of N- and O-glycans in a beta-1,4- linkage, developed IgA nephropathy (IgAN)-like disease. Urinary albumin levels were significantly increased in the beta4GalT-I-deficient mice. Hematuria was detected in some of the beta4GalT-I-deficient mice, suggesting impaired renal function. Furthermore, histological and immunohistochemical examination showed expanded mesangial matrix, IgA deposition with mesangial pattern and electron-dense deposits in the paramesangial regions in the beta4GalT-Ideficient mice. These results demonstrate that the beta4GalT-I-deficient mice developed IgANlike disease. Furthermore, high serum IgA levels with increased polymeric forms were detected. In humans, serum IgA derived from patients with IgAN has aberrant beta3-galactosylation and sialylation on its O-linked glycans of the hinge region. Mouse IgA does not have O-glycans of the hinge region and has several N-glycans. As expected, beta4-galactosylation on the N-glycans of the serum IgA of the beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We suggest that aberrant beta4-galactosylation of serum IgA participates in the Nishie/Miyaishi/Azuma/Kameyama/Naruse/Hashimoto/Yokoyama/Narimatsu/Wada/Asano 126 development of IgAN, including deposition of IgA, polymerization of IgA, and glomerular injury after IgA deposition.

Occlusal tooth wear in female F344/N rats with aging.

OBJECTIVES: This study was conducted to ascertain whether laboratory rats are an adequate animal model for aging oral cavity research, especially on occlusal tooth wear (OTW), which progresses with aging and causes abnormal occlusions. Mastication has been reported to relate to cognition in the elderly. Thus, it is important to care for the oral cavity, especially in the frail elderly, for the maintenance of all-round quality of life. Adequate and appropriate animal models are essential for basic and clinical research on the oral cavity. METHODS: Dried maxilla and mandible specimens from 98 young, aging or aged female F344/N rats were used. RESULTS: The levels of OTW of all molars were monitored with aging. The molar tooth began to wear at 1-month old (M) and progressed rapidly till 12M. Subsequently, OTW progressed slowly till 30M, and then rapidly again after 35M. CONCLUSIONS: This study showed that progress of OTW is well correlated with the entire life span of the rat, and suggested that the rat aged over 12M would be an adequate animal model for research on OTW in middle-aged and elderly people.

Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.

Versican interacts with hyaluronan (HA) at its N-terminus and with fibrillin-1 at its C terminus. As versican in the dermis connects microfibrils to the HA-rich matrix for viscoelasticity, dermal diseases may involve destruction of these complexes. A recombinant versican protein, rVN, covering the HA binding region (HABR) of human versican and a polyclonal antibody, 6084, against rVN were prepared and characterized. Blotting analyses of skin extracts with 6084 and biotin-conjugated HA revealed that versican was a major HA-binding component in the dermis. Matrix metalloprotease-12, which is expressed in areas of solar elastosis, degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084, but positive for 2B1, an antibody recognizing the C-terminus of versican, indicating loss of the HABR in the aggregated elastic fibers. This loss of the HA-binding ability of versican followed by HA exclusion may be responsible for the pathological and phenotypical changes observed in solar elastosis.

Development of immunoglobulin A nephropathy- like disease in beta-1,4-galactosyltransferase-I-deficient mice.

Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.

Natural dental caries in molars of osteogenic disorder Shionogi rats.

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.

Lateral canthal support system in Japanese.

The present study aimed to elucidate microscopically the precise structure of the generally termed 'lateral canthal tendon' (LCT). Specimens from 9 post-mortem lower eyelids of 6 Japanese aged from 72 to 91 years old at death were fixed in 10% buffered formalin, and microscopically examined. Specimens were excised as exenterated samples including an area 5 mm wider than the orbital aperture. The removed contents were further incised longitudinally on the central eyelid and also incised parallel to the upper eyelid margin on the site 3 mm from its margin. After the preparation of microscopical examination, sections of all 9 eyelids were stained with Hematoxylin and Eosin. We found that the structure generally termed LCT consisted of two definitive different layers microscopically. The superficial layer was only an orbital septum (septal band). It was mainly constituted of thick fibers between adipose-rich tissues. The deep layer continued from the tarsus and projected posteriorly; which was a ligament (tarsoligamentous band). This tissue was constituted by thin, minute fibers with little adipose tissues. The structure generally termed LCT is not a tendon but a complex constitution of an orbital septum and a ligament; which we named, in a mass, 'lateral canthal bands', cooperatively supporting the lateral canthus.

Microscopic anatomy of Asian lower eyelids.

PURPOSE: To elucidate the microscopic anatomy of the Asian lower eyelid. METHODS: Specimens (full-thickness sections of lower eyelids from 19 postmortem lower eyelids) from 11 Asians aged 73 to 96 years at death were fixed in 10% buffered formalin and microscopically examined. After pretreatment, sagittal sliced sections of the central part were stained with Masson trichrome. RESULTS: The distinct junction of the orbital septum to the capsulopalpebral fascia (CPF) was confirmed in 7 eyelids in which orbital septum was clearly stained, with an average distance from the tarsus to the junction of 2.38 mm. The other 12 eyelids did not show a distinct junction, and the orbital septum was poorly defined anteriorly and indistinct posteriorly. There was a distinct layer between the orbicularis oculi muscle and the orbital septum. The inferior and the posterior attachments of the CPF to the tarsus were seen in all eyelids. Seventeen of the 19 eyelids had attachment of the CPF on the anterior aspect of the tarsus, from which an extension of the CPF through the pretarsal orbicularis oculi muscle was observed. All eyelids had anterior extension of the CPF through the preseptal orbicularis oculi muscle, which was overridden on the pretarsal orbicularis oculi muscle. CONCLUSIONS: The microscopic findings of Asian lower eyelids, especially fascial components, were mostly similar to those of non-Asian eyelids, but differences existed in higher or indistinct septum fusion, anterior and superior orbital fat projection, and the overriding of the preseptal orbicularis oculi muscle.

Comparison of rat mandible bone characteristics in F344 substrains, F344/Du and F344/N.

The characteristics of the mandible bone were compared through DXA methods between two major substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc at around 60 days of age. Since these two substrains are clearly different in survival and mandible morphology, some genetic differences are supposed to exist. In contrast to a previous microsatellite analysis, clear and significant differences were detected in the body and mandible weights, the mandible bone mineral contents (BMC), bone area (AREA), bone mineral density (BMD) and bone mineral ratio (BMR), between F344/DuCrlCrlj and F344/NSlc, with the mandible molar teeth intact in the bone. Thus, care is needed in the experimental use of these substrains, as results may differ between them. The newly proposed parameter, BMR, may especially contribute to the comparison of bone characteristics among species.

The lower eyelid retractor consists of definite double layers.

PURPOSE: To examine whether the lower eyelid retractor consists of a single layer or multiple layers. DESIGN: Retrospective clinical case series and dissectional study. PARTICIPANTS: Fifty-one lower eyelid retractors (31 right, 20 left) of 44 patients (ages, 63-95 years) during entropion surgeries and 10 lower eyelids (5 right, 5 left) of 5 Oriental cadavers (73-91 years old at death) were observed macroscopically. Specimens from 20 postmortem lower eyelids of 12 Orientals (11 right, 9 left; 66-96 years old at death) were used for microscopic observations. METHODS: Macroscopically, we bluntly or, in parts, sharply dissected lower eyelid retractors into 2 layers during entropion surgeries. Cadaveric lower eyelids also were used to investigate relationships between the lower eyelid retractor and the Lockwood ligament. Cadaveric lower eyelids with sagittal full-thickness sections of the central part were examined microscopically using Masson trichrome staining. MAIN OUTCOME MEASURES: Anatomical findings in the lower eyelid retractor. RESULTS: Lower eyelid retractors easily were detached into 2 layers. Macroscopically, the anterior layer was defined as a coarse tissue continuing from the Lockwood ligament, which joined with the lower orbital septum, and constituted the lower conjoined fascia. The posterior layer appeared to be a dense fibrous sheet with a lustrous surface. Microscopically, the lower eyelid retractor consisted of a definite double layer. The anterior layer, a coarse fibrous tissue, consisted of the suborbicularis fascial layer, orbital septum, and superficial part of the capsulopalpebral fascia, which continued to the anterior lamellae of the lower eyelids. The posterior layer consisted of dense fibers of the capsulopalpebral fascia with smooth muscle continuing to the tarsus. CONCLUSIONS: The lower eyelid retractor consists of a definite double layer. The posterior dense layer containing smooth muscle is the main tractional component of the lower eyelid retractor.

An anomalous muscle linking superior and inferior rectus muscles in the orbit.

Dissections of the bilateral orbits in a 45-year-old female cadaver, who had no ocular movement disorders in her lifetime, revealed anomalous muscles linking the superior and inferior rectus muscles. The muscles, situated between the optic nerve and the lateral rectus muscle, originated from the annulus of Zinn and branched off two heads; one inserted into the medial inferior side of the superior rectus muscle and the other inserted into the central superior side of the inferior rectus muscle. Each insertion was located on a distal site of the myoneural junction of each rectus muscle. Histological investigations showed that the muscles had a striated muscle structure. No definite nerve insertion was observed in the muscles. Although this type of anomalous muscle has been reported in a few Caucasian cases, the present study is the first report in an Asian person. Anomalous orbital structures, which are a rare cause of strabismus, are important in the differential diagnosis of intra-orbital space-occupying lesions, rather than the differential diagnosis of strabismus.

Molecular cloning and characterization of chick SPACRCAN.

MY-174, a monoclonal antibody that reacts with specific sialylated O-linked glycoconjugates of chick SPACR (sialoprotein associated with cones and rods), also recognizes another molecule of 300 kDa. Here, we verified that this 300-kDa molecule is chick SPACRCAN (sialoproteoglycan associated with cones and rods), another member of a novel interphotoreceptor matrix molecule family. Screening for chick SPACRCAN was carried out by plaque hybridization using a probe for chick SPACR. Specific polyclonal antibodies raised against chick SPACRCAN were used for the following experiments. To determine whether the 300-kDa molecule detected by MY-174 was identical to 300-kDa chick SPACRCAN, the migrations of these bands were examined after various glycosidase digestions. Furthermore, the expression levels were measured during retinal development and compared with those of chick SPACR. The results demonstrated that the 300-kDa molecule recognized by MY-174 was chick SPACRCAN, and we further identified it as a proteoglycan with chondroitin sulfate chains. SPACRCAN had heavily sialylated N- and O-linked glycoconjugates, and its MY-174 antigenicity was abolished by O-glycanase treatment after neuraminidase treatment, as observed for chick SPACR. During retinal development, the mRNA and core protein expression levels, MY-174 antigenicity, and hyaluronan binding ability of SPACRCAN peaked around embryonic day 17 and then gradually decreased, whereas the corresponding expression levels of SPACR simply increased, but not its hyaluronan binding ability. The MY-174 reactivity of SPACRCAN in the adult retina was decreased compared with that in the newborn retina, whereas that of SPACR was increased. The decreased hyaluronan binding of SPACR was induced by an inhibitory effect of the excess of sialic acids in the adult stage. Thus, with similar core protein structures and specific sialylated glycoconjugates but distinct chondroitin sulfate chains, SPACRCAN and SPACR may have separate roles in the retina due to their differing expression profiles during development.