Microvasculature of the California sea lion (Zalophus californianus) eye and its functional significance.

OBJECTIVE: To examine the ocular circulation in California sea lions (Zalophus californianus). ANIMALS STUDIED: Eyes were obtained postmortem from three sea lions that died while in captivity. PROCEDURES: Specimens from sea lions were investigated using scanning electron microscopy (SEM) of vascular corrosion casts. The thermal characteristics of live animal eyes were measured using an infrared imaging system. RESULTS: The major orbital artery of the sea lion was the ophthalmic artery. The artery was remarkably thick in diameter, showed a marked convolution and formed an ophthalmic rete around the optic nerve at the posterior pole of the eyeball. The long posterior ciliary artery terminates to form a prominent inner arterial circle at the pupillary margin. The iridial arteries originated from the arterial circle showing either a crimped or somewhat coiled course, extending toward the root of the iris and formed a root supplying a large amount of blood to the iris and ciliary bodies. The venules in the conjunctiva formed a well-developed venous plexus. The vortex veins showed a dilation and constriction at the site passing through the sclera. Thermographic examination revealed that the eye showed a higher degree of thermal emission than adjacent skin areas. CONCLUSIONS: These characteristics suggest that the ocular vasculature might play roles in thermoregulation as well as in hemodynamics by draining a large amount of blood so that the appropriate operating temperature for the eye can be maintained in a deep and cold aquatic environment.

Comparative anatomy of the ophthalmic rete and its relationship to ocular blood flow in three species of marine mammal.

OBJECTIVE: To examine the blood supply to the eyes of bottlenose dolphin (Tursiops truncatus), spotted seal (Phoca largha), and California sea lion (Zalophus californianus). Emphasis is placed on exploring the anatomic function in the context of aquatic life. PROCEDURE: Methyl methacrylate casts were prepared and studied using a scanning electron microscope. Infrared images of the eye were recorded using a thermocamera. RESULTS: In all three marine species, blood is supplied to the ophthalmic rete. The main source of blood supply to the rete is the basilar rete via the spinal rete in the dolphin and via the ophthalmic artery in the seal and sea lion. The retinal and choroidal arteries are derived from the rete. The dolphin rete showed a very well-developed arterial network occupying most of the orbit. The rete in pinnipeds was less developed with several entwining arteries, unlike that in cetaceans. Thermographic examination revealed that the eye shows a higher degree of thermal emission than adjacent areas of the skin in these 3 species. DISCUSSION: The role of the rete in aquatic mammals appears to conserve ocular temperature so that the appropriate operating temperature for photoreceptors and ocular muscles can be maintained in a cold ambient temperature. Additionally, the rete might have a flow-damping effect by maintaining resistance to blood flow in the orbit. This study highlights the special nature of ocular vascular anatomy and function that enabled the unique adaptation of aquatic mammals to life in aquatic habitats.

Functional anatomy of the footpad vasculature of dogs: scanning electron microscopy of vascular corrosion casts.

Dogs are well adapted to cold climates and they can stand, walk and run on snow and ice for long periods of time. In contrast to the body trunk, which has, dense fur, the paws are more exposed to the cold due to the lack of fur insulation. The extremities have a high surface area-to-volume ratio, so they lose heat very easily. We offer anatomical evidence for a heat-conserving structure associated with dog footpad vasculature. Methylmethacrylate vascular corrosion casts for scanning electron microscopy, Indian ink-injected whole-mount and histological specimens were each prepared, in a series of 16 limbs from four adult dogs. Vascular casts and Indian ink studies showed that abundant venules were arranged around the arteries supplying the pad surface and formed a vein-artery-vein triad, with the peri-arterial venous network intimately related to the arteries. In addition, numerous arteriovenous anastomoses and well-developed venous plexuses were found throughout the dermal vasculature. The triad forms a counter-current heat exchanger. When the footpad is exposed to a cold environment, the counter-current heat exchanger serves to prevent heat loss by recirculating heat back to the body core. Furthermore, the arteriovenous anastomoses shift blood flow, draining blood to the skin surface, and the venous plexuses retain warm blood in the pad surface. Hence, the appropriate temperature for the footpad can be maintained in cold environments.

An Assessment of Mutagenic Effect of 3, 3', 4, 4', 5 Pentachlorobiphenyl (PCB126) in Muta Mouse Fetuses.

PCBs are persistent environmental agents that induce multiple impairments in living beings. In this study we used a transgenic mouse model (Muta(TM) Mouse), carrying bacterial lacZ genes for mutation assays and for assessment of the genotoxic effect of PCB126 on fetal mice. Mothers of experimental groups were subjected to a single oral dose of PCB126 (125, 250 and 500 microg/kg) on the 10th day of pregnancy, respectively. Fetuses were autopsied on the 18th day of gestation. Cleft palate was observed in 2 out of 11 fetuses from 3 litters in 500 microg/kg treated group. Other external malformations were not observed. The DNA mutation frequencies (MF) of fetuses in each group were 1.15 +/- 0.24 x 10(-5), 0.90 +/- 0.20 x 10(-5) and 1.08 +/- 0.24 x 10(-5) in fetuses of 125, 250 and 500 microg/kg treated groups, respectively. The MF of controls was 0.81 +/- 0.22 x 10(-5). There were no significant differences among the groups. However, the MF of each treated group was a little highter than that of control group. Possible relationships between PCB and its mutagenic effects in the offspring of mice are discussed.

Development of external genitalia in fetal and neonatal domestic cats.

Development of the external genitalia of fetal and neonatal cat were studied macroscopically, paying attention to the formation of the labia and the sexual differentiation. The female urogenital folds budded from each side of the genital tubercle and, gradually extended to the tip of the genital tubercle by the 6.8 cm stage in crown-rump length. Then, the well-developed urogenital folds ensheathed completely the genital tubercle to form the prepuce of clitoris and the labia, flanking the external opening of vagina as the folds of skin which were equivalent to the labia minora in humans. The genital swellings known to become the labia majora in humans were clearly recognized in the caudolateral region of the genital tubercle during the fetal stage. These swellings became flat and obscure after birth. Thus, in cats the genital swellings did not join to the formation of the labia in the same way as in humans. The sex difference in the external genitalia was first observed at the 3.2-3.3 cm stages. In the male, the anogenital raphe appeared and the caudal portion of the genital swellings moved and fused each other at the caudal region of the genital tubercle. In the female, both features were not easy to observe.

Developmental changes of Müllerian and Wolffian ducts in domestic cat fetuses.

At 0.8 cm of crown-rump length (CRL) in sexually undifferentiated cat fetuses, the Wolffian duct is already observable on the ventro-lateral side of the mesonephros. At 1.2 cm CRL, the anlage of the Müllerian duct growing caudally in close parallel with the Wolffian duct was first observed. At 2.8 cm CRL in sexually differentiated fetuses, the Müllerian duct reached the urogenital sinus but remained indiscernible. Subsequently, at 3.2 cm CRL, regression of the upper part of the Müllerian duct was visible in males, while both ducts continued to grow in females. This suggests that the Müllerian inhibiting substance is produced before 3.2 cm CRL. At 7.0 cm CRL, male and female Wolffian ducts were reduced in diameter by about 50%, accompanied by involution of the mesonephros. Thereafter, the Wolffian duct was retained in the male; however, at 8.5 cm CRL in the female, the Wolffian duct was greatly reduced, by about 80% in diameter, then disappeared completely at 9.0 cm CRL.

Microvascular architecture of the rabbit eye: a scanning electron microscopic study of vascular corrosion casts.

The microvasculature of the eyes of 5 rabbits was investigated using scanning electron microscopy on corrosion casts. The study revealed that the pars plana vessels draining blood from the iris and ciliary body coursed directly into the anterior vortex venous system constituting the scleral venous plexus (the venous circle of Hovius). The episcleral vasculature was found to possess a specialized morphology, with channels draining the aqueous humor. The capillaries of the third palpebral, bulbar and palpebral conjunctiva formed a single-layered capillary network approximately parallel to the epithelium and formed a well-developed venous plexus in the stroma. The retina was found to be merangiotic, meaning that vessels were present only in a small part of the retina, extending in a horizontal direction to form bands on either side of the optic disc. Channels representing the aqueous veins that drained blood mixed with aqueous humor were found to derive directly from the suprachoroidal space and communicate with the scleral venous plexus via the anterior vortex veins. The functional significance of the microvasculature of the iris, cilia, retina and choroid is discussed in this report as well. The elaborate microvasculature of the conjunctiva may be a prerequisite for the exchange of nutrients and gasses between the cornea and the vessels across the conjunctival epithelium when the eyelids are shut during sleep, and possibly for the dynamics of eye drop delivery. The scleral venous plexus in rabbits may be analogous to the scleral venous sinus (Schlemm's canal) in rats, primates and humans.

Functional anatomy of the ocular circulatory system: vascular corrosion casts of the cetacean eye.

OBJECTIVE: To examine the functional anatomy of the ocular circulation in four bottlenose dolphins (Tursiops truncatus) and five melon-headed whales (Peponocephala electra). PROCEDURE: Eyes were obtained postmortem from whales that died while in captivity and/or were found beached. Specimens from whales were investigated using scanning electron microscopy (SEM) of vascular corrosion casts and histology. The thermal characteristics of live dolphin eyes were measured using an infrared imaging system. RESULTS: The whale eye receives its primary blood supply from the ophthalmic rete, which extends just behind the eyeball. The ophthalmic rete diverges from the basilar rete and the cervical rete via the posterior thoracic artery. The iris and ciliary processes are supplied by iridic arteries via the major arterial circle that is located around the iris edge. The retinal vessels show the holangiotic type. Choroidal arteries run in parallel arrays so as to interdigitate the densely packed choroidal veins. The venules in the conjunctival fold and palpebral conjunctiva form a well-developed venous plexus. Thermographic examination revealed that the eye shows a substantially higher degree of thermal emission than adjacent skin areas. CONCLUSIONS: The cetacean eye is characterized by a unique vascular pattern and multivessel plexuses, which are quite different from those of terrestrial mammals. This suggests that the ocular vasculature might function as a thermoregulatory system so that the appropriate operating temperature for the photoreceptors can be maintained in a deep and cold aquatic environment. The distinctive plexuses in the orbit might also be for pooling blood in the eye to conserve oxygen during dives. The ophthalmic rete might play a role in a pressure-damping effect on cetacean ocular circulation as well.

Microvascular anatomy of the pig eye: scanning electron microscopy of vascular corrosion casts.

The microvasculature of the eye of 10 pigs was investigated using scanning electron micrographs of corrosion casts. The ciliary body, iris and bulbar conjunctiva were supplied by the iridociliary ring artery via the long posterior ciliary artery. Capillaries of the ciliary process were of large diameter (23.2-27.5 microm) with an irregular bore, forming a thoroughfare channel draining blood in the ciliary arterioles into the pars plana venous vessels. Arterioles and venules in the iris exhibited a zigzag or spiral features. The third palpebra was supplied by the anterior ciliary artery. The capillary bed of the third palpebra was dense and was formed by many rows of fine hair-pin loops. Capillaries in the bulbar conjunctiva formed a sparse network disposing approximately parallel to the epithelium and formed a well-developed venous plexus, draining into the vortex veins. Retinal arterioles formed a slender and long course to capillaries. Retinal capillaries were extremely thin (3.0-4.0 microm in diameter). The choroid was supplied by the short posterior ciliary arteries. Choroidal arterioles exhibited a thick and short course to the choriocapillaris. The choriocapillaris was flat and sinusoid-like (8.9-13.9 microm in diameter), forming a dense sheet-like network. Blood from the choroid emptied into the episcleral vein via the vortex vein. Blood from the retina was drained by the posterior ciliary veins. The functional significance of this vascular architecture was discussed.

Vascular changes associated with chorioretinal and optic nerve colobomas in rats (Crj: CD(SD), IGS).

OBJECTIVE: Three female adult rats (Crj: CD(SD) IGS) with colobomatous anomalies were investigated. MATERIALS AND METHODS: The microvascular changes of the coloboma were studied using the techniques of fluorescein angiography, histology and scanning electron microscopy (SEM) of vascular corrosion casts. RESULTS: Fluorescein angiography revealed the pits of the optic disk as a dark hole with some abnormalities in vessel arrangement. Light microscopy confirmed the presence of attenuated lamina cribrosa, retinal dysplasia and marked dilation of the retinal veins. SEM revealed that the optic disk coloboma formed a crater-like pit and that central retinal vessels ran a tortuous course along the bottom and side of the crater. Capillaries in the optic nerve head were missing in the affected area. The central retinal veins were thick and had various changes such as strangulation, rough surface structures, mural voids and evaginations, which represent loss of integrity of the vascular wall. CONCLUSIONS: These vascular changes that are associated with colobomatous anomalies may impede the retinal circulation and be responsible for the fluctuating fluorescein pattern during fluorangiogram of affected animals. The lesions of the vascular wall may increase the subretinal fluid due to the leakage of fluid, thus causing the maculopathy or serous retinopathy, which is frequently associated with posterior pole coloboma.

Hypervitaminosis A resulting in DNA aberration in fetal transgenic mice (Muta Mouse).

Treatment with excessive amounts of Vitamin A during maternity induces fetal malformations. However, it is unclear whether these malformations are due to gene mutations or not. Using transgenic mice (containing lacZ gene showing beta-galactosidase enzymatic activity), we planned to observe whether gene mutations occur in the fetal tissues after treatment during maternity with Vitamin A (retinol palmitate). On the 11th day of pregnancy, mothers were given 30 mg (group 2), 150 mg (group 3) and 300 mg (group 4) of Vitamin A/kg body weight orally. Fetuses obtained on the 18th day of gestation showed malformations, such as cleft palate, origodactyly, brachydactyly and ectromeria. Most notably, cleft palate occurred dose dependently. The incidental rates were 100% in group 4, 58% in group 3 and 6% in group 2. The number of dead and absorbed fetuses also increased dose dependently with the treatments. DNA (integrated vectors containing lacZ genes) extracted from each fetus showed Vitamin A-induced lacZ mutations, especially in the malformed fetuses. The mutation frequencies were 4.99x10(-5) in group 4, 5.28x10(-5) in group 3 and 4.26x10(-5) in group 2. The frequencies of group 3 were significantly higher (p<0.05) than that of the controls (group 1), 2.79x10(-5). Maternal treatment with Vitamin A (150 mg/kg of body weight) was carried out on the 11th day of pregnancy. Fetuses obtained on the 14th day of gestation showed a much higher incidence of mutation, approximately 8.91x10(-5) (group 6) that was significantly higher (p<0.0001) than those from the controls (group 5), 2.94x10(-5). The present study indicates a possibility that hypervitaminosis A-induced fetal malformation and death might be caused by gene mutations.

Microvasculature of the retina, ciliary processes and choroid in the North American raccoon (Procyon lotor) eye: a scanning electron microscopic study of corrosion casts.

We have studied the vasculature of the retina, ciliary processes and choroid in the North American raccoon (Procyon lotor), a nocturnal mammal, using light and scanning electron microscopic examination of corrosion casts. We carried out an identical study in the crab-eating macaque (Macaca fascicularis), which forages only during the daytime, in order to compare the ocular vasculature with that of nocturnal mammals. Our observations in raccoons demonstrated a photoreceptor layer associated with rich lymph and a poorly vascularized retina. The meridian region of the eye, which lies in the horizontal plane and pass around the optic disc, had a markedly sparse capillary network. This horizontal sparse vascular band may correspond to a visual streak. Ciliary process capillaries were delicate, and formed a well-developed and compact network. Choriocapillaries were quite thin and formed a coarse capillary network. This contrasted with the dense retinal and well-extended choroidal capillary networks noted in the macaques. Our findings suggest that the sparse retinal capillary network in raccoons is extremely beneficial for photon capture, thereby allowing the raccoon to see well at night, as the retinal vessels restrict the inflow of photons toward the photoreceptors. The well-developed lymph probably compensates for the sparse retinal capillaries and choriocapillaries and nourishes the retina in the nocturnal raccoon.

Sarcoptic mange in free-ranging raccoon dogs (Nyctereutes procyonoides) in Japan.

Sarcoptes scabiei infestation was diagnosed in three freshly dead free-ranging raccoon dogs (Nyctereutes procyonoides) in Kanagawa Prefecture, Japan. The dogs presented with an alopecic pruritic skin disease, with signs of alopecia on the ears, muzzle, around the eyes, elbow, thigh and the neck, and hyperpigmented and crusted skin lesions, which had a severe malodour. Skin scrapings revealed the presence of the mite Sarcoptes scabiei. Histopathology of lesions demonstrated marked acanthosis, hyperkeratosis, parakeratosis and fungal elements, which were subsequently identified as Acremonium sp., Alternaria sp. and an unknown fungus. Mite segments were located mainly in the stratum corneum and also in the stratum granulosum. Tunnels could be observed in the hyperkeratotic stratum corneum. Scanning electron microscopy (SEM) revealed the tortoise-like Sarcoptes scabiei with four long bristles, suckers and blade-like claws on legs 1 and 2, cuticular spines, prominent body striations and a terminal anus. SEM also revealed an adult female mite digging a tunnel with the head wedged into the very end of the closed burrow. Tunnels filled with eggshells, corneocyte debris and faecal pellets were also observed.

Microanatomy of the terminal air spaces of Baird's beaked whale (Berardius bairdii) lungs.

The terminal airways and microvasculature of five adult Baird's beaked whales (Berardius bairdii) lungs have been examined by means of light and scanning electron microscopy of corrosion casts. The respiratory system of the Baird's beaked whale has various anatomical features which allow them to attain great depths and remain submerged for long periods. The whale lung has components including hyaline cartilage and smooth muscle throughout, reaching as far as the peripheral bronchi, sphincters surrounding the terminal bronchioles, the thick alveolar septa with a connective tissue core and a bi-layer capillary bed, and a distinctive venous plexus of the pulmonary veins. The well-developed venous plexuses of the pulmonary vein are found in the interlobular connective tissue, and around the airways and pulmonary arteries with close apposition. The hyaline cartilage throughout the airways may increase the effective dead air space that accommodates most of the air forced from the collapsed alveoli during a dive. The sphincter might serve as a cock for regulating buoyancy and for trapping air in the alveoli to prevent their complete collapse and a sucking in of alveolar tissue as the dive becomes deeper. The venous plexuses might be for pooling the large volume of blood in the lung to conserve oxygen for deep and prolonged diving.

A simple method of intestinal anastomosis (ileocolostomy) in rats.

A simple method of ileocolostomy was performed in rats. The colon near the cecum was ligated, including its arteries and veins. Main artery and vein of the cecum were ligated. Then, the cecum was cut out. A longitudinal incision was made on the anti-mesenteric side of the proximal end of the colon, approximately 7-8 mm long. A 21-G needle was inserted toward the incision 2 cm away from the proximal end of the anti-mesenteric side of the colon. A nylon suture was knotted once to the distal end of the ileum and was introduced into the tip of the needle which had previously been passed through the colon. Then, the needle was removed. The suture was pulled to introduce the distal end of the ileum into the colonic lumen. Then, the suture was knotted once on the colon again to fix the ileum to the colon. The incision in the proximal end of the colon was not closed. At the 2nd week after the operation, X-ray examinations demonstrated that the ileocolonic passages with no leakage at the anastomotic site were quite satisfactory. At the 4th week after the operation, there were no macroscopic or microscopic complications at the anastomotic site. The mucosal and serosal epithelia of the ileum and colon continued smoothly. This simple method may be very effective in preparing anastomosis in the gastrointestinal tract, especially in small laboratory animals for nutritional and surgical experiments.

Microvasculature of the hamster eye: scanning electron microscopy of vascular corrosion casts.

The architecture of the retina, choroid and anterior eye segment was investigated in 12 Syrian hamsters using scanning electron micrographs of methylmethacrylate corrosion casts. The hamster eye receives its primary blood supply from the long posterior ciliary artery, which directly enters the optic nerve head, and divides into three branches: the central retinal artery and medial and lateral long posterior ciliary arteries. In the retina the central retinal artery divides into six radiating branches. Retinal arterioles form a slender and long course to capillaries. Retinal capillaries are extremely thin in diameter and form a sparse capillary network. The choroid is supplied by the long posterior ciliary arteries. Choroidal arterioles exhibit a thick and short course to the choriocapillaris. The choriocapillaris is thick and sinusoid-like, forming a dense network in the choroid. The ciliary body, iris and bulbar conjunctiva are supplied by the anterior ciliary arteries, which are branches of the long posterior ciliary arteries. Capillaries supplying the anterior margin of the ciliary process are large in diameter with an irregular bore, forming a thoroughfare channel draining blood in the ciliary arterioles into the pars plana vessels. Blood from the retina is drained by the central retinal veins. Venules from the anterior eye segment empty into the vortex veins via the pars plana vessels. Venous blood from the choroid is drained only by vortex veins via the choroidal veins. The functional significance of the vascular architecture and species differences are discussed.

JSAAE activities in alternatives to animal experimentation for undergraduate schools: forum for citizens in primary and secondary schools.

The aim of this paper is to introduce some educational activities by the Japanese Society of Alternatives to Animal Experiments (JSAAE). The JSAAE is an academic society to promote the Three Rs in Japan and also the international biomedical community. Our activities include the education of not only scientists but also the public. In particular, activities should be focused on the education in schools regarding alternatives. The JSAAE organised a forum for citizens in alternatives education in primary and secondary schools during their 14th annual meeting.

Microvasculature in the terminal air spaces of the lungs of the Baird's beaked whale (Berardius bairdii).

Lungs were obtained from five adult Baird's beaked whales (Berardius bairdii) and examined by means of light microscopy and scanning electron microscopy of corrosion casts. The alveolar septa of these whales are thick with a connective tissue core and a bi-layer capillary bed. A double capillary network is regularly found in the alveolar duct and alveolar septa. Occasionally, septa adjacent to alveoli and alveoli themselves show only a single capillary layer. The distance between the two capillary layers has a tendency to decrease toward the end of airspaces, suggesting an end result of capillary fusion. Vascular replicas of venous vessels have annular furrows at regular intervals of 50 to 100 microm, which are caused by focal aggregations of collagen fibers circularly oriented and located immediately underneath the endothelium. The first valves appear in the collecting venules gathering alveolar capillaries. These valves are quite characteristic of flap-, funnel-and/or chimney like structures.

Notoedric mange in free-ranging masked palm civets (Paguma larvata) in Japan.

Fifty-one masked palm civets (Paguma larvata) were trapped as part of a nuisance wildlife control programme between July 2001 and August 2002 in Fujisawa City, Kanagawa Prefecture, Japan. Eleven civets had characteristic mange lesions with marked alopecia and crusts, caused by the burrowing epidermal mite Notoedres cati. The diagnosis was confirmed by histology and examination of mites obtained from skin scrapings of affected civets. Histopathology of lesions demonstrated moderate hyperplastic epidermis, parakeratosis and acanthosis. Occasionally microvascular angiogenesis was observed in the epidermis. Tunnels were excavated in the hair follicles, reaching the hair roots. The tunnels were located between the hair shaft and the inner root sheath. Female mites, eggs and nymphs were demonstrated in their tunnels using scanning electron microscopy.

A simple method for sutureless gastrointestinal anastomosis in rat.

A simple tubing stent was attempted to test a model for sutureless gastrointestinal anastomosis in 6 male rats at the age of 15 weeks. In the 3rd and 4th weeks after the operation, X-ray examination demonstrated that the gastrointestinal passage in the anastomotic site was quite satisfactory. There was no incidence of anastomotic leakage. In the 6th week after the operation, there were no macroscopic or microscopic ruptures, nor were there any obstructions at the anastomotic site. This simple sutureless method was effective at preparing anastomosis in the gastrointestinal tract in the rat and could be applied to other small experimental animals.