The anatomy of the parotoid gland in Bufonidae with some histochemical findings. II. Bufo alvarius.

The gross and microscopic anatomy of the venom producing parotoid glands of Bufo alvarius has been studied by light and electron microscopy. Histochemical reactions for the presence of venom constituents and of components in biochemical pathways in the synthesis and release of venom were performed. The gland is composed of numerous lobules. Each lobule is an individual unit with a lumen surrounded by a double cell layer. Microvilli of the outer layer interdigitate with microvilli of the inner layer. Cells of the outer layer resemble smooth muscle cells, are rich in adenosine triphosphatase and glucose-6-phosphatase, and contain numerous pinocytotic vesicles, glycogen granules and various organelles. These organelles include "crystalloids" of what seem to be highly organized agranular reticulum. These outer layer cells probably function in some aspects of venom synthesis, active cellular transport and contraction in the discharge of the secretory product. The inner cell layer demonstrates a positive chromaffin reaction, contains steroid material, various organelles, some pinocytotic vesicles and glycogen granules, and appears devoid of a plasmalemma on its inner surface. This layer is probably involved in venom formation and release via an apocrine type of secretion. Bufo alvarius parotid gland shows significant morphological and histochemical differences from that of B. marinus and more nearly resembles a typical steroid producing organ.     

Investigation of dorsal/ventral skin and the parotoid region of Lyciasalamandra billae and Lyciasalamandra luschani basoglui (Urodela: Salamandridae)

In the present study, the parotoid region, dorsal and ventral integuments of Lyciasalamandra billae and Lyciasalamandra luschani basoglui were investigated in terms of localization of hyaluronic acid (HA) and histochemical characteristics. HA immunoreactivity was carried out using biotinylated hyaluronic acid binding protein (B-HABP) labelled with streptavidin-fluorescein isothiocyanate (FITC). HA was mainly localized in the stratum spongiosum of L. billae and L. luschani basoglui for water homeostasis and skin functionality. Light microscopic observations revealed that the dorsal and ventral integuments of L. billae and L. luschani basoglui exhibited basic morphological characteristics of other amphibians: the epidermis was composed of a stratified squamous epithelium and the dermis subdivided into stratum spongiosum and stratum compactum. Two different types of dermal glands (mucous and granular glands) were identified in the spongious dermis of the dorsal and ventral integuments whereas in the parotoid region, three different types of glands (mucous, granular and parotoid glands) were examined.     

Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications

The composition of indolealkylamines of Bufo rubescens cutaneous secretions was compared to those from six other Brazilian bufonids. Skin, parotoid and tibial gland secretions were obtained for analysis by thin-layer chromatography. A triple-quadrupole mass spectrometer was used to confirm the indolealkylamines standards (serotonin, 5-HT; bufotenin, BTN; dehydrobufotenin, DHB and bufotenidin, BTD). We observed clear variation in the composition of indolealkylamines of the cutaneous secretions studied and also between those found in the skin and parotoid gland secretions of the same species. We discuss the utility of indolealkylamines to the phylogeny of this group of toads.     

Cutaneous blood supply of the penis

Twelve male cadaver specimens were injected with a latex solution to define the cutaneous blood supply of the penis. The cutaneous blood supply of the penile shaft is derived solely from a pair of axial arteries running in the dartos layer. Additional deep perforating arteries from the dorsal penile artery and corporal vessels supply the glans and subcoronal region. An understanding of this anatomy allows one to develop safely a variety of penile skin flaps for difficult reconstructive problems.     

Sex and weaponry: The distribution of toxin‐storage glands on the bodies of male and female cane toads (Rhinella marina)

The parotoid macroglands of bufonid anurans store (and can expel) large volumes of toxic secretions and have attracted detailed research. However, toxins also are stored in smaller glands that are distributed on the limbs and dorsal surface of the body. Female and male cane toads (Rhinella marina ) differ in the location of toxin‐storage glands and the extent of glandular structures. Female toads store a larger proportion of their toxins in the parotoids than males as well as (to a lesser extent) in smaller glands on the forelimbs. Males have smaller and more elongate parotoids than females, but glands cover more of the skin surface on their limbs (especially hindlimbs) and dorsal surface. The delay to toxin exudation in response to electrostimulation varied among glands in various parts of the body, and did so differently in males than in females. The spatial distribution of toxin glands differs between the sexes even in toads that have been raised under standardized conditions in captivity; hence, the sexual dimorphism is due to heritable factors rather than developmentally plastic responses to ecological (e.g., habitat, predation risk) differences between the sexes. The selective advantages of this sexual dimorphism remain unclear. A priori, we might expect to see toxin widely dispersed across any part of the body likely to be contacted by a predator; and a wide distribution also would be expected if the gland secretions have other (e.g., male–male rivalry) functions. Why, then, is toxin concentrated in the parotoids, especially in female toads? That concentration may enhance the effectiveness of frontal displays to deter predation and also may facilitate the transfer of stored toxins to eggs.     

The anatomic basis for the platysma skin flap

Meticulous anatomic dissection of the vasculature of the superficial anterolateral neck indicates that the platysma and overlying skin are supplied by direct cutaneous arteries measuring 0.5 mm in diameter. The small arteries are branches of the postauricular and occipital arteries in the upper lateral neck, the facial and submental arteries in the upper medial neck, the superior thyroid artery in the middle of the neck, the subclavian artery in the lower medial neck, and the transverse or superficial cervical arteries in the lateral aspect of the neck. These vessels traverse the undersurface of the platysma muscle to provide blood flow to the overlying skin. As opposed to this direct cutaneous system, the myocutaneous blood supply perforating through the sternocleidomastoid is scant. The platysma skin flap will survive if the blood supply from at least one region is preserved. In addition, it may be beneficial to include the external jugular and/or the communicating veins in the flap. By following these guidelines, the platysma flap has been successfully used for facial reconstruction in 7 of 8 consecutive patients.     

花背蟾蜍和中华蟾蜍皮肤腺体及耳后腺体的组织学研究

目的了解花背蟾蜍和中华蟾蜍皮肤腺和耳后腺的显微结构特点。方法采用大体解剖学方法观察耳后腺的外部形态及皮肤分泌物特征,采用常规石蜡包埋组织切片技术、HE和PAS染色方法观察皮肤腺和耳后腺的结构。结果花背蟾蜍和中华蟾蜍相比,皮肤黏液腺和耳后腺的显微结构差异有显著性,皮肤颗粒腺结构相似,推测当颗粒腺中的分泌物排出后,腺体外的上皮肌细胞变厚,细胞界限明显。花背蟾蜍皮肤黏液腺有两类分泌细胞相间排列成花环型。根据黏液腺分泌细胞的高矮（即中等高柱状、矮柱状和极矮柱状）,中华蟾蜍皮肤黏液腺相应地可见到三种形态,第三种黏液腺的分泌细胞极矮腺腔较大,数量极少,基本上只在前后肢皮肤中观察到。花背蟾蜍和中华蟾蜍的耳后腺也有差别：一是,中华蟾蜍耳后腺中大瓶状腺体内含物明显不同;二是,中华蟾蜍耳后腺中大瓶状腺体的大导管外侧有不同腺体存在,花背蟾蜍则无;三是,中华蟾蜍耳后腺表皮下大瓶状腺体上有钙化层存在,花背蟾蜍则无。结论两种蟾蜍皮肤腺和耳后腺均存在一定的差异,这种形态结构的差异与其生活习性和防御机能密切相关。     

Localisation by immunofluorescence of thyrotropin-releasing hormone in the cutaneous glands of the frog, Rana ridibunda.

Using the technique of indirect immunofluorescence, thyrotropin-releasing hormone (TRH) has been localised in certain cells of the cutaneous glands of the frog, $\text{Rana ridibunda}$. No specific fluorescence was found in other cells in the dermis, nor were any positive cells found in the epidermis. Previous studies have shown by radioimmunoassay that large amounts of TRH are present in frog skin, particularly in the dorsal region. Since dorsal frog skin contains many more glands than its ventral counterpart, this non-homogeneous distribution of TRH can be explained by its presence in a population of cells found in one type of cutaneous gland.     

Localization of water channels in the skin of two species of desert toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.     

Anatomical consideration of reverse-flow island flap transfers from the midpalm for finger reconstruction.

Primary soft-tissue coverage for large palmar defects of the fingers is a difficult problem for cases in which homodigital or heterodigital flaps cannot be used. The aim of this study was to explore the vascular and neural anatomy of the midpalmar area to assess the possibility of reverse island flaps from this area. In 24 cadaver hands perfused with a silicone compound, the arterial pattern of the superficial palmar arch and common palmar digital artery was examined. The cutaneous perforating arteries and nerve branches supplying the midpalmar area were dissected, and the number, location, and arterial diameter of these branches were measured. In six other specimens, the common palmar digital artery was injected to determine the skin territory supplied by the artery. The superficial palmar arch contained the three common palmar digital arteries and its terminal branch coursed along the radial margin of the index metacarpus. This terminal branch had three to six cutaneous perforators (diameter range, 0.1 to 0.5 mm) and supplied the radial aspect of the midpalmar area located over the ulnar half of the adductor pollicis muscles. The midpalmar area was divided into two regions-the proximal and distal-according to the vascular distributions. The proximal region contained dense aponeurosis and thin subcutaneous tissue, and the cutaneous perforators were rather sparse (between three and nine) and had a small diameter (0.1 to 0.3 mm). The distal region, which had loose aponeurosis and abundant subcutaneous tissue, had a rich vascular supply from the common and proper digital artery. Perforating arteries of this region coursed frequently in an oblique fashion and the number of perforators (between eight and 15) and their arterial diameters (diameter range, 0.1 to 0.5 mm) were higher than those of the proximal region. The area of skin perfused by the common palmar digital artery was 5 x 3 cm at the distal midpalmar region. There were three to five cutaneous nerve branches from the palmar digital nerve supplying the midpalmar area. From this study, two different reverse flaps were proposed. First, a 5 x 2 cm flap from the distal midpalmar region was elevated on the basis of the common and proper palmar digital artery. Measurement of the rotation arc revealed that the pivot point of this flap was located at the proximal interphalangeal joint level and could cover the finger pulp of the digits. The second flap candidate was that from the radial aspect of the midpalm, which was supplied by the terminal branch of the superficial palmar arch. In studies with cadaver hands, connection of this artery with the deep arterial system enabled this flap to reach the thumb pulp. These flaps may be a useful reconstruction option for significant palmar soft-tissue loss of the fingers.     

Reversed forearm island flap supplied by the septocutaneous perforator of the radial artery: anatomical basis and clinical applications

The cutaneous perforators of the radial artery adjacent to the superficial branch of the radial nerve and the lateral antebrachial cutaneous nerve were investigated, and the vascular anatomical features of the reversed forearm island flap supplied by those accompanying perforators were documented. Ten fresh cadavers were systemically injected with lead oxide, gelatin, and water. Twenty forearms were then dissected, and an overall map of the cutaneous vasculature and source vessels was constructed. The accompanying arteries were observed to lie along the lateral antebrachial cutaneous nerve and the superficial branch of the radial nerve and to nourish the skin through cutaneous branches. Vascular communication among these cutaneous vessels was evaluated, to determine the cutaneous vascular territory of the radial forearm flap. This anatomical information facilitates flap design in the forearm region. Clinical experience regarding the usefulness of the reversed forearm island flap for hand reconstruction for a series of five patients is presented.     

Morphological and biochemical characterization of the cutaneous poison glands in toads (<Emphasis Type="Italic">Rhinella marina</Emphasis> group) from different environments

Background

Amphibian defence against predators and microorganisms is directly related to cutaneous glands that produce a huge number of different toxins. These glands are distributed throughout the body but can form accumulations in specific regions. When grouped in low numbers, poison glands form structures similar to warts, quite common in the dorsal skin of bufonids (toads). When accumulated in large numbers, the glands constitute protuberant structures known as macroglands, among which the parotoids are the most common ones. This work aimed at the morphological and biochemical characterization of the poison glands composing different glandular accumulations in four species of toads belonging to group Rhinella marina (R. icterica, R. marina, R. schneideri and R. jimi). These species constitute a good model since they possess other glandular accumulations together with the dorsal warts and the parotoids and inhabit environments with different degrees of water availability.

Results

We have observed that the toads skin has three types of poison glands that can be differentiated from each other through the morphology and the chemical content of their secretion product. The distribution of these different glands throughout the body is peculiar to each toad species, except for the parotoids and the other macroglands, which are composed of an exclusive gland type that is usually different from that composing the dorsal warts. Each type of poison gland presents histochemical and biochemical peculiarities, mainly regarding protein components.

Conclusions

The distribution, morphology and chemical composition of the different types of poison glands, indicate that they may have different defensive functions in each toad species.

     

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine (l-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of l-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP((8-37)), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K(+) (K(+)(ATP)) channel inhibitor. Levcromakalim, a K(+)(ATP) channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K(+)(ATP) channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.     

Blood supply of the rat hypothalamus. VI. Posterior region of the hypothalamus (nucleus hypothalamicus posterior, nuclei praemammillares, nucleus supramammilaris, mammilary body).

It was shown by the double ink-filling technique that the arteries of the rat premammillary region and mammillary body arise from the a. communicans posterior while these areas are drained by the anterior interpeduncular vein. Disregarding some minor overlaps and anastomoses, the blood supplies of the two territories are independent of each other and from the neighbouring areas of the hypothalamus, diencephalon and mesencephalon. Arteries of the premammillary region arise from the premammillary artery, except for some branches of the posterior tuberal and interpeduncular arteries. The mammillary body is supplied by three mammillary arteries (anterior, posterior and lateral). The premammillary region drains into the anterior and posterior premammillary veins. Venous blood of the mammillary body is collected by the anterior and posterior mammillary veins which end in the anterior interpeduncular vein. The circulation of individual premammillary and mammillary nuclei is described in detail.     

The anterior tibial artery flap: anatomic study and clinical application

Satisfactory replacement of skin defects over the lower leg remains a difficult problem. Various forms of coverage, including, local rotation flaps, muscle flaps, and fascial and free flaps, have their specific indications and inherent disadvantages. In this work, a new axial skin flap based on perforating vessels in the territory of the anterior tibial artery is described. A series of 50 lower leg dissections was carried out in 25 fresh cadavers after latex injection into the femoral artery. Detailed studies of the cutaneous distribution of the anterior tibial artery showed that three main arteries perfuse the anterior lateral portion of the lower leg. The superior lateral peroneal artery and the inferior lateral peroneal artery interseptal cutaneous perforators arise at an average of 25.6 and 17.2 cm from the lateral malleolus, respectively. The superior lateral peroneal artery was present in 100 percent of the specimens, whereas the inferior lateral peroneal artery was present in 70 percent of the specimens. In their course, they give several muscular branches to the peroneus longus and brevis prior to perforating the fascia and arborizing in the subcutaneous tissues of the anterolateral portion of the leg. The average external diameter was 1.6 cm for the superior and 1.4 cm for the inferior lateral peroneal artery. The superficial peroneal nerve accessory artery is the third artery which contributes to the skin of the lower leg. It arises from the superior lateral peroneal artery in 30 percent of cases, from the inferior lateral peroneal artery in 40 percent, and from both in 30 percent. The artery runs along with the superficial peroneal nerve and gives several cutaneous perforators along its descending course. Several cutaneous axial flaps can be fashioned around this anatomy. The operative technique along with demonstrative clinical cases is presented followed by pertinent discussion.     

Digeneans (Plathelminthes: Trematoda) of seven sympatric species of anurans from Los Tuxtlas,Veracruz, Mexico

Digeneans of sympatric Bufo marinus, B. valliceps, Hyla picta, H. ebraccata, Smilisca baudini, Rana berlandieri, and R. vaillanti from Los Tuxtlas, Veracruz, Mexico, are reported. A total of 7 adult trematode species and 3 unidentified metacercariae was found in the different host species examined: Gorgoderina attenuata, G. parvicava, Cephalogonimus americanus, Haematoloechus medioplexus, Glypthelmins californiensis, Mesocoelium monas and Langeronia macrocirrus. Prevalence, mean intensity and abundance of the trematodes in each host species are given.     

Vascularization of the myocutaneous latissimus dorsi flap. Injection study on the thoracodorsal artery

This study reports on investigations of the thoracodorsal artery by injection studies. This artery has a long proximal extramuscular course before it enters the muscle. A consistent neurovascular hilum was found at a considerably constant location on the inferior muscle surface, 2-3 cm medial to its lateral edge and about 5 cm distal to the inferior scapular border. A constant branching pattern of the thoracodorsal artery into a lateral and a medial vessel stem was found. A high number of muscle-perforating arteries from a dense network in the cutaneous and subcutaneous layer. The thoracodorsal artery supplies the whole cutaneous area adjacent to the latissimus dorsi muscle plus a streak of about 2 cm at the medial and distal muscle borders. The presented anatomical landmarks are useful for locating the neurovascular hilum, and the intramuscular course of the thoracodorsal artery for exploration of the vessel by Doppler sonography or dissection. The length of the pedicle and the relatively big vessel gauge are good anatomical markers for the free transfer of a latissimus dorsi flap.     

Histochemically demonstrable catecholamines and cholinesterases in nerve fibres of rat dorsal skin

Summary Histochemically demonstrable cholinesterases of rat skin and cutaneous nerves hydrolyze acetylthiocholine iodide and butyrylthiocholine iodide. Cholinesterase activity of the skin was located in the epidermis, in the hair follicles at the level of the sebaceous glands, in adjacent parts of the sebaceous glands, in erector pili muscles and their nerves, in cutaneous and subcutaneous nerves and nerve trunks, including some nerves accompanying cutaneous blood vessels, and in the membranes of fat cells. No encapsulated nerve endings were found. In the nerves of erector pili muscles there was some neurilemmal non-specific cholinesterase activity, demonstrated in the presence of 10^–5 M BW 284C 51, and specific acetylcholinesterase activity resistant to 10^–5 M iso-OMPA. The cholinesterase activity in other cutaneous nerves was inhibited by 10^–5 M iso-OMPA but was resistant to 10^–5 M BW284 C 51, thus representing mainly non-specifc cholinesterase (nsChE) activity.The adrenergic nerves of the dorsal skin, as revealed by glyoxylic acid-induced fluorescence (GIF), were located in association with erector pili muscles and surrounded arteries and arterioles. Small fluorescent nerves were situated in subcutaneous nsChE-positive nerve trunks.Using GIF and cholinesterase techniques performed either simultaneously or consecutively, it was found that the nsChE-positive, probably sensory, nerves accompanying blood vessels were fewer in number than the fluorescent adrenergic nerves and ran a course independent of them. No cholinesterase reaction was seen in the fluorescent adrenergic nerves when short incubation times were used. When the incubation time was prolonged overnight, the nsChE reaction closely followed the course of fluorescent adrenergic nerves.     

Microbial biotransformation as a source of chemical diversity in cane toad steroid toxins

The cane toad is an invasive pest that is rapidly colonising northern Australia. The cane toad parotoid gland secretes cardiotoxic steroids (bufadienolides) that are poisoning native predator species. This study reveals bufadienolide diversity within the secretions of Australian cane toads is different to cane toads from overseas, being far more structurally diverse than previously assumed. It is proposed that this variation is mediated by in situ bacterial biotransformation.