Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.     

The Pattern of Nipple Use Before Weaning Among Littermates of the Domestic Dog

Suckling provides a well‐defined and functionally significant context in which to study mammalian sibling competition and the possible consequences of this for long‐term differences in individual behavior, physiology, and life histories. There have, however, been surprisingly few studies of suckling behavior, even in species as accessible as the domestic dog. In a first approach, we filmed the suckling behavior of 10 litters (47 puppies) of various dog breeds in their owners' homes during the first postnatal month before the start of weaning. The pattern of nipple use was very similar across litters but very different to that of the better‐studied pig or cat. Puppies showed only weak preference for particular (central) nipple rows, they often switched nipples during nursing sessions and showed no evidence of teat consistency in which each littermate uses primarily one or two particular nipples. They also showed very little agonistic behavior that could be interpreted as contesting access to nipples. The present findings provide a reminder of the diversity of suckling patterns among altricial mammals and of the need for more comparative information to better understand the reasons for and functional significance of such differences.     

Fighting by Kittens and Piglets during Suckling: What Does it Mean?

Newborn kittens and piglets show some notable similarities in suckling‐associated behaviours. Both develop a pattern of teat use in which each member of the litter predominantly occupies one or two particular nipples or teats, both show agonistic behaviour towards littermates during suckling, and both are born with ‘weapons’–kittens with sharp claws and piglets with sharp needle teeth. Traditionally, the differential use of nipples or teats has been thought to be due to differential quality of the mammary glands. However, neither our findings in kittens nor an examination of the literature on piglets support this view. As mammary glands usually are of equal quality and as there are normally more glands than young, there seems little reason for littermates to fight. Furthermore, there is also little evidence that individual performance in suckling contests or use of ‘weapons’ is related to weight gain or to dominance relations. Given the present lack of satisfying adaptive explanations for the seemingly aggressive behaviour and use of weapons by kittens and piglets, we suggest these to be epiphenomena of developmental processes anticipating the use of these behaviours and structures in later adaptive contexts.     

Light on the moth-eye corneal nipple array of butterflies

The outer surface of the facet lenses in the compound eyes of moths consists of an array of excessive cuticular protuberances, termed corneal nipples. We have investigated the moth-eye corneal nipple array of the facet lenses of 19 diurnal butterfly species by scanning electron microscopy, transmission electron microscopy and atomic force microscope, as well as by optical modelling. The nipples appeared to be arranged in domains with almost crystalline, hexagonal packing. The nipple distances were found to vary only slightly, ranging from about 180 to 240 nm, but the nipple heights varied between 0 (papilionids) and 230 nm (a nymphalid), in good agreement with previous work. The nipples create an interface with a gradient refractive index between that of air and the facet lens material, because their distance is distinctly smaller than the wavelength of light. The gradient in the refractive index was deduced from effective medium theory. By dividing the height of the nipple layer into 100 thin slices, an optical multilayer model could be applied to calculate the reflectance of the facet lenses as a function of height, polarization and angle of incidence. The reflectance progressively diminished with increased nipple height. Nipples with a paraboloid shape and height 250 nm, touching each other at the base, virtually completely reduced the reflectance for normally incident light. The calculated dependence of the reflectance on polarization and angle of incidence agreed well with experimental data, underscoring the validity of the modelling. The corneal nipples presumably mainly function to reduce the eye glare of moths that are inactive during the day, so to make them less visible for predators. Moths are probably ancestral to the diurnal butterflies, suggesting that the reduced size of the nipples of most butterfly species indicates a vanishing trait. This effect is extreme in papilionids, which have virtually absent nipples, in line with their highly developed status. A similar evolutionary development can be noticed for the tapetum of the ommatidia of lepidopteran eyes. It is most elaborate in moth-eyes, but strongly reduced in most diurnal butterflies and absent in papilionids.     

强壮水螅的特征及其与寡水螅的种间差别问题

一种广泛分布于黑龙江省的大型具柄水螅被暂定为强壮水螅Hydra robusta(IT(?) 1947)。这一中国品系与IT(?)(1947)在日本报告的新种主要特征相同,包括具有精巢乳头。只是精巢乳头不稳定。在实验室条件下第一次有性生殖时每个雄体都发生精巢乳头,但第二次有性生殖时,同一群的后代可全部失去精巢乳头。这是介于强壮水螅与寡水螅Hydra oligactis Pallas(1766)之间的中间性状。从而导致作者做出结论,认为过去其他学者所主张的,以精巢乳头的有无做为鉴别特征来区分强壮水螅和寡水螅是不可靠的。因此,本文又检验了此种水螅的体细胞染色体,证明2n=30,其中第二对染色体上有明显近中位置的次缢痕,最小染色体的长度为最大染色体长度的二分之一以上。这些可做为此种水蝗的鉴别特征。对Niiyama(1944)和Datta(1970)在寡水螅上所做的染色体研究也做了比较及讨论。     

The inverted nipple: its grading and surgical correction

Inverted nipples have been treated by various methods by many authors, but the relationship between the grade of the deformity and the appropriate surgical procedure is not clearly described. One hundred seven inverted nipples in 60 patients were treated from 1993 to 1997. They were divided into three groups by the authors' system of grading. The grade was made by preoperative evaluation of severity of inversion and was confirmed by the surgical findings. In grade I, the nipple is easily pulled out manually and maintains its projection quite well. Grade I nipples are believed to have minimal fibrosis; thus, manual traction and a single, buried purse-string suture are enough for the correction. The majority of inverted nipples belong to grade II, i.e., the nipples can be pulled out but cannot maintain projection and tend to go back again. These nipples are thought to have moderate fibrosis beneath the nipple. Blunt dissections for surgical release were carried out until the inversion did not recur after releasing the traction. The lactiferous ducts could be identified and preserved, permitting proper release of fibrotic bands in the grade II group. The purse-string suture was used. In grade III, to which the least number of inverted-nipple cases belong, the nipple can hardly be pulled out manually. Severe fibrosis made it impossible to reach optimal release of the fibrotic band with the preservation of the ducts. The fibrotic bands are widely dissected, and the lactiferous ducts are cut, especially in the central portion. Two or three deepithelialized dermal flaps may be used to make up for soft-tissue deficiency; a purse-string suture is also used. This grading system will be useful for patient classification and analysis, systematic planning, and application of the proper surgical procedures.     

Change of nipple preference between successive offspring in Japanese macaques

Selective use of nipples among free-ranging infant Japanese macaques (Macaca fuscata) was studied. Each infant held only one of its mother's two nipples in the mouth during an average of 90.9% ± 1.5% (SEM) of total nipple contact time. The next offspring born to the same mother tended to use the nipple that its previous sibling had not used. Maternal behavior did not cause use of one nipple or the other.     

An alternative operation for inverted nipple.

An operation to correct inverted nipples is described. Its main advantage is that reinversion of the nipple is prevented, because two "dermal" flaps not only increase the density of tissue underneath the nipple but also act as slings.     

The contribution of the olfactory and tactile modalities to the nipple-search behaviour of newborn rabbits

By performing uni- and bilateral olfactory bulb lesions and uni- and bilateral transsections of the infraorbital branches of the trigeminal nerves in 2-day-old rabbits, it could be shown that: Both the olfactory and tactile modalities are essential for the successful performance of nipple-search behaviour. While bilateral bulbectomy completely eliminates searching, and hence suckling, unilateral bulbectomy has relatively little effect. Bilateral denervation of the muzzle does not eliminate searching, but pups are unable to suckle as they fail to show the mouth-opening component necessary for nipple attachment. In contrast to unilateral bulbectomy, unilateral denervation of the muzzle results in a lateralization of head movements during searching, nipple grasping and nipple release.     

A direct surgical approach to correct the inverted nipple

Inverted nipples are cosmetically unpleasing to the patient and can become inflamed due to mechanical difficulty with cleaning the nipple-areola complex. A surgical technique for the permanent repair of inverted nipples is described. The rationale for the surgical approach is that the major pathophysiologic basis for nipple inversion is shortened lactiferous ducts. Briefly outlined, under local anesthesia, the nipple is everted with a skin hook and held in gentle traction while a small incision is made on each side at the nipple-areola junction. Breast ducts are then divided by sharp dissection, and a drain is inserted through the tunnel under the nipple. The drain is removed in 7 to 10 days. The patient must be informed before the procedure that breast-feeding will not be possible afterward because breast ducts will be permanently divided. Advantages to the procedure are (1) no scars on the areola, (2) no stricture from sutures, (3) adequate blood and nerve supply to the nipple, and (4) decreased risk of hematoma.     

Does the aquatic invertebrate nipple array prevent bubble adhesion? An experiment using nanopillar sheets

The nipple array is a submicrometre-scale structure found on the cuticle surfaces of various invertebrate taxa. Corneal nipples are an antiglare surface in nocturnal insects, but the functional significance of the nipple array has not been experimentally investigated for aquatic organisms. Using nanopillar sheets as a mimetic model of the nipple array, we demonstrated that significantly fewer bubbles adhered to the nanopillar surface versus a flat surface when the sheets were hydrophilic. Many more bubbles adhered to the hydrophobic surface than the hydrophilic surfaces. Bubbles on the body surface may cause buoyancy problems, movement interference and water flow occlusion. Here, bubble repellence is proposed as a function of the hydrophilic nipple array in aquatic invertebrates and its properties are considered based on bubble adhesion energy.     

Effect of accommodating sucking and nosing on the behaviour of artificially reared piglets

Neonatal piglets are often used in biomedical research applications that require artificial rearing. Social housing can be problematic because the piglets develop belly nosing, navel and ear sucking that can result in injury. Our objective was to determine the effectiveness of using feeding devices that provide various opportunities for sucking and nosing behaviour on reducing piglet-directed behaviour of group-housed laboratory piglets. Fifteen piglets were used in each of four trials. The piglets nursed their dam for approximately 72 h to obtain passive immunity before transfer to a laboratory facility where they were allotted, five per group, to one of three stainless steel isolator units. Each unit featured a different style of feeding system for the delivery of milk replacer: a plastic trough (T), a nipple (N) mounted on a smooth plexiglass wall, or a nipple mounted on a pliant bag of sterile water (artificial udder [AU]). Each system had five feeding spaces so that all piglets fed simultaneously. Milk was provided at 6-h intervals, and behaviour was recorded on alternate days for 12 days post-weaning. Although trough-fed piglets began to eat much sooner than those piglets fed from nipples, time spent nosing, chewing or sucking on pen-mates and belly nosing were markedly higher in T piglets than in either N or AU, overall (mean: P<0.05) and over time (quadratic: P<0.05). Over time, N piglets developed a stereotypic snout rubbing on the wall behind the nipples, while AU piglets massaged and often fell asleep in contact with the udder from day 2 of the trial. Resting patterns were also affected. N and AU piglets settled down to rest more quickly (P<0.01) and spent significantly more time resting in the hour following feeding than T piglets (P<0.05). A feeding device that accommodates both sucking and massage can significantly reduce piglet-directed behaviour and may facilitate social housing of artificially reared piglets.     

Role of nipple stimulation in the suppression of estrous cyclicity during extended lactation in the rat

The role of nipple stimulation in the suppression of the estrous cycle during extended lactation was studied in rats subjected to either total, partial, or sham excision of the nipples. Each female cohabited with 4 pups, 4-14 days old, over a period of 70 days postpartum, during which vaginal smears were recorded daily. Initially, regardless of the presence of nipples, all rats exhibited a postpartum diestrus that lasted for 12-20 days. Intact females (bearing 6 pairs of nipples) continued to exhibit successive prolonged diestrous phases over 70 days of lactation. A comparable result was obtained with females bearing only the anterior pair of nipples, which, in a separate experiment, was found to be the most frequently suckled pair. However, females devoid of nipples resumed regular (4-day) estrous cycles between Days 12 and 27 postpartum, in spite of their continuous contact with pups. Thus, when lactation is prolonged beyond the normal time of weaning (Day 21 postpartum), stimulation of the nipples by sucking becomes indispensable for the continued arrest of the estrous cycle. The possible mechanisms underlying this phenomenon are discussed.     

Growing pigs’ drinking behaviour: number of visits,duration, water intake and diurnal variation

Individual drinking patterns are a potential tool for disease monitoring in pigs. However, to date, individual pig drinking behaviour has not been described, and effects of external factors have not been examined. The aim of this study was to perform detailed quantification of drinking behaviour of growing pigs and to examine effects of period of day and effects of competition for access to the drinking nipple on the drinking behaviour, amount of water used and water wastage. In all, 52 cross-bred castrated male pigs (live weight 20.5±1.7 kg; mean±s.d.) maintained as either 3 (N3) or 10 (N10) pigs per pen and water nipple (four groups/treatment) were used. All pigs were fitted with a transponder ear tag. A radio frequency identification reader recorded and time stamped visits at the nipple. In each pen, water flow was logged every second. The drinking behaviour was recorded for 4 consecutive days and analysed using a linear mixed model. Overall, the pigs spent 594 s at the nipple during 24 h distributed among 44 visits. During this period, 5 l of water were used, of which >30% was wasted. Social competition did not affect the drinking behaviour over 24 h, except for the proportion of interrupted visits where pigs, kept with recommended nipple availability (N10), showed an increased proportion of interrupted drinking bouts compared with pigs kept at very low level of competition (N3) (0.18±0.01 v. 0.11±0.01; P<0.01). However, splitting data into 8-h periods (P1, P2, P3) starting from 0600 h revealed differences between treatments, showing that in N3, water use per visit was lower in P1 than P2 and P3 (110±10 v. 126±7 and 132±7 ml; P<0.05), whereas in N10, the water used per visit was higher during P3 than during the other periods (P1: 107±14 ml, P2: 112±10 ml v. P3: 151±10 ml; P<0.001). A similar pattern was found for visit duration. In N3, fewer nipple visits were observed in P2 than P1 (15.6±1.2 v. 22.0±1.2; P<0.001), whereas no difference was found between P1 and P2 in N10. The results demonstrate that growing pigs at the two levels of competition maintained a comparable level of 24 h water intake by changing behavioural variables involved in drinking. This dynamic characteristic of drinking behaviour means that if individual drinking patterns are to be used as disease monitoring tools, it is important to consider effects of external factors and include data on period level to allow rapid detection of behavioural changes.     

The reactivity of neonatal rabbits to the mammary pheromone as a probe for viability

Newborn rabbits depend on a daily nursing interaction with the mother to gain milk and to survive. During this interaction, they localise and seize the nipples displaying a typical behaviour triggered by maternal odour cues. The mammary pheromone constitutes such a signal in domestic rabbits: it elicits sucking-related movements in more than 90% of the pups. However, some newborns remain unresponsive to the presentation of the pheromone, even pups apparently healthy and highly motivated to suck. The main goal of the present study was therefore to explore the link between the unresponsiveness of rabbit pups to the mammary pheromone and their growth and survival in breeding conditions. To that end, 293 newborns from 30 litters were tested for their head searching-oral grasping responses to the mammary pheromone on days 1 and 3, and their milk intake and mortality were followed up from days 1 to 21. It was hypothesised that unresponsive newborns would have subsequent difficulties in finding nipples, sucking and surviving. Early weight and success in milk intake were further considered as mediating factors in growth and viability. The results showed that pups that were unresponsive to the mammary pheromone on day 1 were less successful in gaining milk and had a higher rate of mortality than the responsive pups. However, this impact was modulated by the weight of pups: it appeared only in the lightest newborns. Moreover, this impact vanished on day 3. On the other hand, the pup weight and sucking success on days 1 to 3 strongly influenced viability and growth during the period extending from days 1 to 21. Taken together, the results show that the day-1 responsiveness of rabbit pups to the mammary pheromone can be considered as an indicator of individual viability in pups having a small weight (<48 g on day 1). The predictive validity of the pups’ pheromonal reactivity seems however time-limited as it works only during the first, but crucial, postnatal days.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the rat mammary gland

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP) family of peptides, is present in the brain and in neuronal elements of a number of peripheral organs. Since no information on PACAP in the mammary gland exists, we have investigated, by radioimmunoassay and immunohistochemistry, the occurrence and distribution of PACAP immunoreactivity in the mammary gland of lactating and non-lactating rats. A specific monoclonal mouse anti-PACAP antibody'has been used to show that the peptide is located in nerve fibres associated with bundles of circular and longitudinal smooth muscle surrounding the lactiferous duct of the nipple. PACAP-immunoreactive nerve fibres and nerve bundles are present in the subepidermal connective tissue of the nipple and in the mammary parenchyma, some of the fibres being in close contact with blood vessels. Occasionally, a few delicate varicose fibres are associated with secretory alveoli and lactiferous ducts. The majority of PACAP-positive nerve fibres are, however, located in the glabrous skin of the nipple and the hairy skin adjacent to the nipple forming a subepithelial plexus from which delicate varicose nerve fibres enter the overlying epithelium. Double immunostaining for PACAP and a marker for sensory neurons, calcitonin gene-related peptide, has disclosed that the two peptides are almost completely co-localized. A minor population of the PACAP-immunoreactive nerve fibres shows co-existence with VIP. Although no obvious changes at the immunohistochemical level could be observed during pregnancy or lactation, elevated concentrations of immunoreactive PACAP-38 in mammary extracts have been found during lactation. Our data suggest that PACAP is involved in the nervous control of mammary gland function, probably in the transmission of suckling stimuli.     

Characterization of the autonomic innervation of mammary gland in lactating rats studied by retrograde transynaptic virus labeling and immunohistochemistry

The aim of experiments was to characterize the neurons of the autonomic chain that innervates the nipple and the mammary gland of lactating rats using retrograde transynaptic virus labeling and neurotransmitter and neuropeptide immunohistochemistry. Two days after injection of green fluorescence protein labeled virus in two nipples and underlying mammary glands, labeling was observed in the ipsilateral paravertebral sympathetic trunk and the lateral horn. Three days after inoculation the labeling appeared in the brain stem and the hypothalamic paraventricular nucleus. Above the spinal cord the labeling was bilateral. A subpopulation of virus labeled cells in the paraventricular nuclei synthesized oxytocin. Labeled neurons in the lateral horn showed cholinergic immunoreactivity. These cholinergic neurons innervated the paravertebral ganglia where the virus labeled neurons were partially noradrenergic. The noradrenergic fibers in the mammary gland innervate the smooth muscle wall of vessels, but not the mammary gland in rats. The neurons in the lateral horn receive afferents from the brain stem, and paraventricular nucleus and these afferents are noradrenergic and oxytocinergic. New findings in our work: Some oxytocinergic fibers may descend to the neurons of the lateral horn which innervate noradrenergic neurons in the paravertebral sympathetic trunk, and in turn these noradrenergic neurons reach the vessels of the mammary gland.     

The male genital tract and the nipples of male and female offspring of rats given the non-steroidal antiandrogens DIMP and Sch 13521, during pregnancy.

Male and female offspring of rats given antiandrogens, the steroidal BOMT or the non steroids DIMP or Sch 13521, daily during the last third of pregnancy were studied. Detailed examinations were made of the genital tract of male, and of the nipples of male and female offspring. A) Male offspring. 1) Genital tract of newborn and 31-91 day old males : Modifications of the development of accessory sexual tissues were found in all treatment groups. As indicated by the severity of deviations from normal (morphology and weight of sex accessories), the antiandrogenic effect of the preparations, in the doses given to the mother rats, increased from BOMT (50 or 75 mg/day) via Sch 13521 (30 mg/day) and DIMP (50 or 60 mg/day) to Sch 13521 (60 mg/day). 2) Nipples of 10-60 day old males : Whole mount preparations were made unilaterally of the row of 6 mammary glands with nipples. The number of intact and abnormal nipples, respectively, was recorded. The relation between intact and abnormal nipples served as indicator of the efficiency of the antiandrogenic substances studied. The result showed that the antiandrogenic effect increased from BOMT to Sch 13521, 60 mg, in the same order as that arrived at from studies of the genital tract. The combined results obtained from the male offspring indicated that the tissues of the genital region, the growth and differentiation of which was most readily impaired by antiandrogens, were the same as those known from other work to be stimulated most easily in female rat fetuses by testosterone. B) Female offspring. Nipples of 31-60 day old females were judged from whole mount preparations and recorded as in the males. The nipples of adult virginal females were examined macroscopically. The same procedure was applied to lactating females, but the results were controlled in consecutive lactational periods and at autopsy. The 3 groups of females showed uniformly that 1) offspring of rats given BOMT during pregnancy had many (about 50 per cent) malformed nipples and 2) the treatment of mother rats with DIMP or Sch 13521 did not influence the development of nipples in female offspring. The result was assumed to be due to an androgenic effect of the steroidal antiandrogen, BOMT, on the nipple anlage.     

Autologous fat graft in nipple reconstruction

Reconstruction of the nipple is the penultimate step in breast reconstruction after mastectomy. A number of reconstructive techniques have been described for nipple reconstruction including skin grafts, composite grafts, and various local flaps. The authors' preferred reconstructive technique is the local C-V or modified star flap. This flap produces an excellent reconstruction, but it is dependent on underlying subcutaneous fat to provide bulk to the reconstructed nipple. In most instances, the subcutaneous tissue is adequate. However, under certain circumstances, the subcutaneous fat may be insufficient to produce a nipple of adequate projection. Two cases of bilateral nipple reconstruction after soft-tissue expansion and implant placement and subsequent nipple reconstruction with local flaps provided inadequate nipple projection. These instances, as well as a retrospective review of reconstructed nipples after mound restoration using a variety of techniques, led the authors to conclude that a more predictable alternative to sustain nipple projection was necessary. The authors identified two broad categories of breast reconstruction patients in whom this new technique would be beneficial. In the first category of patients, breast mounds are reconstructed with tissue expansion and implant insertion, and in the second category, breast mounds are reconstructed by any technique in which the nipple reconstruction subsequently flattens. This article describes the indications, techniques, and experience in 13 patients treated over a 10-month period with fat grafting for nipple reconstruction.