Evaluating the Reproducibility of Mouse Anatomy under Rotation in a Custom Immobilization Device for Conformal FLASH Radiotherapy.

The observation of an enhanced therapeutic index for FLASH radiotherapy in mice has created interest in practical laboratory-based FLASH irradiators. To date, systems capable of 3D conformal FLASH irradiation in mice have been lacking. We are developing such a system, incorporating a high-current linear accelerator to produce a collimated X-ray beam in a stationary beamline design, rotating the mouse about a longitudinal axis to achieve conformal irradiation from multiple beam directions. The purpose of this work was to evaluate the reproducibility of mouse anatomy under rotation at speeds compatible with conformal FLASH delivery. Three short-hair mice and two hairless mice were immobilized under anesthesia in body weight-specific contoured plastic molds, and subjected to three rotational (up to 3 revolutions/s) and two non-rotational movement interventions. MicroCT images were acquired before and after each intervention. The displacements of 11 anatomic landmarks were measured on the image pairs. The displacement of the anatomical landmarks with any of the interventions was 0.5 mm or less for 92.4% of measurements, with a single measurement out of 275 (11 landmarks × 5 interventions × 5 mice) reaching 1 mm. There was no significant difference in the displacements associated with rotation compared to those associated with moving the immobilized mouse in and out of a scanner or with leaving the mouse in place for 5 min with no motion. There were no significant differences in displacements between mice with or without hair, although the analysis is limited by small numbers, or between different anatomic landmarks. These results show that anatomic reproducibility under rotation speed corresponding to FLASH irradiation times appears to be compatible with conformal/stereotactic irradiation in mice.

Arterial and microvascular supply of cerebral hemispheres in the nude mouse revealed using corrosion casting and scanning electron microscopy.

Morphological analyses of cerebral vascularization are not only important for the characterization of the anatomical and physiological relationships between vascular and nervous tissue, but also required to understand structural modifications that occur in many pathological conditions affecting the brain. The aim of this study was to generate a three-dimensional vascular map of the cerebral hemispheres in the nude mouse brain, a widely used animal model for studying tumour biology. We used the corrosion casting (CC) technique to isolate blood vessels from 30 nude mouse brains. All casts were analysed using scanning electron microscopy (SEM), which generated quantitative data regarding vessel length and diameter as well as inter-vascular and inter-branching distances. We identified three different topographical regions: (i) the cortical region, characterized by a superficial wide sheet of vessels giving rise to terminal perforant vessels that penetrate the grey matter; (ii) the inner part of the grey matter, in which dense capillary nets form many flake-like structures extending towards the grey-white matter boundary, where perforant vessels finally change direction and form a well-defined vascular sheet; and (iii) the white matter layer, characterized by a more disorganized vascular architecture. In this study, we demonstrate the accuracy of the CC-SEM method in revealing the 3D-topographical organization of the vascular network of the normal nude mouse brain. These baseline data will serve as a reference for future anatomical investigations of pathological alterations, such as tumour infiltrations, using the nude mouse model.

The regional distribution and relative frequency of gastrointestinal endocrine cells in the nude mice, Balb/c-nu/nu: an immunohistochemical study.

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal (GI) tract - fundus, pylorus, duodenum, jejunum, ileum, cecum, colon and rectum of the nude mouse, Balb/c-nu/nu were studied with immunohistochemical method using six types of anti-sera against serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (hPP). All of six types of immunoreactive (IR) cells were identified. Most of IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) while cells showing round in shape (close type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies were varied according to each portion of GI tract. Serotonin-IR cells were detected throughout the whole GI tract and they showed the highest frequency in the pylorus. Gastrin-IR cells were restricted to the pylorus and duodenum with numerous and a few frequencies, respectively. CCK-8-IR cells were also restricted to the pylorus, duodenum and jejunum with numerous, a few and rare frequencies, respectively. Somatostatin-IR cells were demonstrated throughout the whole GI tract except for the colon and rectum, and they showed the highest frequency in the fundus. In addition, glucagon- and hPP-IR cells were restricted to the fundus and rectum, respectively with a few frequencies. In conclusion, the general distribution patterns and relative frequency of GI endocrine cells of the nude mouse, Balb/c-nu/nu was similar to that of other strains of mice. However, some strain and/or species-dependent unique distributions and frequencies of endocrine cells were also observed especially for somatostatin- and hPP-IR cells.

Learning from nudity: lessons from the nude phenotype.

In mice, rats, and humans, loss of function of Foxn1, a member of the winged helix/forkhead family of transcription factors, leads to macroscopic nudity and an inborn dysgenesis of the thymus. Nude (Foxn1(nu)/Foxn1(nu)) mice develop largely normal hair follicles and produce hair shafts. However, presumably because of a lack of certain hair keratins, the hair shafts that are generated twist and coil in the hair follicle infundibulum, which becomes dilated. Since hair shafts fail to penetrate the epidermis, macroscopic nudity results and generates the - grossly misleading - impression that nude mice are hairless. Here, we provide an overview of what is known on the role of Foxn1 in mammalian skin biology, its expression patterns in the hair follicle, its influence on hair follicle function, and onychocyte differentiation. We focus on the mechanisms and signaling pathways by which Foxn1 modulates keratinocyte differentiation in the hair follicle and nail apparatus and summarize the current knowledge on the molecular and functional consequences of a loss of function of the Foxn1 protein in skin. Foxn1 target genes, gene regulation of Foxn, and pharmacological manipulation of the nude phenotype (e.g. by cyclosporine A, KGF, and vitamin D3) are discussed, and important open questions as well as promising research strategies in Foxn1 biology are defined. Taken together, this review aims at delineating why enhanced research efforts in this comparatively neglected field of investigative dermatology promise important new insights into the controls of epithelial differentiation in mammalian skin.

Optically imageable metastatic model of human breast cancer.

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease.

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in BALB/c +/+, nu/+, and nu/nu mice.

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.

Neurobiology of human neurons (NT2N) grafted into mouse spinal cord: implications for improving therapy of spinal cord injury.

Emerging data suggest that current strategies for the treatment of spinal cord injury might be improved or augmented by spinal cord grafts of neural cells, and it is possible that grafted neurons might have therapeutic potential. Thus, here we have summarized recent studies of the neurobiology of clonal human (NT2N) neurons grafted into spinal cord of immunodeficient athymic nude mice. Postmitotic human NT2N neurons derived in vitro from an embryonal carcinoma cell line (NT2) were transplanted into spinal cord of neonatal, adolescent and adult nude mice where they became integrated into the host gray and white matter, did not migrate from the graft site, and survived for > 15 months after implantation. The neuronal phenotype of the grafted NT2N cells was similar in gray and white matter regardless of host age at implantation, and some of the processes extended by the transplanted NT2N neurons became ensheathed by oligodendrocytes. However, there were consistent differences between NT2N processes traversing white versus gray matter. Most notably, NT2N processes with a trajectory in white matter extended over much longer distances (some for > 2 cm) than those confined to gray matter. Thus, NT2N neurons grafted into spinal cord of nude mice integrated into gray as well as white matter, where they exhibited and maintained the morphological and molecular phenotype of mature neurons for > 15 months after implantation. Also, the processes extended by grafted NT2N neurons differentially responded to cues restricted to gray versus white matter. Further insight into the neurobiology of grafted human NT2N neurons in the normal and injured spinal cord of experimental animals may lead to novel and more effective strategies for the treatment of spinal cord injury.

Postnatal and postpartal morphology of the mammary gland in nude mice.

The object of this work was to compare the postnatal and postpartal morphology of the mammary gland of nu/nu with that of nu/(+)-mice. All studies were carried out on groups of female (athymic) nude mice with NMRI genetic background, their nu/(+)-siblings and dams. The various age groups (3, 21, 40, 55, 70 and 120 days) each consisted of 6 nu/nu- and 6 heterozygous nu/(+)-mice respectively. The morphological examination of the mammary gland tissue were made on histological sections and whole mounts. Body weights, total areas of the mammary glands and the number of the terminal end buds were compared. The mammary gland of the athymic nude mouse exhibited no essential morphological differences from the normal developing mammary gland of the hairy euthymic nu/(+)-animal. The area of the mammary gland increased with increasing body weight. Both collectives of mice differed only in their rate of mammary gland development. As a result, the terminal end buds appeared numerously as growth points of mammary gland in nu/(+)-animals as early as the 21st day of life. The athymic nude mice showed a maximum only on the 40th day of life and a lower degree of density and differentiation of specific mammary gland structures (lateral buds, lobulo-alveolar glandular endings) until the 70th day of life. The mammary gland of 120-day-old animals and dams of both animal groups reached the same state of maturity. Thus it is not the rate of development of the dam, but other, yet unidentified factors, which determine, if successful breeding of nude mice with homozygous parents is possible.

Methionine-enkephalin immunoreactivity in Merkel cell dense-core granules of nude mice sinus hair. A post-embedding immunogold electron-microscopic study.

The ultra-immunocytochemical technique applied in the present study revealed the occurrence of methionine-enkephalin (met-enkephalin)-like substance in the dense-core granules of Merkel cells of nude mice sinus hair. Incubation of ultra-thin sections of sinus hair with met-enkephalin antisera conjugated with gold particles showed specific association of gold particles on the dense-core granules of the Merkel cells. Gold particles were heavily and specifically located on the dense-core granules as well as in the adjacent cytoplasm. Dense-core granules of degenerating Merkel cells also exhibit met-enkephalin immunoreactivity. The nerve terminals associated with the Merkel cell did not show met-enkephalin immunoreactivity. Therefore, it is concluded that a met-enkephalin-like substance is present and stored in nude mice Merkel cell dense-core granules and it might act as a neurotransmitter or neuromodulator which could be involved in the functioning of the cell. Non-osmicated tissue should be used to locate this substance because of the possibility of cross-linkage of the amino acid sequence with osmium tetroxide.

Ossification in nude mice. 1. Macroscopical study.

Ossification was studied in cleared fetuses, newborns, 1- and 6-week-old nu/nu and nu/+ mice of the B 10 LP background. The same ossification pattern was observed in nu/nu and nu/+ in the embryonal period as well as in newborn animals and mice aged 1 or 6 weeks. On the other hand, 6-week-old nu/nu mice exhibited a lower X-ray density of the skeleton and a lower thickness of the proximal tibial growth plate than 6-week-old nu/+ litter mates. The results indicate the influence of postnatal factors on the skeletal development of nu/nu mice.

Vascularization of corneas of hairless mutant mice.

During the course of experiments examining the immunobiology of corneal transplants, the corneas of athymic, nude mice (nu/nu) were found to contain blood vessels that extended through the entire superficial stroma into the centermost portion of the cornea. The presence of corneal vessels was not related to the immunodeficient condition of the nude mouse since corneas from the severe combined immunodeficiency (SCID) mutant mouse strain were avascular and indistinguishable from corneas obtained from immunocompetent BALB/c mice. Furthermore, Langerhans cells were not found to accompany the blood vessels in the corneas of any of the nude mice examined. Corneal vascularization that was similar to that seen in the nude mouse was found in the cuthymic, hairless mutant mouse strain (SKH1; hr/hr). Although vascularization of the corneal stroma was associated with the heritable loss of hair, the genes responsible for hair loss in these two mutant mouse strains reside on different chromosomes. Understanding the processes involved in either promoting or preventing corneal vascularization may have significant impact in preventing corneal allograft rejection and in controlling inflammatory diseases of the corneal surface. The two mutant mouse strains described here may serve as valuable tools for such investigations.

Phenotype of intraepithelial lymphocytes in euthymic and athymic mice: implications for differentiation of cells bearing a CD3-associated gamma delta T cell receptor.

This study was carried out to determine the exact phenotype of intraepithelial lymphocytes (IEL) in euthymic and athymic nude mice. The phenotype of IEL in euthymic and athymic mice is mainly CD3+CD8+. However, based on Thy-1- and CD3-associated receptor expression we can subdivide the CD3CD8 population into different subpopulations in euthymic and athymic mice. In euthymic and athymic mice several CD3CD8 populations can be defined. One population expressing Thy-1 and the T cell receptor (TcR) alpha beta is absent in athymic mice. Two other CD3+CD8+ populations can be detected in euthymic and athymic mice. Based on Northern blot and flow cytometric analysis we have to conclude that these populations express the CD3-associated TcR gamma delta. One of the TcR gamma delta-expressing populations also expresses Thy-1 at low surface density. This is in contrast to the CD3CD8 population expressing the TcR alpha beta, which expresses Thy-1 at high surface density. There are also, however, especially in athymic nude mice, significant numbers of CD3-CD8+ cells present with the same localization as IEL. The function of these cells is yet unknown. Using a probe for the delta chain we have shown that IEL preferentially express 2-kb mRNA, while nearly no delta chain 1.7-kb mRNA is expressed by these cells. This is in contrast to delta mRNA in thymocytes. Equal quantities of the 1.7- and 2.0-kb delta chain mRNA species were found in RNA isolated from thymocytes. The results imply that CD3+CD8+ intestinal IEL expressing the CD3-associated TcR gamma delta can differentiate in absence of the thymus and represent a thymus-independent lineage of cells bearing this receptor.

Nude mice are not hairless. A morphological study.

In the present study, the morphological aspect of the skin and the hairs of athymic, macroscopically nude mice (NMRI, nu/nu) was investigated by descriptive light- and electron-microscopical methods and compared with the appearance of the skin and the hairs in normally haired mice (NMRI). These morphological studies revealed that athymic, macroscopically nude mice are not at all hairless, but have about the same number of hair bulbs, embedded in the hypodermis, as normally haired animals. However, within the hair follicles of athymic mice, the keratinization processes are obviously deeply impaired, resulting in the formation of short, crippled and bent hair shafts which only seldom emerge from the hair follicles. The cuticles of the inner root sheath and the hair are not built up, the cortex of the hair being composed by abnormal globular aggregates. The epidermis shows similar disturbances of keratinization, which are reflected by the presence of only few and thin bundles of tonofilaments in the basal, spinous and granular layers of the epidermis and, in the stratum corneum, by bizarrely formed and irregularly arranged lamellae of corneocytes, separated from one another. These results demonstrate that athymic, nude mice are not hairless but that the development and differentiation of hairs are severely injured in this mouse mutant. Analogously, the keratinization of the epidermis is also impaired. In view of the previously shown ectodermal defect as primary cause for the dysgenesis of the thymus, it seems to be probable that defects of the ectoderm are actually the common reason for both the thymus dysgenesis and the severe disturbances of hair development in 'athymic, nude' mice.

Changes in histological differentiation of human tumors transplanted to athymic nude mice: a morphometric study.

Differing conclusions have been reached regarding the phenotypic stability of human tumors transplanted to athymic nude mice. Since previous conflicting studies of tumor histology have been largely subjective, quantitative methodology was applied to an analysis of 13 human adenocarcinoma tumor lines that were originally derived directly from surgical specimens. Glandular differentiation was quantitated, both in the original human tumor (OHT) and in a minimum of 6 serial passages of the nude mouse-grown tumors (MGT), by means of point counting. A significant change in differentiation was noted in 12 lines, with 9 showing a decrease. Variance from the OHT was most commonly noted in the initial MGT, but additional changes were also noted in 8 lines during subsequent passages. Most of the lines also showed increased necrosis in the MGTs. Since histological differentiation and necrosis are related to tumor aggressiveness, it would appear that the predominant tendency was to evolve toward a more malignant phenotype. These changes may mimic those seen in human tumor metastases.

Structural and cell population changes in the lymph nodes of the athymic nude mouse.

A recent tridimensional analysis of the lymph node demonstrated that its deep cortex is composed of grossly hemispherical "units," adjoining a portion of its peripheral cortex. Each deep cortex unit can be distinguished into a center and a periphery. The periphery was concluded to be a site for migration of circulating lymphocytes, the center, a site where T cells would participate in cellular immune responses. The aim of the present work was to determine the influence of the congenital athymic state on the development of the units and of other components in the lymph nodes of the nude mouse. For this, the lymph nodes at various anatomical locations in adult athymic nude mice were analyzed. The present study revealed that the athymic state did not inhibit the development of the units but severely depleted the lymphocyte population of their center only. However, it did inhibit the development of an area of peripheral cortex located over the middle part of a unit. Such an area of peripheral cortex is, thus, concluded to be thymus dependent, as is the center of a deep cortex unit. The athymic state also prevented the development of the cells of the nodules (germinal centers) and of much of the plasmocytes. On the other hand, it yielded to the enlargement of the follicles, the formation of new structures: medullary "lymphocyte clusters" and the transformation of the medullary venules into high endothelial venules. The various modifications of the nodal structures resulting from the congenital athymic state are discussed in relation to some functions of the organ.

Pituitary and cerebellum of nude mice.

The anterior pituitary of athymic homozygous "nude" (nu nu) mice shows signs of reduced activity of the acidophilic cells, nuclear volumes of which lag significantly behind normal values. Also there is a significant deficiency in the number of basophilic cells. A similar deficiency is found in the numbers of Purkinje cells of the cerebellum, which in addition also show a reduction in size, as part of an overall reduction of cerebellum size.

Preferential sites of growth of human tumors in nude mice following subcutaneous transplantation.

The growth characteristics and biological behavior of human tumors transplanted s.c. into two different anatomical regions of nude mice were studied. It was observed that tumors trnasplanted in the anterior lateral thoracic wall grew faster than did tumors transplanted in the posterior aspect of the trunk. Anteriorly growing tumors, in contrast to the posteriorly transplanted ones, were better vascularized, showed less necrosis, invaded the tumor bed, and metastasized to the regional lymph nodes. These findings were independent of their histogenetic and morphological characteristics. It is concluded that regional vascular supply is a key factor influencing the biological behavior of the transplanted tumors and that it may affect tumor response to treatment as well.