A simultaneous ex vivo model of embryogenesis: I. Organogenesis.

In vivo culture of chick embryo was carried out to develop an experimental interphase between in vitro and in vivo study of embryonic physiology. In the process, a simultaneous model of vasculogenesis and organogenesis has been worked out, which is impossible to achieve in mammalian system. Both early (40 hours of incubation) and late (64 & 88 hours of incubation) hours of cultures were conducted for morphological and morphometric studies. A new combination of stains was used in place of conventional haematoxylin and eosin in 40 hours old whole-mount of embryos. Semithin plastic sections were etched for haematoxylin/pyronin stain in addition to paraffin (both normal and enblock) sections. Specific stains (histological, enzyme histochemical or immunohistochemical) were chosen according to the specific organs/areas studied. Immunohistochemistry and NADPH-diaphorase activity were standardized in whole-mount of embryos. Morphometry was done using camera lucida and quantitative image analysis system. A parallel preparation of extra embryonic whole-mounts, paraffin and semithin plastic sections with different types of stainings provides evidence for the scope of the simultaneous study of vasculogenesis. Thus the morphological and morphometric data presented in this and the succeeding article describe the scope and avenues for the use of ex vivo model in various aspects of embryonic physiology, preliminary drug trials/metabolism, radiology, teratology and toxicology.

A simultaneous ex vivo model of embryogenesis: II. Vasculogenesis.

Vasculogenesis was simultaneously studied with embryogenesis in in ovo chick embryo culture, which was harvested at 40 hours. Endodermal cells and vascular endothelial cells were studied using a new combination of stains, immunohistochemistry (for nuclei and basement membrane) and NADPH-diaphorase activity in whole-mounts, paraffin sections and etched semithin sections. The model can be used for the study of developmental process of blood vessels as well as embryonic physiology of blood vessels vis-a-vis organogenesis in response to different angiogenic agents, drug trials, cancer therapy by angiostatic chemicals/radiations and toxins. Considering that vasculogenesis/angiogenesis as one of the fundamental phenomena in physiology, pathophysiology, toxicology and pharmacology of developmental sciences, the model in developing embryo is presented.

UV-dependent melanocyte plasticity--the structure-function relationship.

The UV response of marginal melanocytes in vitiliginous skin was studied using a whole skin organ culture technique. This method assesses the plasticity of melanocytes in response to UV. It is observed that there is a sequential increase in catecholoxidase production and in the volume and dendricity of the melanocytes on exposure to a single pulse of UV, reaching a peak at 3 1/2 h. From this study it is evident that the melanocyte shows a prominent structural and functional plasticity in response to UV. Implicit is the utilisation and transduction of UV energy by the melanocyte, for transcriptional and translational activity, enhancing catecholoxidase and cell structural protein production.

ACTH acts directly on melanocytes to stimulate melanogenesis--an in vitro study.

A total of 108 whole skin organ cultures taken from vitiliginous skin were incubated in MEM containing ACTH. It was observed that 53.7% that is 58 showed a positive response with an increase in pigment production and enzyme activity, as observed on frozen sections stained for dopaoxidase activity. On immunohistochemical staining for locating ACTH binding, it is observed that 27.3% control skin and 72.7% ACTH treated skin show positivity. The ACTH is seen to bind with the melanocyte membrane as well as the cytoplasm. This indicates that ACTH can bind to the MSH-receptors expressed by the melanocyte. Thus, ACTH acts directly on the melanocyte to enhance melanogenesis and does not require to act via the adrenal-pituitary axis. This also indicates that the response is not associated with immune suppression by ACTH.

Aspirin by virtue of its acidic property may act as teratogen in early chick embryo.

Aspirin (acetylsalicyclic acid) was dissolved either in normal saline or in phosphate buffer and was used in two doses to find out whether teratogenic potential of aspirin in chick blastoderm model is due to its acidic property or due to drug action. Drug was injected sub-blastodermally by window technique in fresh embryonated eggs after 17 hours of incubation at 39 degrees C. Eggs were re-incubated and harvested at 40 hours. Normal development of embryos was seen with normal saline and percentage of normal embryos with 30 micrograms (pH-3.19) and 120 micrograms (pH-2.64) aspirin was 31.7 and 4.9 respectively. Buffer produced 80.8% normal embryos and buffered 30 micrograms (pH-6.87) and 120 micrograms (pH-6.69) aspirin produced 67.7% and 30.8% normal embryos respectively. Changing the pH of aspirin to near neutral decreased the defect induced by aspirin but a significant effect of aspirin was observed at higher dose which could be independent of pH action.

Mechanism of aspirin induced neural tube defect in chick embryo.

The effect of acetyl salicylic acid (aspirin) on neural tube development in chick embryo was studied, using the chick embryo blastoderm model. Aspirin was injected in four different doses sub-blastodermally into fresh embryonated eggs. The role of PGE1 and PGE2 alpha in the defect induced by aspirin on neural tube development in chick embryo was studied. PGE1 (5 micrograms) given after aspirin (30 micrograms) treatment was found to produce greater defect in development. All the four doses of aspirin used (i.e., 6, 30, 60 and 120 micrograms/embryo) produced significant changes (P < 0.01) in the neural tube development of chick embryo. Pre-treatment with PGE1 did not modify the defect induced by aspirin, whereas pre-treatment with PGF2 alpha prevented neural tube defects induced by aspirin. It appears that aspirin (in the doses used) affects neural tube formation by decreasing PGF2 alpha synthesis in chick embryo blastoderm.

Hair cycle and the histogenesis of pillar tumours.

In a study of 50 tumours of pilar origin, it was observed that tumours arise from each cell type depending upon the phase of the hair cycle. Thus nevoid lesions arise from the pluripotent cells of the early anagen phase; tumours from the hair matrix, outer root sheath and inner root sheath arise during the anagen phase, and the keratoacanthomas during the interphase between anagen and telogen. Basal cell epitheliomas can arise at any phase. It was observed that the tumours arising during the anagen phase from the fully differentiated follicles formed the bulk (88%) and the keratoacanthoma simulating the catagen/telogen phase was rare in consonance with the length of each phase. It is proposed that the inner root sheath tumours be named trichilemmomas and the outer root sheath tumours trichochlamydomas to distinguish them from one another.