Long-term corticosteroid-induced chronic glaucoma model produced by intracameral injection of dexamethasone-loaded PLGA microspheres.

PURPOSE: To evaluate a new chronic glaucoma model produced by intracameral injection of dexamethasone-loaded poly lactic-co-glycolic acid microspheres (Dex-PLGA-Ms) over six months. METHODS: Healthy rats received two injections (at baseline and Week 4) of Dex-PLGA-Ms into the anterior chamber of the right eye. Clinical signs and intraocular pressure (IOP) were weekly recorded. The structure of the retina and optic nerve was in vivo evaluated using optical coherence tomography (OCT) every two weeks and functionally using dark- and light-adapted electroretinography at 0-12-24 weeks. Histological studies were also performed. RESULTS: IOP progressively increased up to hypertension (23.22 ± 3.63 mmHg) in both eyes but did so later in left eyes. OCT quantified a decrease in full-thickness retina posterior pole (R), retinal-nerve-fiber layer (RNFL), and ganglion-cell layer (GCL) thickness up to 24 weeks. Right eyes showed higher neuroretinal thickness loss up to week 8. RNFL experienced the highest percentage thickness loss at the inferior-superior axis, while in GCL the inner sectors of the horizontal axis (Nasal-Temporal) suffered the greatest decrease in thickness. Retinal ganglion cell, photoreceptor, and intermediate cell functionality decreased over time. Increased deposition of collagen IV was also found in zonular fibers and the ciliary body. CONCLUSIONS: This work shows the usefulness of drug delivery systems, not to treat pathology but to induce it. Only two injections of Dex-PLGA-Ms in the anterior chamber of rat eyes were enough to progressively create ocular hypertension and subsequent functional and structural neuroretinal degeneration, at least over 6 months.

Brimonidine-LAPONITE® intravitreal formulation has an ocular hypotensive and neuroprotective effect throughout 6 months of follow-up in a glaucoma animal model.

Intravitreal administration is widely used in ophthalmological practice to maintain therapeutic drug levels near the neuroretina and because drug delivery systems are necessary to avoid reinjections and sight-threatening side effects. However, currently there is no intravitreal treatment for glaucoma. The brimonidine-LAPONITE® formulation was created with the aim of treating glaucoma for extended periods with a single intravitreal injection. Glaucoma was induced by producing ocular hypertension in two rat cohorts: [BRI-LAP] and [non-bri], with and without treatment, respectively. Eyes treated with brimonidine-LAPONITE® showed lower ocular pressure levels up to week 8 (p < 0.001), functional neuroprotection explored by scotopic and photopic negative response electroretinography (p = 0.042), and structural protection of the retina, retinal nerve fibre layer and ganglion cell layer (p = 0.038), especially on the superior-inferior axis explored by optical coherence tomography, which was corroborated by a higher retinal ganglion cell count (p = 0.040) using immunohistochemistry (Brn3a antibody) up to the end of the study (week 24). Furthermore, delayed neuroprotection was detected in the contralateral eye. Brimonidine was detected in treated rat eyes for up to 6 months. Brimonidine-LAPONITE® seems to be a potential sustained-delivery intravitreal drug for glaucoma treatment.

ΔNp63α promotes adhesion of metastatic prostate cancer cells to the bone through regulation of CD82.

ΔNp63α is a critical mediator of epithelial development and stem cell function in a variety of tissues including the skin and breast, while overexpression of ΔNp63α acts as an oncogene to drive tumor formation and cancer stem cell properties in squamous cell carcinoma. However, with regards to the prostate, while ΔNp63α is expressed in the basal stem cells of the mature gland, during adenocarcinoma development, its expression is lost and its absence is used to clinically diagnose the malignant state. Surprisingly, here we identify a sub-population of bone metastatic prostate cancer cells in the PC3 cell line that express ΔNp63α. Interestingly, we discovered that ΔNp63α favors adhesion and stem-like growth of these cells in the bone microenvironment. In addition, we show that these properties require expression of the target gene CD82. Together, this work uncovers a population of bone metastatic prostate cancer cells that express ΔNp63α, and provides important information about the mechanisms of bone metastatic colonization. Finally, we identify metastasis-promoting properties for the tetraspanin family member CD82.

Imaging of cellular aging in human retinal blood vessels.

To date two main aging vascular lesions have been reported in elderly human retinas: acellular capillaries and microaneurysms. However, their exact mechanism of formation remains unclear. Using high resolution microscopy techniques we revise cellular alterations observed in aged human retinal vessels, such as lipofuscin accumulation, caveolae malfunction, blood basement membrane disruption and enhanced apoptosis that could trigger the development of these aging vascular lesions. Moreover, we have generated a set of original images comparing retinal vasculature between middle and old aged healthy humans to show in a comprehensive manner the main structural and ultrastructural alterations occurred during age in retinal blood vessels.

Experimental diabetes in neonatal mice induces early peripheral sensorimotor neuropathy.

Animal models of diabetes do not reach the severity of human diabetic neuropathy but relatively mild neurophysiological deficits and minor morphometric changes. The lack of degenerative neuropathy in diabetic rodent models seems to be a consequence of the shorter length of the axons or the shorter animal life span. Diabetes-induced demyelination needs many weeks or even months before it can be evident by morphometrical analysis. In mice myelination of the peripheral nervous system starts at the prenatal period and it is complete several days after birth. Here we induced experimental diabetes to neonatal mice and we evaluated its effect on the peripheral nerve 4 and 8 weeks after diabetes induction. Neurophysiological values showed a decline in sensory nerve conduction velocity at both time-points. Morphometrical analysis of the tibial nerve demonstrated a decrease in the number of myelinated fibers, fiber size and myelin thickness at both time-points studied. Moreover, aldose reductase and poly(ADP-ribose) polymerase activities were increased even if the amount of the enzyme was not affected. Thus, type 1 diabetes in newborn mice induces early peripheral neuropathy and may be a good model to assay pharmacological or gene therapy strategies to treat diabetic neuropathy.

Mimicking microvascular alterations of human diabetic retinopathy: a challenge for the mouse models.

Although it has become acceptable that neuroretinal cells are also affected in diabetes, vascular lesions continue to be considered as the hallmarks of diabetic retinopathy. Animal models are essential for the understanding and treatment of human diabetic retinopathy, and the mouse is intensively used as a model because of its similarity to human and the possibility to be genetically modified. However, until today not all retinal vascular lesions developed in diabetic patients have been reproduced in diabetic mice, and the reasons for this are not completely understood. In this review, we will summarize retinal vascular lesions found in diabetic and diabetic-like mouse models and its comparison to human lesions. The goal is to provide insights to better understand human and mice differences and thus, to facilitate the development of new mouse models that mimic better human diabetic retinopathy.

Dilated cardiomyopathy and mitochondrial dysfunction in Sirt1-deficient mice: a role for Sirt1-Mef2 in adult heart.

The deacetylase Sirtuin-1 (Sirt1) is involved in the cardiac hypertrophic responses and cardiac embryo morphogenesis. However, the physiological function of Sirt1 deficiency in the postnatal development of the heart remains to be characterized. The aim of the study was to investigate the relevance of Sirt1 in the development and function of the myocardium. Hearts from Sirt1-deficient mice partially or totally lacking Sirt1 protein activity were analyzed. Loss of Sirt1 activity led to dilated cardiomyopathy in adult hearts, a phenotype accompanied by reduced cardiomyocyte size and the absence of fibrosis. Morphological and functional mitochondrial abnormalities were observed in the adult hearts lacking Sirt1, suggesting that mitochondrial dysfunction contributes to the progression of the observed cardiomyopathy. Moreover, gene expression analyses revealed that mitochondrial genes were the most affected in Sirt1-deficient mice, showing a reduction in their expression. No overt cardiac dilatation was observed in neonates lacking Sirt1 activity, but first signs of mitochondrial alterations were already present. Immunoblot analyses revealed that Sirt1 is highly expressed in the heart after birth, indicating the importance of Sirt1 in the neonatal period. Finally, Sirt1 deficiency affected the acetylation pattern of the myocyte enhancer factor 2 (Mef2) transcription factors, which are critical for normal heart development and mitochondrial integrity. Collectively, our findings indicate that Sirt1 is essential for the maintenance of cardiac mitochondrial integrity and normal postnatal myocardium development.

Chronically increased glucose uptake by adipose tissue leads to lactate production and improved insulin sensitivity rather than obesity in the mouse.

AIMS/HYPOTHESIS: In adipocytes, triacylglycerol synthesis depends on the formation of glycerol 3-phosphate, which originates either from glucose, through glycolysis, or from lactate, through glyceroneogenesis. However, glucose is traditionally viewed as the main precursor of the glycerol backbone and thus, enhanced glucose uptake would be expected to result in increased triacylglycerol synthesis and contribute to obesity. METHODS: To further explore this issue, we generated a mouse model with chronically increased glucose uptake in adipose tissue by expressing Gck, which encodes the glucokinase enzyme. RESULTS: Here we show that the production of high levels of glucokinase led to increased adipose tissue glucose uptake and lactate production, improved glucose tolerance and higher whole-body and skeletal muscle insulin sensitivity. There was no parallel increase in glycerol 3-phosphate synthesis in vivo, fat accumulation or obesity. Moreover, at high glucose concentrations, in cultured fat cells overproducing glucokinase, glycerol 3-phosphate synthesis from pyruvate decreased, while glyceroneogenesis increased in fat cells overproducing hexokinase II. CONCLUSIONS/INTERPRETATIONS: These findings indicate that the absence of glucokinase inhibition by glucose 6-phosphate probably led to increased glycolysis and blocked glyceroneogenesis in the mouse model. Furthermore, this study suggests that under physiological conditions, when blood glucose increases, glyceroneogenesis may prevail over glycolysis for triacylglycerol formation because of the inhibition of hexokinase II by glucose 6-phosphate. Together these results point to the indirect pathway (glucose to lactate to glycerol 3-phosphate) being key for fat deposition in adipose tissue.

The suppressor of cytokine signalling 2 (SOCS2) is a key repressor of insulin secretion.

AIMS/HYPOTHESIS: Suppressor of cytokine signalling (SOCS) proteins are powerful inhibitors of pathways involved in survival and function of pancreatic beta cells. Whereas SOCS1 and SOCS3 have been involved in immune and inflammatory processes, respectively, in beta cells, nothing is known about SOCS2 implication in the pancreas. METHODS: Transgenic (tg) mice were generated that constitutively produced SOCS2 in beta cells (betaSOCS2) to define whether this protein is implicated in beta cell functioning and/or survival. RESULTS: Constitutive production of SOCS2 in beta cells leads to hyperglycaemia and glucose intolerance. This phenotype is not a consequence of decreased beta cell mass or inhibition of insulin synthesis. However, insulin secretion to various secretagogues is profoundly altered in intact animals and isolated islets. Interestingly, constitutive SOCS2 production dampens the rise in cytosolic free calcium concentration induced by glucose, while glucose metabolism is unchanged. Moreover, tg islets have a depletion in endoplasmic reticulum Ca(2+) stores, suggesting that SOCS2 interferes with calcium fluxes. Finally, in betaSOCS2 mice proinsulin maturation is impaired, leading to an altered structure of insulin secretory granules and augmented levels of proinsulin. The latter is likely to be due to decreased production of prohormone convertase 1 (PC1/3), which plays a key role in proinsulin cleavage. CONCLUSIONS/INTERPRETATIONS: SOCS2 was shown to be a potent regulator of proinsulin processing and insulin secretion in beta cells. While its constitutive production is insufficient to induce overt diabetes in this mouse model, it causes glucose intolerance. Thus, increased SOCS2 production could be an important event predisposing to beta cell failure.

Constitutive expression of suppressor of cytokine signalling-3 in skeletal muscle leads to reduced mobility and overweight in mice.

AIMS/HYPOTHESIS: Due to their ability to regulate various signalling pathways (cytokines, hormones, growth factors), the suppressor of cytokine signalling (SOCS) proteins are thought to be promising therapeutic targets for metabolic and inflammatory disorders. Hence, their role in vivo has to be precisely determined. METHODS: We generated transgenic mice constitutively producing SOCS-3 in skeletal muscle to define whether the sole abundance of SOCS-3 is sufficient to induce metabolic disorders and whether SOCS-3 is implicated in physiological roles distinct from metabolism. RESULTS: We demonstrate here that chronic expression of SOCS-3 in skeletal muscle leads to overweight in mice and worsening of high-fat diet-induced systemic insulin resistance. Counter-intuitively, insulin sensitivity in muscle of transgenic mice appears to be unaltered. However, following constitutive SOCS-3 production, several genes had deregulated expression, among them other members of the SOCS family. This could maintain the insulin signal into skeletal muscle. Interestingly, we found that SOCS-3 interacts with calcineurin, which has been implicated in muscle contractility. In Socs-3 transgenic muscle, this leads to delocalisation of calcineurin to the fibre periphery. Relevant to this finding, Socs-3 transgenic animals had dilatation of the sarcoplasmic reticulum associated with swollen mitochondria and decreased voluntary activity. CONCLUSIONS/INTERPRETATION: Our results show that constitutive SOCS-3 production in skeletal muscle is not in itself sufficient to induce the establishment of metabolic disorders such as diabetes. In contrast, we reveal a novel role of SOCS-3, which appears to be important for muscle integrity and locomotor activity.

IGF-I mediates regeneration of endocrine pancreas by increasing beta cell replication through cell cycle protein modulation in mice.

AIMS/HYPOTHESIS: Recovery from diabetes requires restoration of beta cell mass. Igf1 expression in beta cells of transgenic mice regenerates the endocrine pancreas during type 1 diabetes. However, the IGF-I-mediated mechanism(s) restoring beta cell mass are not fully understood. Here, we examined the contribution of pre-existing beta cell proliferation and transdifferentiation of progenitor cells from bone marrow in IGF-I-induced islet regeneration. METHODS: Streptozotocin (STZ)-treated Igf1-expressing transgenic mice transplanted with green fluorescent protein (GFP)-expressing bone marrow cells were used. Bone marrow cell transdifferentiation and beta cell replication were measured by GFP/insulin and by the antigen identified by monoclonal antibody Ki67/insulin immunostaining of pancreatic sections respectively. Key cell cycle proteins were measured by western blot, quantitative RT-PCR and immunohistochemistry. RESULTS: Despite elevated IGF-I production, recruitment and differentiation of bone marrow cells to beta cells was not increased either in healthy or STZ-treated transgenic mice. In contrast, after STZ treatment, IGF-I overproduction decreased beta cell apoptosis and increased beta cell replication by modulating key cell cycle proteins. Decreased nuclear levels of cyclin-dependent kinase inhibitor 1B (p27) and increased nuclear localisation of cyclin-dependent kinase (CDK)-4 were consistent with increased beta cell proliferation. However, islet expression of cyclin D1 increased only after STZ treatment. In contrast, higher levels of cyclin-dependent kinase inhibitor 1A (p21) were detected in islets from non-STZ-treated transgenic mice. CONCLUSIONS/INTERPRETATION: These findings indicate that IGF-I modulates cell cycle proteins and increases replication of pre-existing beta cells after damage. Therefore, our study suggests that local production of IGF-I may be a safe approach to regenerate endocrine pancreas to reverse diabetes.

Overexpression of Il6 leads to hyperinsulinaemia, liver inflammation and reduced body weight in mice.

AIMS/HYPOTHESIS: IL-6 is released by the adipose tissue and increased circulating levels in obesity are associated with hyperinsulinaemia and insulin resistance. Short-term experiments suggest that increased IL-6 release by the skeletal muscle following exercise may improve insulin sensitivity. METHODS: In order to examine the effect of chronically elevated IL-6 levels, we overexpressed Il6 in skeletal muscle in mice using an electro-transfer procedure. RESULTS: Circulating IL-6 levels were increased and the animals rapidly lost both weight and body fat, but food intake was unchanged, which is consistent with the finding that IL-6 increased energy expenditure. Insulin levels were inappropriately elevated and combined with hypoglycaemia in spite of reduced 2-deoxy-D: -glucose uptake by skeletal muscle. Insulin-stimulated glucose uptake by skeletal muscles ex vivo was reduced, probably due to the decreased amounts of glucose transporter (GLUT)-4. Beta cell insulin content was increased, while apparent beta cell mass was unchanged. Circulating serum amyloid A cluster levels were increased tenfold due to a pronounced proinflammatory state in the liver with infiltration of inflammatory cells. However, no liver steatosis was found, which may be accounted for by concomitant AMP kinase activation. CONCLUSIONS/INTERPRETATION: Chronically elevated IL-6 levels lead to inappropriate hyperinsulinaemia, reduced body weight, impaired insulin-stimulated glucose uptake by the skeletal muscles and marked inflammation in the liver. Thus, the pleiotrophic effects of chronically elevated IL-6 levels preclude any obvious usefulness in treating obesity or its associated metabolic complications in man, despite the fact that weight reduction may be expected.

Mophometric study of the aortic arch and its major branches in rat fetuses on the 21st day of gestation.

The anatomy and embryology of the aortic arch and its branching tributaries (brachiocephalic trunk, left common carotid artery and left subclavian artery) in man and animals are well substantiated. However, the anatomical variations and morphometry of the aortic arch and its branching tributaries in rat fetus at the 21st gestation day have not been studied. Pregnant rats were hysterectomized and the arterial systems of 114 fetuses were injected with a polymerisable resin through the umbilical artery. After maceration, the vascular casts were dissected out and prepared for observations under a scanning electron microscope (SEM). The resulting SEM pictures were studied with a picture analyser and different vessel parameters (diameters, lengths and angles) were measured. The success rate of the microvascular cast injection was 46.5%. Out of the 53 observed aortic arch casts, 98.1% showed the classical branching pattern and one (1.9%) had no brachiocephalic trunk. Morphological analysis showed many differences, which were not linked to the litter. The statistical processing of the measurements enabled us to determine that the aorta diameter after the branching of the left subclavian artery was the most replicable parameter. Moreover, the results revealed some strong correlations between different parameters. There are probably no discrete categories among the various observed parameters as diameters and angles. Some parameters show very little variability and can thus be used as reference points for further studies such as the comparison of a control population with a population treated with a relevant xenobiotic.

Albendazole sulphoxide enantiomers in pregnant rats' embryo concentrations and developmental toxicity.

Three single oral doses (8.5, 10, and 14 mg/kg) of a racemic formulation of albendazole sulphoxide (ABZSO) were administered to pregnant rats on day 10 of gestation. Mother plasma and embryo concentrations of ABZSO enantiomers and albendazole sulphone (ABZSO(2)) were determined 9 h after administration. The (-)-ABZSO enantiomer showed higher peak concentrations in both maternal plasma and embryo than the (+) enantiomer. An increase in embryo concentrations of ABZSO enantiomers and ABZSO(2) was only observed when dose rose to 14 mg/kg. There was an increase in resorption when the dose increased, but significant differences were only found in the higher dose group when compared with the other groups. The incidence of external and skeletal malformations (mostly of the tail, vertebrae and ribs) rose significantly in the 10 mg/kg group, producing almost 20% and 90% of malformed fetuses, respectively, and gross external and skeletal abnormalities in the thoracic region and limbs were also found.

Vasculogenesis and angiogenesis in the subcardinal venous plexus of quail mesonephros: spatial and temporal morphological analysis.

Vasculogenesis and angiogenesis are involved in a coordinated program for the development of the mesonephric subcardinal venous plexus of quail embryo. Vasculogenesis occurs between days 3 and 4 of incubation, while angiogenesis takes place from day 5 to day 7. Examination of vascular corrosion casts and whole mounts, and tissue sections labelled with specific markers to hemangioblast lineage (QH1, LEP100 and AcPase activity), allowed us to distinguish six phases in the formation of subcardinal plexus. (1) Appearance of isolated angioblast-like cells where the subcardinal plexus will form. (2) Alignment of angioblast-like cells into cellular strands. (3) Formation of compact vascular cords by association of angioblast-like strands. (4) Polygonal interconnection of vascular cords to constitute the primary subcardinal plexus. In this stage, isolated angioblast-like cells were present inside inter-vascular spaces. (5) The splitting of primary inter-vascular spaces by angiogenic sprouts to form secondary subcardinal plexus (outward angiogenesis). Isolated angioblast-like cells were not present in this stage. (6) Expansion of the secondary subcardinal plexus by insertion of slender transcapillary tissue pillars (inward angiogenesis) and angiogenic sprouts. We also describe three morphogenetic gradients during the development of the subcardinal plexus: ventral-to-dorsal, cranial-to-caudal and lateral-to-medial.

Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin.

Netobimin (NTB) is a prodrug of albendazole (ABZ) and is used as a broad-spectrum anthelmintic both in human and veterinary medicine. Pregnant Sprague-Dawley rats were treated po with 50, 59.5 and 70.7 mg/kg of NTB on Gestational Day (GD) 10. The results, observed on GD 20, demonstrated that NTB induced a significant increase of resorptions. Moreover, decreased fetal body weight and an increase in skeletal malformations were observed in treated groups. We report the first study in which vascular malformations are described in rats after the administration of a benzimidazole compound. An interesting relationship between intercostal vessel and rib malformations was found.

The lack of genital ridge vascularization in the early chick embryo: implications in the migration of the primordial germ cells.

It is known that chick primordial germ cells (PGCs) in early embryonic development migrate via the blood vascular system to colonize the gonadal anlagen. Classically, two factors have been involved in the extravasation of PGCs from the blood stream: chemotactic and mechanical factors. An accurate knowledge of the vascular system of the genital ridge is therefore necessary. However, development of gonadal vascularization in bird embryos has been scarcely studied. Our previous studies have shown that the gonadal arteries develop from the mesonephric arteries. The purpose of this work was to study the implications of the development of the vascular system of the chick genital ridge on PGCs colonization. We selected the Hamburger and Hamilton (H-H) stage 18, since the genital ridge is well developed and PGCs actively extravasate. Forty chick embryos of this stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point-dried specimens as well as light microscopy. Our results are conclusive. We could not find any vessel or capillary network supplying the genital ridge; the dorsal aorta and the primordia of the mesonephric arteries were the closest vessels. However, numerous interendothelial spaces were found in the dorsal aorta at the level of the genital ridge. It is suggested that the interendothelial gaps may be very important in the exchange of substances between the avascular genital ridge and the aortic endothelium at this developmental stage. Two different routes are thought to be involved in PGC migration to the gonadal anlage at this stage: the aortic endothelium and the mesonephric arteries. Whereas mechanical factors may be important for extravasation of PGCs in the mesonephric arteries, no reasons have been found from the morphological point of view to support a slowness of the blood flow in the dorsal aorta at the level of the genital ridge facilitating the extravasation.

Injection technique and scanning electron microscopic study of the arterial pattern of the 20 gestation days (G20) rat fetus.

A technique to obtain microvascular corrosion casts of the G20 rat fetus and the normal pattern of the main arteries of the G20 rat fetus are described. The casts were studied by means of scanning electron microscopy (SEM). The arterial pattern is similar to that described in the adult; however, several variations have been found. It is concluded that the use of vascular corrosion casts studied by SEM may be particularly helpful to observe the extremely small arteries of rat fetuses. Moreover, we suggest that this technique may be useful in practical teratological studies.

Anthelmintic induced congenital malformations in sheep embryos using netobimin.

Benzimidazole compounds have teratogenic effects in domestic and experimental animals. In this study, 14 Manchega ewes were treated orally, under controlled conditions, with 20 mg netobimin (a prodrug of a benzimidazole compound) per/kg bodyweight on the 17th day of pregnancy. Congenital malformations and abortions affected 60 per cent of the lambs. The main malformations were skeletal and renal, but vascular malformations were observed for the first time. The abnormalities were investigated using radiological, dissection and vascular injection techniques, and associations among them were recorded. The anomalies are discussed in terms of embryological considerations.

Study of the distribution of albendazole-sulphoxide (ABZ-SO) in fertilized egg compartments.

In order to use the chicken embryo in teratogenic studies, it is necessary to know the internal volume in which a xenobiotic distributes. The inoculation of a xenobiotic in one of the compartments of the fertilized egg is the usual technique used in these studies. Neither the concentration nor the moment in which the xenobiotic comes into contact with the chicken embryo have been considered. Predicting the internal volume of distribution in the egg from some of the external parameters that do not interfere with the normal development is necessary. A simple method to calibrate these external parameters and their correlation with the different compartments of the fertilized eggs as well as the different distribution of the xenobiotic in these compartments has been successfully demonstrated. After injection of ABZ-SO, the maximum concentration in the embryo is reached by 36 h. The mean AUC for the albumen (sharp and obtuse end), yolk, and embryo were 78.4, 40.7, 79.2, and 10.8 micrograms.h/ml respectively. The results obtained about the kinetics of the diffusion of ABZ-SO indicate that this compound does not have a homogeneous distribution in all the compartments of the fertilized egg. These results highlight that whenever fertilized eggs are used as a screening for the possible toxicity of a drug or other substances, the dose of the xenobiotic to be injected has to be precisely determined in accordance with the total volume and the stage of embryonic development selected to be affected, starting from the previous knowledge of when and how much substance accedes to the embryo.