The Endocrine Disruptor Compound Bisphenol-A (BPA) Regulates the Intra-Tumoral Immune Microenvironment and Increases Lung Metastasis in an Experimental Model of Breast Cancer.

Breast cancer (BC) metastasis represents the main physiopathology leading to poor prognosis and death. Bisphenol A (BPA) is a pollutant, classified as an endocrine-disrupting chemical compound with estrogenic properties, their exposure in the early stages of neonatal life leads to an increase in the size and weight of breast tumors and induces cellular changes in the tumoral immune microenvironment where cytokines play a key role. Thus, we used female BALB/c mice exposed neonatally to a single dose of BPA. Once mice reached sexual maturity, a mammary tumor was induced, injecting 4T1 cells in situ. After 25 days of injection, we evaluated endocrine alterations, cytokine expression, tissue alterations denoted by macro or micro-metastasis in the lung, and cell infiltration induced by metastasis. We found that BPA neonatal treatment did not show significant endocrine alterations. Noteworthy, BPA led to an augmented rate of metastasis to the lung associated with higher intratumoral expression of IL-1ß, IL-6, IFN-γ, TNF-α, and VEGF. Our data suggest that cytokines are key players in the induction of BC metastasis and that BPA (an environmental pollutant) should be considered as a risk factor in the clinical history of patients as a possible inductor of BC metastasis.

Biology of primordial germ cells in vertebrates with emphasis in urodeles amphibians.

Primordial germ cells (PGCs) are highly specialized cells that play a relevant role in the maintenance and evolution of the species, since they create new combinations of genetic information between the organisms. Amphibians are a class of amniote vertebrates that are divided into three subclasses, the anurans (frogs and toads), the urodeles (salamanders and newts), and the gymnophiones (caecilians). The study of PGCs in amphibians has been addressed in more detail in anurans while little is known about the biology of this cell lineage in urodeles. Studies in some urodeles species have suggested that PGCs are of mesodermal origin, specifying in the lateral plate mesoderm at the late gastrula stage. With classical experiments it shown that, there is an induction of mesoderm, therefore most likely urodeles PGCs develop from unspecialized mesodermal tissue that responds to extracellular signals. However, some fundamental biological processes of PGCs such as the analysis of their specification, arrival, and colonization to the gonads, and their maintenance and differentiation into mature and fertile gametes remain to be elucidated. Therefore, knowledge about the biology of PGCs is of great importance to ensure the perpetuation of urodeles amphibians, as some species are in danger of becoming extinct.

Interaction between oocytes, cortical germ cells and granulosa cells of the mouse and bat, following the dissociation-re-aggregation of adult ovaries.

It is widely accepted that the oocyte plays a very active role in promoting the growth of the follicle by directing the differentiation of granulosa cells and secreting paracrine growth factors. In turn, granulosa cells regulate the development of the oocytes, establishing close bidirectional communication between germ and somatic cells. The presence of cortical cells with morphological characteristics, similar to primordial germ cells that express specific germline markers, stem cells and cell proliferation, known as adult cortical germ cells (ACGC) have been reported in phyllostomid bats. Using magnetic cell separation techniques, dissociation-cellular re-aggregation and organ culture, the behaviour of oocytes and ACGC was analyzed by interacting in vitro with mouse ovarian cells. Bat ACGC was mixed with disaggregated ovaries from a transgenic mouse that expressed green fluorescent protein. The in vitro reconstruction of the re-aggregates was evaluated. We examined the viability, integration, cellular interaction and ovarian morphogenesis by detecting the expression of Vasa, pH3, Cx43 and Laminin. Our results showed that the interaction between ovarian cells is carried out in the adult ovary of two species, without them losing their capacity to form follicular structures, even after having been enzymatically dissociated.

Mechanisms related to sexual determination by temperature in reptiles.

A number of strategies have emerged that appear to relate to the evolution of mechanisms for sexual determination in vertebrates, among which are genetic sex determination caused by sex chromosomes and environmental sex determination, where environmental factors influence the phenotype of the sex of an individual. Within the reptile group, some orders such as: Chelonia, Crocodylia, Squamata and Rhynchocephalia, manifest one of the most intriguing and exciting environmental sexual determination mechanisms that exists, comprising temperature-dependent sex determination (TSD), where the temperature of incubation that the embryo experiences during its development is fundamental to establishing the sex of the individual. This makes them an excellent model for the study of sexual determination at the molecular, cellular and physiological level, as well as in terms of their implications at an evolutionary and ecological level. There are different hypotheses concerning how this process is triggered and this review aims to describe any new contributions to particular TSD hypotheses, analyzing them from the "eco-evo-devo" perspective.

Expression of the Sox9, Foxl2, Vasa, and TRPV4 genes in the ovaries and testes of the Morelet's crocodile, Crocodylus moreletii.

The Sox9 gene is important for determining sex in vertebrates, as well as for maintaining testis morphology and fertility during adult life. In the same way, Vasa is an important gene for the maintenance of the germinal lineage and has been highly conserved throughout evolution, as it is expressed in germ cells of both vertebrates and invertebrates. In the particular case of crocodiles, the expression of Sox9 during gonadal morphogenesis and in the testes of 3-month-old Alligator mississippiensis has been studied. However, it is interesting to carry out studies on other species of crocodiles in relation to their particular mechanism for sex determination influenced by temperature. In this work, we investigated the expression of the Sox9, Vasa, Foxl2, and TRPV4 genes in the ovaries and testes of 5-year-old juvenile crocodiles from Crocodylus moreletii. As expected, Sox9 expression was found in males, but surprisingly, it was also found in females. For the first time, the expression of Vasa was reported in spermatogonia, oogonia, and oocytes of 5-year-old crocodiles. Foxl2 is important for the development and maintenance of the ovary during adult life in vertebrates; moreover, Foxl2 protein and transcripts are both highly expressed in the ovaries compared to the testes. A possible upstream regulator of the Sox9 gene in reptiles has not yet been discovered; as such, the expression of the TRPV4 ion channel was evaluated. The TRPV4 ion channel was expressed in the cytoplasm of Sertoli and follicular cells and was therefore proposed as a possible regulator of SOX9.

Neo-oogenesis in mammals.

Recently, the existence of a mechanism for neo-oogenesis in the ovaries of adult mammals has generated much controversy within reproductive biology. This mechanism, which proposes that the ovary has cells capable of renewing the follicular reserve, has been described for various species of mammals. The first evidence was found in prosimians and humans. However, these findings were not considered relevant because the predominant dogma for reproductive biology at the time was that of Zuckerman. This dogma states that female mammals are born with finite numbers of oocytes that decline throughout postnatal life. Currently, the concept of neo-oogenesis has gained momentum due to the discovery of cells with mitotic activity in adult ovaries of various mammalian species (mice, humans, rhesus monkeys, domestic animals such as pigs, and wild animals such as bats). Despite these reports, the concept of neo-oogenesis has not been widely accepted by the scientific community, generating much criticism and speculation about its accuracy because it has been impossible to reproduce some evidence. This controversy has led to the creation of two positions: one in favour of neo-oogenesis and the other against it. Various animal models have been used in support of both camps, including both classic laboratory animals and domestic and wild animals. The aim of this review is to critically present the current literature on the subject and to evaluate the arguments pro and contra neo-oogenesis in mammals.

Gonadal morphogenesis and establishment of the germline in the phyllostomid bat Sturnira lilium.

In vertebrates such as the mouse and the human, primordial germ cells (PGCs) arise at the base of the allantois and are carried to the epithelium of the posterior intestine, to later migrate to the primordial gonads. In the case of bats, almost nothing is known about this process. To clarify the dynamics of PGCs during gonadal morphogenesis in the phyllostomid bat Sturnira lilium, the proteins for the Ddx4, Sox9 and Mis genes were detected in the gonads of embryos at different stages of development. We identified 15 stages (St) of embryonic development in Sturnira lilium. We found that the formation of the genital ridge and the establishment of the undifferentiated gonad take place between stages 11 and 14. The onset of morphological differentiation in the gonad is first detected in the male gonads at St17. The first PGCs in meiosis are detected in the ovary at St19, whereas in the testicles, the PGCs were in mitotic arrest. Structural changes leading to testicular and ovarian development in Sturnira lilium are observed to be similar to those described for the mouse; however, differences will be established concerning the time taken for these processes to occur.

Morphological structures for potential sperm storage in poeciliid fishes. Does superfetation matter?

Sperm storage within the female reproductive tract has been reported as a reproductive strategy in several species of vertebrates and invertebrates. However, the morphological structures that allow for sperm to be stored and kept viable for long periods are relatively unknown in osteichthyes. We use histological and stereological tools to identify and quantify sperm storage structures (spermathecae) in 12 species of viviparous Poeciliidae. We found spermathecae in nine species, six of which exhibit superfetation (the ability of females to simultaneously carry within the ovary two or more broods of embryos at different stages of development). These spermathecae are folds of ovarian tissue that close around spermatozoa. We compared the number and size (volume) of spermathecae between species with and without superfetation. Species that exhibit superfetation had a significantly higher number of spermathecae than species that do not exhibit this reproductive strategy. In addition, we found that the mean volume of spermathecae and total volume of spermathecae present in the ovary are marginally higher in species with superfetation. Our results contribute to the understanding of the morphological structures that allow for sperm storage in viviparous osteichthyes and suggest a positive relationship between superfetation and the capacity of females to store sperm.

Characterization of the myoepithelial cells in the major salivary glands of the fruit bat Artibeus jamaicensis.

Bats constitute one of the most numerous mammalian species. Bats have a wide range of dietary habits and include carnivorous, haematophagous, insectivorous, frugivorous and nectivorous species. The salivary glands of these species have been of particular research interest due to their structural variability among chiropterans with different types of diets. Myoepithelial cells (MECs), which support and facilitate the expulsion of saliva from the secretory portions of salivary glands, are very important for their function; however, this cell type has not been extensively studied in the salivary glands of bats. In this study, we characterized the MECs in the major salivary glands of the fruit bat Artibeus jamaicensis. Herein, we describe the morphology of the parotid, submandibular and sublingual glands of A. jamaicensis at the light- and electro-microscopic level and the distribution of MECs in these glands, as defined by their expression of smooth-muscle markers such as α-smooth muscle actin (SMAα) and desmin, and of epithelial cell markers, such as KRT14. We found that the anatomical locations of the major salivary glands in this bat species are similar to those of humans, except that the bat sublingual gland appears to be unique, extending to join the contralateral homologous gland. Morphologically, the parotid gland has the characteristics of a mixed-secretory gland, whereas the submandibular and sublingual glands were identified as mucous-secretory glands. MECs positive for SMAα, KRT14 and desmin were found in all of the structural components of the three glands, except in their excretory ducts. Desmin is expressed at a lower level in the parotid gland than in the other glands. Our results suggest that the major salivary glands of A. jamaicensis, although anatomically and structurally similar to those of humans, play different physiological roles that can be attributed to the dietary habits of this species.

In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis.

Sperm capacitation occurs during the passage of sperm through the female reproductive tract. Once the sperm binds to the pellucid zone, the acrosome reaction to enable penetration of the oocyte is completed. In this study, sperm of Artibeus jamaicensis bat was used to evaluate both capacitation status and the acrosome reaction under in vitro conditions, incubating sperm at 32 and 37°C with and without progesterone. Sperm was incubated at different times to assess sperm cells' functionality in terms of capacitation and acrosome reaction, using the chlortetracycline staining, lectin fluoresceinisocyanate conjugate-Pisum sativum agglutinin (FITC-PSA), and transmission electron microscopy. Sperm cells that presented uniform fluorescence throughout the head and mid-piece were classified as non-capacitated. Subsequently, sperm cells, which were observed with fluorescence only in the anterior portion of the head and mid-piece, were classified as capacitated. Sperm cells with no fluorescence in the head, but fluorescence in the mid-piece, were categorized as sperm cells that have carried out the acrosome reaction. During the acrosome reaction, sperm cells showed changes in their morphology, so it was not possible to distinguish the plasma and acrosomal membranes. Around the entire head, it was not possible to distinguish the fusion points between these membranes that made it possible for the acrosomal reaction to take place and thus to release the enzymes necessary to penetrate the pellucid zone. In conclusion, under appropriate in vitro conditions and by supplementing the culture medium with progesterone, A. jamaicensis bat sperm cells are able to be capacitated in a period from 6 to 8 h and to carry out the acrosome reaction.

The endocrine-immune network during taeniosis by Taenia solium: The role of the pituitary gland.

It is well known that sex hormones play an important role during Taenia solium infection; however, to our knowledge no studies exist concerning the immune response following complete or lobe-specific removal of the pituitary gland during T. solium infection. Thus, the aim of this work was to analyze in hamsters, the effects of lack of pituitary hormones on the duodenal immune response, and their impact on T. solium establishment and development. Thus, in order to achieve this goal, we perform anterior pituitary lobectomy (AL, n = 9), neurointermediate pituitary lobectomy (NIL, n = 9) and total hypophysectomy (HYPOX, n = 8), and related to the gut establishment and growth of T. solium, hematoxylin-eosin staining of duodenal tissue and immunofluorescence of duodenal cytokine expression and compared these results to the control intact (n = 8) and control infected group (n = 8). Our results indicate that 15 days post-infection, HYPOX reduces the number and size of intestinally recovered T. solium adults. Using semiquantitative immunofluorescent laser confocal microscopy, we observed that the mean intensity of duodenal IFN-γ and IL-12 Th1 cytokines was mildly expressed in the infected controls, in contrast with the high level of expression of these cytokines in the NIL infected hamsters. Likewise, the duodenum of HYPOX animals showed an increase in the expression of Th2 cytokines IL-5 and IL-6, when compared to control hamsters. Histological analysis of duodenal mucosa from HYPOX hamsters revealed an exacerbated inflammatory infiltrate located along the lamina propria and related to the presence of the parasite. We conclude that lobe-specific pituitary hormones affect differentially the T. solium development and the gut immune response.

Transient transgenesis of the tapeworm Taenia crassiceps.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). Infestation of the human brain, also known as neurocysticercosis, is the most common parasite disease of the central nervous system worldwide. Significant advances in the understanding of the disease have been achieved using the Taenia crassiceps murine model. We describe here a successful transfection protocol of T. crassiceps cysticerci as the first step to approach a number of currently inaccessible biological questions on cysticercosis. T. crassiceps cysticerci (ORF strain) were microinjected with the plasmid pcDNA3.1/NT-GFP-TOPO, encoding the green fluorescent protein (GFP) driven by a cytomegalovirus promoter (CMV). Twelve hours after the microinjection, GFP fluorescence gradually developed in patches associated to bud structures in the bladder wall of cysts. Fluorescence reached a peak at 24-48 h and lasted up to 72 h after the microinjection. Immunohistochemical studies on tissue sections of transfected cysts using an anti-GFP antibody, demonstrated co-localization of the antibody and the GFP fluorescence in the tegumentary cytoplasm and subtegumentary cytons. To validate at the mRNA level the expression of GFP, we carried out RT-PCR using two pairs of nested primers. Results showed expression of GFP-mRNA at 24 h post-transfection. Moreover, western blot assays of crude extracts of transfected cysts, carried out using the anti-GFP specific antibody, showed the expected protein band of 27 kDa, demonstrating that the GFP expression started at 24 after plasmid microinjection and was maintained up to 72 h. These findings will facilitate the development of functional genomics approaches applied to this model of cysticercosis.

Characterization of gametes in two phyllostomid bat species: Artibeus jamaicensis and Sturnira lilium.

Morphology of gametes is used to understand the physiological processes in reproduction among domestic and wild animals. These gametes are used in assisted reproductive technology (ART) and conservation programs. In the case of Artibeus jamaicensis and Sturnira lilium, few studies have been conducted related to these issues. The aim of this study was to describe the structure of spermatozoa, semen characteristics and also the morphology and quality of cumulus oocyte complexes (COC) of A. jamaicensis and S. lilium. Semen characteristics were: A. jamaicensis had a sperm concentration of 4.26×10(6)sperm/ml, progressive motility of 34.55%; viability of 73.23%; head, tail and mid-piece abnormalities of 12.50%. Head length was 6.26µm, mid-piece 18.61µm and tail 70.92µm. S. lilium, had a sperm concentration of 5.15×10(6)sperm/ml, progressive motility of 60.00%, viability of 83.82%; abnormalities in head, tail and mid-piece of 13.77%. Head length was 7.01µm, mid-piece 20.33µm and tail 70.50µm. On average 12.8 of right ovarian oocytes and 9.9 of left ovarian oocytes of A. jamaicensis were recovered. For S. lilium on average 10.7 oocytes from the right ovary and 10.9 oocytes from the left ovary were recovered, ranging in quality from excellent to poor. Sperm morphology and quality of COC were similar to those for other domestic and wild animals. Bat gametes can be used for the study of reproductive biology, in conservation programs and assisted reproductive technology (ART) among domestic and wild animals.

Regulation of intestinal immune response by selective removal of the anterior, posterior, or entire pituitary gland in Trichinella spiralis infected golden hamsters.

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been previously reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, and reduction of gastric secretion and intestinal absorption, as well as increased susceptibility to bacterial and viral infections. However, to our knowledge, no findings have been published concerning the immune response following HYPOX during worm infection, particularly that which is caused by the nematode Trichinella spiralis. The aim of this work was to analyze the effects of total or partial HYPOX on colonization of T. spiralis in the intestinal lumen, together with duodenal and splenic cytokine expression. Our results indicate that 5 days post infection, only neurointermediate pituitary lobectomy (NIL) reduces the number of intestinally recovered T. spiralis larvae. Using semiquantitative inmunofluorescent laser confocal microscopy, we observed that the mean intensity of all tested Th1 cytokines was markedly diminished, even in the duodenum of infected controls. In contrast, a high level of expression of these cytokines was noted in the NIL infected hamsters. Likewise, a significant decrease in the fluorescence intensity of Th2 cytokines (with the exception of IL-4) was apparent in the duodenum of control and sham infected hamsters, compared to animals with NIL surgeries, which showed an increase in the expression of IL-5 and IL-13. Histology of duodenal mucosa from NIL hamsters showed an exacerbated inflammatory infiltrate located along the lamina propria, which was related to the presence of the parasite. We conclude that hormones from each pituitary lobe affect the gastrointestinal immune responses to T. spiralis through various mechanisms.

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle.

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.

Identification of cortical germ cells in adult ovaries from three phyllostomid bats: Artibeus jamaicensis, Glossophaga soricina and Sturnira lilium.

It is generally considered that, in mammals, the ovary is endowed with a finite number of oocytes at the time of birth. However, studies concerning rodents, lemurs and humans suggest the existence of stem cells from the germline that may be involved in germ-cell renewal, maintaining postnatal follicle development. This type of work on wild species is scarce; therefore the objective of this study was to determine ovarian morphology and the presence of progenitor cells from the germline of three species of phyllostomid bats (Artibeus jamaicensis, Glossophaga soricina and Sturnira lilium). The morphological characteristics of the ovaries and the expression of specific markers of germline cells, stem cells and proliferation cells were analysed. The morphology of the ovaries of the three bat species was similar. A polarised ovary with follicles at different stages of development and groups of cortical cells similar to primordial germ cells were observed. Immunofluorescent analysis showed that these cortical cells express germline, stem-cell and proliferative markers, indicating the identification of germ cells that could maintain pluripotency, as well as being mitotically active. This suggests that in the adult ovary of phyllostomid bats there may be a mechanism for the self-renewal of the germline.

Different levels of testicular organization during gonadal differentiation in B6.Y(Tir) mice manifesting sex reversal.

B6.Y(Tir) (mice with Y chromosome from a strain in Tirano, Italy, and autosomes and X-chromosomes from the B6 strain) mice provide an excellent model for analysing sex development that occurs during gonadal differentiation; however, the molecular mechanisms that contribute to sex reversal are unclear. Our aim has been to establish which molecular events participate in this sex reversal. The pattern of gene expression related to testicular [Sry (sex-determining region of the Y chromosome), Sox9 (Sry-related high-mobility group box gene 9) and Mis (Müllerian-inhibiting substance)] and ovarian [Wnt4 (Wingless-type MMTV (murine-mammary-tumour virus) integration site family, member 4), Rspo1 (cysteine-rich secretory protein containing a thrombospondin type 1 repeat) and Stra8 (stimulated by retinoic acid gene 8)] differentiation was analysed by applying immunofluorescence and real-time RT-PCR (reverse transcription-PCR), focusing on XY gonads from the B6.Y(Tir) mouse, but also analysing the normal strains CD-1 and C57BL/6J (B6). The expression of genes related to the process of sexual differentiation was altered in the case of the B6.Y(Tir) strain, both at the transcript and protein level, inducing differentiation of ovaries and ovotestes, but not the formation of the testes, which were normal. Our results indicate that the expression of testicular genes is inhibited at various levels, permitting the expression of ovarian genes such as Wnt4, Stra8 and Rspo1. However, their activity was not clear when the data were averaged. Correlation analysis indicated that an ovary differentiation pathway is activated when the testicular differentiation pathway is inhibited.

Characterization of Progenitor Cells during Canine Retinal Development.

We identify the presence of progenitor cells during retinal development in the dog, as this species represents a natural model for studying several breed-specific degenerative retinal disorders. Antibodies to detected progenitor cells (Pax6, C-kit, and nestin) and ganglion cells (BDNF, Brn3a, and Thy1) were used in combination with H3 for the purpose of identifying proliferating cells. Pax6, nestin, C-kit, and H3 were localized mainly in the neuroblastic layer of the retina during the embryonic stage. During the fetal stage, proteins were expressed in the inner neuroblastic layer (INL) as well as in the outer neuroblastic layer; BDNF, Thy1, and Brn3a were also expressed in the INL. During the neonatal stage only C-kit was not expressed. Proliferating cells were present in both undifferentiated and differentiated retina. These results suggest that, during canine retinogenesis, progenitor cells are distributed along the retina and some of these cells remain as progenitor cells of the ganglion cells during the first postnatal days.

Effect of allopurinol on damage caused by free radicals to cryptorchid testes.

Cryptorchidism causes apoptosis of germ cells. It has been suggested that the redox regulatory system is involved in this process. The free radicals produced are thought to be generated during the production of uric acid, a reaction catalyzed by xanthine oxidase. This enzyme is inhibited by allopurinol; however, the role of allopurinol in neonate rats with inguinal cryptorchidism has not been assessed yet. Sixty male Wistar rats were used and five groups were formed: a control, a sham, a sham group with allopurinol administration and two groups with surgical unilateral cryptorchidism, which either did not receive, or received, allopurinol. The rats were assessed at 40 days post-partum. Reactive oxygen species concentration and epithelial area were measured and the histopathological, apoptotic and cellular proliferation indexes were determined. We found a decrease in reactive oxygen species, histopathological and apoptotic indexes and an increase in proliferation index and epithelial area in rats with cryptorchidism treated with allopurinol in comparison with rats with untreated cryptorchidism. We suggest that the over-production of reactive oxygen species plays an important role in the damage of the cryptorchid testes. Allopurinol administration decreases reactive oxygen species concentrations as well as the damage to the germ epithelium.

Retrograde axonal transport obstruction of brain-derived neurotrophic factor (BDNF) and its TrkB receptor in the retina and optic nerve of American Cocker Spaniel dogs with spontaneous glaucoma.

OBJECTIVE: To determine the degree of retrograde optic nerve axonal transport obstruction at the scleral lamina cribosa level by examining levels of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor type B (TrkB) in dogs presenting with high intraocular pressure. ANIMALS STUDIED: A total of 10 eyes, four normal and six glaucomatous eyes, from normal and affected American Cocker Spaniels with primary glaucoma were studied. All eyes were assessed by neuro-ophthalmic examination, tonometry, gonioscopy, slit-lamp biomicroscopy, and indirect ophthalmoscopy prior to enucleation. METHODS: Immunocytochemistry analysis was performed to evaluate BDNF and TrkB receptor expression in retina and optic nerve in normal and glaucomatous dogs. RESULTS: In all normal eyes BDNF immunostaining was detected in the cytoplasm of retinal ganglion cells (RGC), inner plexiform layer (IPL), inner nuclear layer (INL), nerve fiber layer (NFL), optic nerve head cells, and lamina cribosa cells. In all glaucomatous eyes BDNF was more evident in RGC, NFL and lamina cribosa cells. TrkB receptor was detected in the cytoplasm of RGC, NFL and ONH bundles in all normal eyes, and in a more intense pattern in all glaucomatous eyes. CONCLUSIONS: BDNF retrograde axonal transport is substantially inhibited by intraocular pressure elevation. TrkB accumulation at the ONH in glaucoma suggests a role for neurotrophin deprivation in the pathogenesis of RGC death in canine glaucoma, as well as a possible paracrine and/or autocrine signaling within the lamina cribosa. Neurotrophin signaling may regulate more than neuronal development, survival and differentiation. BDNF neurotrophin and its TrkB receptor expression by lamina cribosa cells and ONH astrocytes in glaucomatous eyes may help to determine the role of these cells as a paracrine source in terms of retinal ganglion cell survival, during episodes of elevated intraocular pressure.