Immunohistochemical evidence of leptin and its receptor in the carpal glands of domestic pigs and wild boar.

BACKGROUND: In recent years, the protein hormone leptin has been the subject of numerous studies designed to clarify and interpret its functional significance; it has been speculated that this goes well beyond the control of appetite and energy metabolism. In particular, the presence of leptin and its receptor has been observed in various glands anatomically and functionally related to the reproductive apparatus. This has led to the hypothesis that leptin may act directly in the functional control of these glands and, in general, the control of reproductive function. HYPOTHESIS/OBJECTIVES: The presence and distribution of leptin and its receptor in the carpal glands of domestic pigs and wild boar are examined, using immunohistochemical techniques. ANIMALS: Tissue samples were collected from five domestic pigs and five wild boar, following slaughter. RESULTS: The presence of leptin and its receptor was demonstrated in the glands, localized in the dark cells of the glandular secretory epithelium. In addition, no difference was observed between wild boar and domestic pigs. CONCLUSIONS AND CLINICAL IMPORTANCE: We hypothesize that leptin may be produced by the carpal gland and may act on the gland's secretory epithelial cells with an autocrine/paracrine mechanism, thus affecting the secretory activity of the gland itself.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia-bearing mice.

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract.

The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction. Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer. Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control.

Receptors for leptin and estrogen in the subcommissural organ of rabbits are differentially modulated by fasting.

In rabbits, the fasting-dependent reduction of LH secretion is likely mediated by leptin and estrogens via receptors in the brain. For the first time, using immunohistochemistry, the presence and regulation of receptors for leptin (Ob-R) and estradiol-17beta subtype alpha (ERalpha) were studied in the subcommissural organ (SCO) of rabbits, which were fed either ad libitum (control) or fasted for 48 h (treated) to verify whether this brain structure is a potential site of integration for metabolism and reproduction. In control rabbits, the cytoplasm of glial cells lining the SCO evidenced strong Ob-R immunoreactivity, whereas both ependymal and hypendymal cells of this glandular-like structure were negative. The Ob-R positive glial cells were identified as fibrous astrocytes using the phosphotungstic acid-hematoxylin histochemical (PTAH) and glial fibrillary acidic protein (GFAP) immunohistochemical techniques. ERalpha immunoreactive nuclei were detectable exclusively in the specialized cells forming the SCO, whereas surrounding astrocytes and neurons were negative. Compared to controls, in fasted rabbits, the staining of Ob-R immunoreaction was reduced in the cytoplasm of positive astrocytes, but greatly enhanced in plasma membranes, whereas the number of ERalpha immunoreactive SCO cells was increased (13.2+/-2.7 vs. 5.2+/-2.0, P<0.01). Ependymal cells lining the third ventricle were negative for both Ob-R and ERalpha. Our results indicate, although indirectly, that the SCO, together with the astrocytes in close contact with this structure, is a likely target for nutritional and gonadal signals carried by leptin and estrogens, suggesting that these specialized glial cells may regulate reproduction and metabolism through mechanisms still unknown.

Angiogenic and anti-inflammatory properties of micro-fragmented fat tissue and its derived mesenchymal stromal cells.

BACKGROUND: Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population. METHODS: Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1. RESULTS: Our results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1. CONCLUSIONS: Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

Role of the endothelin-1 system in the luteolytic process of pseudopregnant rabbits.

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P

Presence and distribution of leptin and its receptor in the minor salivary glands of the donkey.

Leptin is a hormone widely diffused in the mammalian body in which it plays functions that go far beyond control of appetite and energy metabolism. The finding that it is present in the major salivary glands of various animal species is of interest for the functional implications that it may imply. Since there are no data on the presence of leptin and its receptor in the minor salivary glands, the aim of this study was to demonstrate their presence and distribution in such glands of donkeys. This latter was chosen as species of reference because the monogastric herbivore shows intense salivation during their masticatory activity. Tissue samples were collected from four adult donkeys, of both sexes, following slaughter. Samples were fixed, embedded in paraffin, and processed for immunohistochemical analysis using primary antibodies directed against leptin and its receptor. Controls for non-specific staining were always included. Leptin and its receptor were found in the minor salivary glands. Their distribution was similar to that described in the major salivary glands of animal species that have been investigated to date. We hypothesized that leptin can play a role in regulating gland function, via an autocrine/paracrine mechanism.

Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model.

BACKGROUND: Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth. METHODS: Murine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis. RESULTS: We found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis. CONCLUSIONS: Our results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human.

Ob receptor in rabbit ovary and leptin in vitro regulation of corpora lutea.

We studied leptin involvement in rabbit corpora lutea (CL) activity, and its post-transcriptional signalling pathway. The expression of leptin receptor (Ob-R) in rabbit ovary at day 9 of pseudopregnancy was evaluated by immunohistochemistry and Western blot analysis. The specificity of the Ob-R receptor antibodies was characterised by immunoprecipitation and competition with blocking peptide. Day 9 CL were incubated in vitro with leptin alone or with inhibitors of PLC (phospholipase C), PLD (phospholipase D), AC (adenylate cyclase), JAK (janus kinase), MAPK (mitogen-activated protein kinase) and both cAMP- and cGMP-specific PDE (phosphodiesterase). Prostaglandin F2alpha(PGF2alpha), PGE2 and progesterone levels were measured in the culture medium, while NOS (nitric oxide synthase) and cAMP- and cGMP- specific PDE activities were measured in CL tissue. Positive staining for Ob-R was found within the cytoplasm of large luteal cells of CL as well as in granulosa cells of follicles and oocytes. Immunoblots detected a band of about 99 kDa size in Ob-R immunoprecipitates from CL homogenates. This band was not detectable after pre-incubation of the primary antibody with the immunising leptin peptide. Leptin increased PGF2alphaand cAMP-specific PDE, decreased basal progesterone and did not affect PGE2 and NOS levels. Leptin used the JAK pathway in increasing PGF2alpha, and MAPK and cAMP-specific PDE in decreasing progesterone. This study supports a permissive luteolytic role for leptin in rabbit CL.

Glucidic determinants expressed by the digestive apparatus of Umbrina cirrosa (L.) fries as revealed by lectin histochemistry.

Glycoconjugates present in the various parts of the digestive apparatus (oesophagus, stomach, intestine) of 27, 34 and 44 days-old Umbrina cirrosa (L.) fries were characterised by means of lectin histochemistry in conjunction with sialidase digestion and KOH treatment. High amounts of carbohydrates were detected especially in goblet cells of the oesophagus. Positivity obtained with the various lectins in oesophagus, stomach and intestine did not change during the development of the fries examined. D-Gal and D-GalNAc appeared to occupy a terminal position in oligosaccharidic chains whereas they are linked to sialic acid residues in adult subjects, which suggests an incomplete chemical structure of the glycoconjugates secreted by the digestive apparatus of fries.

Immunohistochemical localization of orexin A and orexin type 2 receptor-positive cells in the placenta of dogs.

The aim of the present study was to examine the presence and distribution of cells that express immunopositivity for orexin A (OXA) and its type 2 receptor (OX2R) in the dog placenta toward the end of pregnancy using immunohistochemical techniques. In the placental fetal portion, a few OXA and OX2R-positive cells were seen scattered in the outermost coating layer of chorionic villi and in the trophoblastic protrusions. Closer to the maternal portion, immunopositive labeling for both peptides was visible in the glandular epithelia and that for OXA also in the endothelium of the capillaries. These observations allow us to hypothesize that the canine placenta may be not only a source of orexin A, but also its target, and that orexin A may play an important role in controlling the function of this important organ for normal fetal development.

Leptin receptor is expressed by epidermis and skin appendages in dog.

Leptin is a polypeptide secreted by adipocytes which binds to a specific receptor (Ob-R) that is expressed in various tissues. The wide distribution of the Ob-R suggests that leptin might exert diverse biological functions, not only by regulating energy metabolism and appetite, but also by acting as a mitogen in many cell types, including keratinocytes. In this study, the presence and localization of Ob-R was investigated in the skin of the dog using RT-PCR and immunohistochemical techniques. RT-PCR revealed the presence of Ob-R m-RNA in the skin specimens collected from the dorsal region of two smooth coat breed dogs. Through immunohistochemistry performed on the skin of five dogs, the expression of the receptor was observed in the basal layer of the epidermis, in the hair follicles as well as in the apocrine sweat and sebaceous glands. No staining for Ob-R was detected in the suprabasal epidermis layers. Strong positive signals were observed in many cells of the outer root sheath of hair follicles in growing and in regressive phases. The identification of Ob-R in the above targets suggests that leptin may play a role in the regulation of cyclic renewal of the epidermis and skin appendages in dog. This study represents an important contribution to understand the complex mechanisms that are involved in the skin biology in this species.

Membrane vesicles mediate pro-angiogenic activity of equine adipose-derived mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSCs) have attracted a great deal of interest, due to several distinctive features, including the ability to migrate to damaged tissue and to participate in tissue regeneration. There is increasing evidence that membrane vesicles (MVs), comprising exosomes and shedding vesicles, represent a key component, responsible for many of the paracrine effects of MSCs. The aim of the present study was to establish whether equine adipose-derived MSCs (E-AdMSCs) produce MVs that are capable of influencing angiogenesis, a key step in tissue regeneration. A morphological study was performed using MSC monolayers, prepared for transmission and scanning electron microscopy and on ultracentrifuged MSC supernatants, to identify production of MVs. The ability of MVs to influence angiogenesis was evaluated by means of the rat aortic ring and scratch assays. The results demonstrated that MVs, constitutively produced by E-AdMSCs, are involved in intercellular communication with endothelial cells, stimulating angiogenesis. Although many questions remain regarding their formation, delivery, content and mechanism of action, the present study supports the concept that MVs released by MSCs have the potential to be exploited as a therapeutic tool for regenerative medicine.

Evaluation of storage conditions on equine adipose tissue-derived multipotent mesenchymal stromal cells.

The transplantation of multipotent mesenchymal stromal cells (MSCs) is a potentially promising therapy for the treatment of tendon and ligament injuries and some forms of articular pathology in horses. This study investigated the effects of storage conditions on MSCs. Equine adipose tissue-derived MSCs (eAd-MSCs) were stored at 4 °C and at room temperature (RT) for 24 and 48 h, and viability, doubling time, expression of CD44 and CD90 antigens, clonogenic/differentiation potentials, and karyotype were subsequently evaluated. The eAd-MSC viability was significantly affected by the storage conditions, while doubling time was not significantly altered. The findings indicate (1) that storage at 4 °C is preferable to RT as this results in greater numbers of viable, morphologically normal cells, and (2) that cells should be used within 24 h.

Decellularized silk fibroin scaffold primed with adipose mesenchymal stromal cells improves wound healing in diabetic mice.

INTRODUCTION: Silk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice. METHODS: The conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse's back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors. RESULTS: We found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15-17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and matrix remodeling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs, DFs and KCs, preferentially enhanced the HUVECs' migration and the release of angiogenic factors stimulating microvessel outgrowth in the aortic ring assay. CONCLUSIONS: Our results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of D-Ad-MSCs-SF patches in chronic diabetic ulcers in humans.

Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery.

Mesenchymal stromal cells (MSCs) have been proposed for delivering anticancer agents because of their ability to home in on tumor microenvironment. We found that MSCs can acquire strong anti-tumor activity after priming with Paclitaxel (PTX) through their capacity to uptake and then release the drug. Because MSCs secrete a high amount of membrane microvesicles (MVs), we here investigated the role of MVs in the releasing mechanism of PTX. The murine SR4987 line was used as MSC model. The release of PTX from SR4987 in the conditioned medium (CM) was checked by HPLC and the anti-tumor activity of both CM and MVs was tested on the human pancreatic cell line CFPAC-1. MVs were isolated by ultracentrifugation, analyzed by transmission (TEM) and scanning electron microscopy (SEM), and the presence of PTX by the Fourier transformed infrared (FTIR) microspectroscopy. SR4987 loaded with PTX (SR4987PTX) secreted a significant amount of PTX and their CM possessed strong anti-proliferative activity on CFPAC-1. At TEM and SEM, SR4987PTX showed an increased number of "vacuole-like" structures and shed a relevant number of MVs, but did not differ from untreated SR4987. However, SR4987PTX-derived-MVs (SR4987PTX-MVs) demonstrated a strong anti proliferative activity on CFPAC-1. FTIR analysis of SR4987PTX-MVs showed the presence of an absorption spectrum in the corresponding regions of the PTX marker, absent in MVs from SR4987. Our work is the first demonstration that MSCs are able to package and deliver active drugs through their MVs, suggesting the possibility of using MSCs as a factory to develop drugs with a higher cell-target specificity.

Immunolocalization of leptin and its receptor in the pancreas of the horse.

The aim of the present study was to demonstrate the presence and the distribution of leptin and its receptor in the pancreas of horses of both sexes by immunohistochemical techniques. The presence and the distribution of leptin receptor were also investigated in the initial portion of the duodenum, near the duodenal ampulla. The immunohistochemical investigation demonstrates the immunolocalization of both leptin and its receptor in the endocrine cells of pancreatic islets, which led us to hypothesize that leptin may possibly exert an autocrine/paracrine action on the endocrine pancreas. Examination of the exocrine pancreas in the same treated sections showed the presence of leptin-positive cells in the wall of the interlobular ducts where, however, the receptor was not found. This observation led us to consider that some cells of the ducts may perform some minimal secretory activity, and that leptin produced by these ductal cells may reach the duodenum in the pancreatic juice. This hypothesis is enhanced by the presence of leptin-receptor in the duodenum of the same animals, where the epithelial cells of the mucosa showed intense immunolocalization in the brush border. Consequently it is possible that the ductular leptin may play a regulatory role on the functionality of the enterocytes.

Keratinisation degree of rumen epithelium and body condition score in sheep grazing on Brachypodium rupestre.

This article describes the result of a study focusing on the keratinisation degree of rumen mucosa and changes in the body condition of sheep that had grazed for 20 days in a pasture densely covered with Brachypodium rupestre. Grazing in this type of pasture can reduce the probability of fires in a Mediterranean mountain setting. However, foraging in areas with a prevalence of Brachypodium rupestre can affect animals' well being. In this respect, it is essential to determine the length of time during which the animals can remain in this environment before their welfare is compromised by this type of pasture. Ewes grazing on a semi-mesophilic pasture were included as a control. On days 1, 10, and 20, five ewes from each group were sacrificed to evaluate the variations of the epithelial keratinization degree of the rumen atrium and ventral sac. Body weight (BW) and body condition score (BCS) were assessed in ten ewes per group. The control animals showed little variation in the keratinisation degree of rumen mucosa without any detrimental effects on the BCS and BW. The experimental animals showed a significant increase in the epithelial keratinisation degree within 10 days and a decrease of BCS and BW within 20 days. The data collected suggest that animals should not remain for longer than 10-12 days on pasture highly covered with Brachypodium rupestre.

Immunolocalization of leptin and its receptor in the placenta of cats.

The aim of the present study was to investigate the presence and the distribution of leptin (Ob) and its receptor (ObR) in the feline placenta at term by means of immunohistochemical techniques. A few Ob-positive cells were observed scattered in the lamellae of the labyrinthine placenta. These cells had the morphological characteristics typical of the very abundant cells in the placenta of cats that can be considered as being decidual and, in some cases, syncytiotrophoblastic cells. A few ObR-positive cells were observed in the same placental portion and were mainly localized in the lamellae, showing morphological features typical of decidual and syncytiotrophoblastic cells. No other structure of the placenta or the uterine wall showed positive reaction to the antibodies used. Our results confirm what has already been demonstrated in humans and laboratory animals, but not in domestic animals. Together with other emerging data on the secretory activities of the feline placenta, our study underlines its relevance in the production of molecules long known to be involved in appetite control and, probably, with potential effects on the developing fetus.

Identification of cannabinoid type 1 receptor in dog hair follicles.

In veterinary medicine, there is an increasing interest in the study of the endo-cannabinoid system and the possible use of the cannabinoids for the treatment of several diseases. Cannabinoid receptors (CB) are widely distributed in human and laboratory animal tissues, justifying the involvement of the endo-cannabinoid system in a great number of metabolic ways. Since there are no data regarding cannabinoid receptors in hair follicles of domestic animals, we investigated the presence and localization of CB1 receptor in dog hair follicles. By using a goat anti-CB1 polyclonal antibody, we observed CB1 receptor in the proximal part of both primary and secondary hair follicles. Staining was localized in the inner root sheath cells. We suppose that the endo-cannabinoid system is involved in the molecular mechanisms regulating hair follicle activity in dog. The identification of CB1 receptor at the level of the inner root sheath may help in the understanding of hair follicle biology and the possibility that cannabinoid molecules could be considered as suitable therapeutic tools in dog.