Allogeneic mesenchymal stem cells improve the wound healing process of sheep skin.

BACKGROUND: Skin wound healing includes a system of biological processes, collectively restoring the integrity of the skin after injury. Healing by second intention refers to repair of large and deep wounds where the tissue edges cannot be approximated and substantial scarring is often observed. The objective of this study was to evaluate the effects of mesenchymal stem cells (MSCs) in second intention healing using a surgical wound model in sheep. MSCs are known to contribute to the inflammatory, proliferative, and remodeling phases of the skin regeneration process in rodent models, but data are lacking for large animal models. This study used three different approaches (clinical, histopathological, and molecular analysis) to assess the putative action of allogeneic MSCs at 15 and 42 days after lesion creation. RESULTS: At 15 days post-lesion, the wounds treated with MSCs showed a higher degree of wound closure, a higher percentage of re-epithelialization, proliferation, neovascularization and increased contraction in comparison to a control group. At 42 days, the wounds treated with MSCs had more mature and denser cutaneous adnexa compared to the control group. The MSCs-treated group showed an absence of inflammation and expression of CD3+ and CD20+. Moreover, the mRNA expression of hair-keratine (hKER) was observed in the MSCs-treated group 15 days after wound creation and had increased significantly by 42 days post-wound creation. Collagen1 gene (Col1α1) expression was also greater in the MSCs-treated group compared to the control group at both days 15 and 42. CONCLUSION: Peripheral blood-derived MSCs may improve the quality of wound healing both for superficial injuries and deep lesions. MSCs did not induce an inflammatory response and accelerated the appearance of granulation tissue, neovascularization, structural proteins, and skin adnexa.

Corneal sequestra in cats: 175 eyes from 172 cases (2000-2016).

OBJECTIVES: To report the clinical outcomes of different surgical treatments used to manage feline corneal sequestra in a large number of cases. MATERIALS AND METHODS: Medical records of 172 cats affected by feline corneal sequestra and surgically managed by different techniques were retrospectively evaluated. Signalment, surgical technique, visual outcomes, postoperative corneal clarity and recurrence were evaluated. RESULTS: One hundred and seventy-two cats (175 eyes) of different breeds, ages and sex were included in the retrospective case series. The Persian was the most represented breed (123/172; 71.5%), followed by Exotic Shorthair breed (21/172; 12.2%), Domestic shorthair breed (21/172; 12.2%) and a smaller group of other different breeds (7/172). Surgical management included a superficial (84/175 eyes) or deep (91/175 eyes) lamellar keratectomy in association with the following covering techniques (tectonic support and/or protective support): nictitans membrane flap (n=84), conjunctival pedicle graft (n=52), BioSISt graft (n=9), corneoconjunctival transposition (n=7), corneoconjunctival transposition+nictitans membrane flap (n=6), conjunctival free island graft (n=6), BioSISt+conjunctival pedicle graft (n=6), BioSISt+nictitans membrane flap (n=4) and bridge conjunctival graft (n=1). At the end of the retrospective case series, visus was present in all patients and no or mild corneal opacity was detected in 86% (151/175 eyes) of the patients. Sequestra recurred in 20 eyes (20/175; 11.4%) within 2 years. CLINICAL SIGNIFICANCE: This paper describes the clinical outcomes of a large number of cases of feline corneal sequestra treated with varying surgical techniques and provides useful insight regarding the prognosis and outcomes of these techniques within feline ophthalmology.

Hyaluronic acid, Manuka honey and Acemannan gel: Wound-specific applications for skin lesions.

Healing of open wounds is of great medical importance. Wound healing is a complex process that aims to restore the function and structure of damaged tissue. This study was conducted to compare secondary intention healing of wounds treated daily with a topical application of commercially available hyaluronic acid (HA), Manuka honey (MH), Acemannan gel (AG), or a placebo. Bilateral wounds were surgically created on the backs of six sheep. At two and six weeks post-wound creation, biopsies were obtained to perform histological, immunohistochemical, and molecular analyses of the wound site. Daily clinical evaluations were performed and weekly photographs were taken of the wounds. HA treatment promoted a physiological progression of the healing process in all wound healing phases, while stimulating an abundant cutaneous adnexa and promoting rapid healing, representing the most compelling treatment. MH-treated wounds were slightly dry. However, the main effect of MH was to promote cell proliferation and neovascularization, with an overall pro-inflammatory effect. Results suggest that MH treatment enhances the healing process. AG treatment dehydrated the wounds and stimulated late granulation tissue and cell proliferation. Moreover, AG-treated wounds produced a mild late pro-inflammatory and neovascularization effect. Our data indicate that AG treatment can have a positive influence on moist wounds with abundant granulation tissue and exudate.

Effect of storage on long-term stability of salivary α-amylase, lysozyme, lactate dehydrogenase, calcium and phosphorus in dogs.

Research or diagnostic conditions may require the storage of salivary samples for long periods before analysis is processed. The aim of this study was to evaluate the stability of canine salivary α-amylase, lysozyme, lactate dehydrogenase (LDH), and total calcium and phosphorus after storage for 1, 3 and 6 months at -20°C. Available saliva samples were 75 immediately after collection (T0) and 46 at 1 month (T1), 31 at 3 months (T3) and 18 at 6 months (T6) of storage, according to the number of aliquots collected from each saliva sample. Compared to T0, LDH declined by 92.3% after 1 month of storage (P<0.001), whereas lysozyme concentration significantly decreased as storage time increased (-29.3%, -43.4% and -59.1% at T1, T3, and T6 respectively, P<0.001). Amylase maintained basically the same concentration for the entire experimental period, whereas total calcium and phosphorus concentration decreased over time (calcium, P<0.001). Assessing the long-term stability of canine salivary analytes stored at -20°C may have important implications in diagnosis and research.

Investigations of the corneal epithelium in Veterinary Medicine: State of the art on corneal stem cells found in different mammalian species and their putative application.

The existence of progenitor cells that can readily differentiate into a specific cell type is a common cellular strategy for physiological tissue growth and repair mechanisms. In the mammalian cornea, many aspects regarding the nature and location of these cells are still unclear. In the human limbus (peripheral area of the cornea) progenitor cells have been found and characterized but in non-human mammals, the picture is not so clear. In this review, we examine current knowledge about the morphology of limbus and the localization of corneal epithelial stem cells in all species studied so far, comparing data with humans. We have also explored different research directions in the veterinary field in order to discuss the: i) currently used protocols and ii) best range of treatments for ocular pathologies in which corneal stem cells are involved.

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse.

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

Immunohistochemical localization of atrial natriuretic factor and autoradiographic distribution of atrial natriuretic factor-binding sites in the brain of the cave salamander Hydromantes genei (Amphibia, Plethodontidae).

The distribution of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the cave salamander Hydromantes genei (Amphibia, Plethodontidae) was investigated by using antisera raised against rat and human ANF(1-28). Concurrently, the location of ANF-binding sites was determined by autoradiography, using radioiodinated human ANF(1-28) as a tracer. In several regions of the brain, including the olfactory bulb, the preoptic area, the ventral thalamus, the tectum of the mesencephalon, and the choroid plexuses inside the ventricles, a good correlation was observed between the distribution of ANF-immunoreactive elements and the location of ANF-binding sites. Mismatching was found in the habenular nucleus, the commissura habenularis, the fasciculum retroflexus, and the interpeduncular nucleus, which contained high levels of binding sites but were devoid of ANF-immunoreactive structures. In contrast, a few other regions, such as the pineal gland and the subcommissural organ, showed a high concentration of ANF-like immunoreactivity but did not contain ANF-binding sites. This study provides the first localization of ANF-like immunoreactivity and ANF-binding sites in the brain of an urodele amphibian. The results show that the ANF peptidergic system in the cave salamander has an organization more simple than the organizations described for the brain of frog or other vertebrates. This feature is probably related to the expression of highly pedomorphic characters in plethodontids. The anatomical distribution of ANF-immunoreactive elements and ANF-binding sites suggests that ANF-related peptides may act as hypophysiotropic hormones as well as neurotransmitters and/or neuromodulators in the salamander brain.

Metabolic fate of zetidoline, a new neuroleptic agent, in man.

Healthy volunteers administered orally a single dose (20 mg) of [2-14C]zetidoline, a new dopamine antagonist, exhibited rapid absorption of radioactivity with peak plasma levels of 250-300 ng/ml achieved in 1 h. The compound underwent intensive metabolic first-pass so that plasma radioactivity was represented mostly by two products, metabolite B endowed with neuroleptic activity, and metabolite D inactive, while unchanged zetidoline was not detected. Disappearance of radioactivity from plasma was rapid with a half-life of 1.78 +/- 0.20 h. The simultaneous assay of plasma prolactin showed increased levels of the hormone (+ 464% at the peak time) up to the 6th h after dosing, with plasma concentration profile which mimic those of metabolite B. The radioactive test-dose was eliminated mainly via the kidneys with an average urinary recovery of 84.7 +/- 1.7% in 4 days (73.4 +/- 1.1% within 8 h). The main urinary metabolite (metabolite G) and two minor ones (metabolites B and D) were purified and their structures assigned by IR, MS and NMR spectroscopy, they are: 1-(3-chloro-4-hydroxyphenyl)-3 [2-(3,3-dimethyl-1-azetidinyl)ethyl]imidazolidin-2-one, metabolite B; 1-[2-(3,3-dimethyl-1-azetidinyl)ethyl]-imidazolidin-2-one, metabolite D and the 4'-O-sulphate ester of metabolite B, metabolite G. The metabolic fate of zetidoline in man follows the same phase I reactions demonstrated in rats and dogs, while the phase II reaction is sulphoconjugation instead of the glucuronidation observed in animals.

Studies of binding C3-substitute rifamycins to human and bovine serum albumin.

The interactions of a series of C3-substituted rifamycins with human and bovine serum albumins were studied in order to find possible correlations between the degree of binding and the structural features of the various molecules. The results obtained indicate some of the physicochemical properties and, therefore, of the structural requirements which appear to determine or influence the bonding mechanisms of this series of rifamycins. Two types of interaction were found to exist, ionic and hydrophobic types. The findings suggest that the inhibition by protein of the antibacterial activities of these antibiotics depends on the type of bonding mechanism rather than the degree of binding.

Pharmacokinetics-activity relationships of a new non-hormonal antifertility agent: 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1, 2,4-triazole, in the rat and the hamster.

The pharmacokinetic profiles of a new non-hormonal anti-fertility agent, DL 111-IT, were studied in rats and hamsters given the 14C labelled compound parenterally dissolved in aqueous or oily vehicles. In both species, DL 111-It was rapidly metabolized and excreted when given intravenously or subcutaneously in aqueous vehicles (half-lives = 15-45 min.), whereas the kinetics were prolonged when it was administered in oily formulations (half-lives = 7-10 h). Binding studies revealed a high affinity of DL 111-IT for rat serum albumin (Ka = 6 X 10(5) 1/mole). The radioactivity concentrations in different tissues of pregnant rats appeared to be uniform with the excretory organs and lungs being the main target tissues. At the site of action, the utero-placental complex, the levels of total 14C were comparable to those in plasma, whereas the concentration of unchanged DL 111-IT was higher and remained so for a longer time. A comparison between the kinetic profiles and the activity data after single or multiple dose administration in different formulations, clearly indicates a close relationship between activity and plasma and tissue (utero-embryo placental complex) levels of DL 111-IT, and also makes clear the influence of the formulation and of the treatment schedule on the anti-fertility activity of the compound.

Pharmacokinetics and metabolism of tomoxiprole, a new analgesic antiinflammatory agent, in the rat.

The pharmacokinetics and the metabolic profile of tomoxiprole, a new analgesic antiinflammatory agent belonging to the class of 3-alkyl-2-aryl-3H-naphth (1,2-d)imidazoles, were studied in the rat. After oral administration (5 mg/kg) to male rats, tomoxiprole was rapidly absorbed, mostly by the gut, and reached maximum plasma levels of about 0.5 microgram/ml in 0.25-2 h. A metabolic first pass reduced the extent of oral bioavailability of the parent compound to about half, while absorption (total 14C data) was estimated to be complete. After intravenous injection (2.5 mg/kg), the plasma kinetics of tomoxiprole in male rats showed a bi-exponential profile, and the terminal elimination half-life was 4.2 h. The apparent volume of distribution was high, suggesting a wide distribution of the drug. Increasing the oral dose by ten times (50 mg/kg), resulted in linear kinetics with a proportional increase of the C max and AUC values and the same value of terminal elimination half-life. In females given a 5 mg/kg dose, the plasma levels of 14C, tomoxiprole and AUC values were somewhat higher than in males. The plasma levels of total 14C after iv or po treatments were higher and more sustained than those of tomoxiprole. The kinetic profile after iv administration was described by a three exponential terms equation and the terminal elimination half-life was 38.7 h. Upon iv administration, total 14C was rapidly distributed in highly vascularized tissues while in others, like the bone, fat, gonads, pancreas and skin the equilibrium with the central compartment was attained later. Target organs were the adrenals, liver, lungs, pancreas, thyroid, stomach and above all the fat tissue. Elimination from tissues was almost complete 48 h after the treatment. 14C was eliminated mainly in the feces (80% of dose) as metabolites. In the bile, five polar metabolites were detected; one of them, desmethyl tomoxiprole glucuronide, accounting alone for more than 80% of the total biliary radioactivity; was purified and its structure assigned.

Competition between diftalone and other drugs for the binding site(s) on human serum albumin.

The influence of a series of drugs on the binding of diftalone to human serum albumin was investigated by equilibrium dialysis. The results indicated that among the different drugs tested phenylbutazone, indomethacin, warfarin and tolbutamide were, in the order listed, the most effective in displacing diftalone from its binding site(s). Additional investigation showed that there was no consistent inhibition by diftalone of warfarin and tolbutamide binding to albumin. The information obtained might prove useful in clinical therapy whenever a combination of drugs is to be administered.

Metabolism of 5-isopropyl-1-methyl-2-nitro-1H-imidazole. Identification of some urinary metabolites in the dog.

The metabolism of 5-isopropyl-1-methyl-2-nitro-1H-[2-14C] imidazole in dogs has been investigated after oral administration of 50 mg/kg. Three main metabolites, still containing the nitro group and accounting for about 50% of the total radiocarbon, together with a small amount of the unchanged drug, were isolated from the urine within 48 hr. The structures were determined by mass, infrared, and nuclear magnetic resonance spectroscopy. The biotransformations giving rise to the metabolites isolated involve the isopropyl chain of the molecule, either at the tertiary carbon atom or at one of the two methyl groups, or both. Thus, the metabolic behavior of this 2-nitroimidazole derivative appears to be similar to that previously demonstrated for the class of the isomeric 5-nitroimidazoles.

Metabolism of deflazacort in the rat, dog and man.

The metabolism of [2'-14C]deflazacort, (11 beta, 16 beta)-21-(acetoxyl)-11-hydroxy-2'-methyl-5'H-pregna-1, 4-dieno[17,16-d]oxazole-3,20-dione, orally given to rats, dogs, and humans, has been studied. From the urine of the three species and from rat bile and liver preparations, five main metabolites I-V have been isolated and their structures investigated by physicochemical analysis: 1,(5 beta,11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregn-1-eno[17,16-d]oxazole-3,20-dione; II, (11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; III, (6 beta,11 beta,16 beta)-6,11,21-trihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; IV, (3 epsilon,11 beta,16 beta)-3,11,21-trihydroxy-2'-methyl-5'H-pregn-5-eno[17,16-d]oxazol-20-one. Metabolites II and III are quantitatively the most important in the urine of the rat, dog, and man; metabolite V, whose structure is uncertain, has been found in human and rat urine. In the formation of metabolites I-V the fused 2-methyloxazoline ring is unmetabolized, whereas the steroid moiety follows the general metabolic pathways reported for other related corticosteroids.

Influence of metabolism on the activity of a new anti-fertility agent, 2-(3-ethoxyphenyl)-5,6-dihydro-s-triazolo [5,1-a]isoquinoline (DL 204-IT), in the rat and the hamster.

The activity of a new non-hormonal anti-fertility agent, 2-(3-ethoxyphenyl)-5,6-dihydro-s-triazolo[5,1-a]isoquinoline (DL 204-IT), effective in terminating pregnancy after i.m. or s.c. treatment, was tested after single and multiple oral doses given to rats and hamsters. Although the compound was absorbed well from the gastrointestinal tract and the plasma levels of the radioactivity administered were rather high and sustained, the oral activity was by two orders of magnitude lower than the parenteral one. The plasma profile of the metabolites found after both p.o. and i.m. administration indicates that the compound is rapidly metabolized. Seven metabolites (I-VII) were isolated from the urine of pregnant rats and fully characterized by MS, IR and NMR spectroscopy. They are 2-(3-hydroxyphenyl)-5,6-dihydro-s-triazolo[5,1-a]isoquinoline (I); 2-(3,4-dihydroxyphenyl)-5,6-dihydro-s-triazolo[5,1-1]isoquinoline (II); 2-(3-hydroxyphenyl)-5,6-dihydro-6 beta-hydroxy-s-triazolo[5,1-a]isoquinoline (III); metabolite I-sulphate ester (IV); metabolite III-3-sulphate ester (V); metabolite I-beta-D-glucuronide (VI) and metabolite II-3-beta-D-glucuronide (VII). Metabolite I was shown to be from 1/10 (rat) to 1/30 (hamster) as active as the parent compound, while metabolites II and III were completely inactive. The very low oral activity of DL 204-IT seems to be due mainly to its rapid biotransformation.

Disposition and metabolism of a new steroidal anti-inflammatory agent, deflazacort, in cynomolgus monkeys.

The kinetics and metabolic fate of 2'-14C-deflazacort, a new steroidal antiinflammatory agent, were studied in the cynomolgus monkey after both p.o. and i.v. administration (5 mg/kg). There is no unchanged deflazacort in the plasma or urine after either p.o. or i.v. treatment. As judged from the plasma AUC and urinary elimination values, the oral availability of both total 14C and metabolites seems to be lowered because of a route-dependent first-pass. Both radioactivity and the main metabolite (21-desacetyl deflazacort) are eliminated from the plasma with half-lives of 2--3-5 h. The i.v. administered 14C is eliminated mainly in the urine (52--55% of dose), but biliary excretion is also quantitatively important. Six metabolites were isolated from urine and identified by physico-chemical analysis. Among them desacetylated deflazacort and its 6 beta-hydroxy derivative were shown to be the major radioactive products in plasma and urine, respectively. Minor metabolites were: 21-desacetyl, 6 alpha-hydroxy deflazacort; 21-desacetyl, 5 alpha, 1-eno, deflazacort; 21-desacetyl, 20 beta hydroxy deflazacort; and 21-desacetyl, 11-keto deflazacort.

Pharmacokinetics of a new antihypertensive pyrrolyl pyridazinamine (MDL-899) in the rat and the dog.

The pharmacokinetics of N-(2,5-dimethyl-1H-pyrrol-1-yl)-6-(4-morpholinyl)-3-pyridazinamine hydrochloride (MDL-899), a new antihypertensive agent, was studied in rats and dogs. The 14C-labelled compound was synthesized by a microscale procedure with 45% chemical yield and 98% radiochemical purity. In both animal species, MDL-899 was rapidly absorbed from the gastro-intestinal tract, achieving peak plasma levels in 0.5-2 h. The ratio between the plasma concentrations of 14C and of unchanged MDL-899 indicates rapid metabolic transformation and, especially in the rat, a marked first-pass effect. MDL-899 binds to serum proteins with a very low affinity and rapidly enters the tissue compartment, with large distribution volumes (1.6 l/kg rat, 2.0 l/kg dog). Target tissues in the rat were the liver, kidneys, adrenals, lungs, ovaries, uterus and the arterial walls, which constitute a deep-compartment. The plasma half-life of unchanged MDL-899 was 0.5 h in the rat and 1.4 h in the dog, while the terminal half-lives for total radioactivity were much longer (two elimination phases). Within the range of doses tested (1 and 40 mg/kg) there is evidence of non-linear kinetics (dog). The plasma kinetics profiles of both MDL-899 and 14C were the same in both males and females (rat). In both rats and dogs, elimination of the test dose was preferentially via the kidneys, as metabolites. The time course of the pharmacological response seems to be correlated to the kinetics of the active species in deep-compartment(s) rather than to the plasma concentration.