Comparative and quantitative morphology of the pig and wild boar cerebellum for identifying possible effects of domestication.

The domestic pig (Sus scrofa domesticus) stems from the Eurasian wild boar (Sus scrofa): this offers an appealing window to study microanatomical changes related to the process of domestication, the symbiotic relationship between human and animal. In this light, we quantitatively demonstrated significant microanatomical differences between pig and wild boar cerebella. Calbindin D-28, a calcium binding protein, was employed as immunohistochemical marker of the Purkinje cells. Our results showed that: (i) the foliation index, expressing the rate of cerebellar cortical folding, and the number of granular cells were not significantly different between pigs and wild boars; (ii) area of the granular layer and the molecular layer, and area of white matter were lower in pigs; (iii) the fraction area, grey matter/white matter, was higher in pigs; (iv) the Purkinje cell linear density and their soma area were higher in wild boars. Despite the morphological data alone are not sufficient to draw any final conclusions, our findings on Purkinje cells may represent good indicators of a reduction of the pig cerebellum motor and cognitive functions during the process wild boar-to-pig domestication.

From the Semen Collection Method to the Hatchlings: The Use of Cryopreserved Sperm from Pheasants Fed an Antioxidant-Enriched Diet.

A widely used approach to preserving genetic diversity in birds involves the cryopreservation of semen. In this process, cells are subjected to physical and chemical stresses, but not all cell species respond equally. Many studies have been published on the freezing-thawing of sperm cells from a wide variety of domestic and wild species, on issues ranging from the sperm quality to different protocols, fertilisation success rates, etc. Nevertheless, very little information is available on the common pheasant. To fill this gap, the aim of this study was to describe the pheasant semen collection method, evaluate some qualitative parameters of sperm from males fed an antioxidant-enriched diet, and to test the in vivo fertilising capacity of the cryo-preserved semen. The freezing protocol employed involved pellets thawed by the hotplate method. Dimethylacetamide was used as a cryoprotectant at a final concentration of 6%. A total of six AIs were performed at 3-4-day intervals on a total of 40 females with doses of 35 × 106 of normal live thawed sperm. Males receiving the enriched diet produce more abundant and concentrated ejaculates. Freeze-thawed sperm lost 85% of their initial mobility, and diet influenced neither sperm mobility nor viability. The enriched diet did improve the number of normal freeze-thawed cells and was associated with a lower sperm fracture incidence. Regardless of the dietary group, frozen-thawed sperm resulted in a fertility rate of 30%, with 8-9 chicks hatching for every 100 eggs incubated.

Optimization of a Protocol for the Cryopreservation of Sperm in Pellets for the Common Pheasant (Phasianus colchicus mongolicus).

The sperm of each avian species and breed have unique characteristics that render them more or less susceptible to the freezing-thawing process; therefore, a suitable cryopreservation protocol that is specific for the sperm of each type of bird is needed. In this context, little information about the common pheasant's sperm is available. Therefore, the aim of this study was to test different parameters at each step of the process of freezing into pellets and thawing to detect the least deleterious parameter settings. Sixteen different protocols were tested by studying two levels in each of the four steps (dilution, equilibration at 5 °C, final dimethylacetamide concentration, and dimethylacetamide equilibration time) comprising the freezing process. The pheasant sperm exhibited a high susceptibility to the damage caused by freezing into pellets; however, the survival of the sperm reached 29%, and the greatest recovered mobility was 22%. The mobility of the sperm was affected by the dilution and the dimethylacetamide concentration, and the viability of the sperm was affected by the equilibration at 5 °C and the dimethylacetamide equilibration. The protocols that caused the least damage to the pheasant sperm were found to be those with higher dilution rates, 10 min of equilibration at 5 °C, and 6% dimethylacetamide equilibrated for 1 or 5 min. In the present study, we individualise some applicable parameters for certain critical steps of the freezing-thawing process; however, further investigations are needed in order to improve upon and complete a suitable protocol for the cryopreservation and thawing of pheasant sperm.

Histochemical Characterisation and Gene Expression Analysis of Skeletal Muscles from Maremmana and Aubrac Steers Reared on Grazing and Feedlot Systems.

This study aimed to characterise the fibre composition of Triceps brachii (TB) and Semimembranosus (SM) muscles from 20 Maremmana (MA) and 20 Aubrac (AU) steers, and the effect of grazing activity in comparison with feedlot system. The histochemical method was performed with the m-ATPase method with an acid pre-incubation, thus allowing to distinguish type I, IIA, and IIB fibres. Additionally, on total RNA extracted from SM muscle, the expressions of atp1a1, mt-atp6, and capn1 genes were evaluated, in order to find potential associations with muscle fibre histochemical characteristics. In SM muscle, the MA steers had the greater frequency of oxidative fibres (type I and IIA) and the higher atp1a1 expression, in comparison to AU steers. Conversely, AU steers had a greater frequency of type IIB fibres, and the higher capn1 expression. A similar histochemical pattern was observed in TB muscle. The grazing activity was probably insufficient to determine differences both for fibre proportion and size, and gene expressions, except for mt-atp6 expression that was surprisingly highest in feedlot MA in comparison to other steers. These findings further the knowledge of muscle properties belonging to these breeds, and the effect of voluntary physical activity since few studies were available in this regard.

The path from trigeminal asymmetry to cognitive impairment: a behavioral and molecular study.

Trigeminal input exerts acute and chronic effects on the brain, modulating cognitive functions. Here, new data from humans and animals suggest that these effects are caused by trigeminal influences on the Locus Coeruleus (LC). In humans subjects clenching with masseter asymmetric activity, occlusal correction improved cognition, alongside with reductions in pupil size and anisocoria, proxies of LC activity and asymmetry, respectively. Notably, reductions in pupil size at rest on the hypertonic side predicted cognitive improvements. In adult rats, a distal unilateral section of the trigeminal mandibular branch reduced, on the contralateral side, the expression of c-Fos (brainstem) and BDNF (brainstem, hippocampus, frontal cortex). This counterintuitive finding can be explained by the following model: teeth contact perception loss on the lesioned side results in an increased occlusal effort, which enhances afferent inputs from muscle spindles and posterior periodontal receptors, spared by the distal lesion. Such effort leads to a reduced engagement of the intact side, with a corresponding reduction in the afferent inputs to the LC and in c-Fos and BDNF gene expression. In conclusion, acute effects of malocclusion on performance seem mediated by the LC, which could also contribute to the chronic trophic dysfunction induced by loss of trigeminal input.

Immunolocalization of cannabinoid receptor 1 (CB1), monoglyceride lipase (MGL) and fatty-acid amide hydrolase 1 (FAAH) in the pig claustrum.

The claustrum (Cl) is a subcortical nucleus present in all mammalian species examined so far, whose function is still a puzzling problem. There is a wealth of data on its general anatomy, cytoarchitecture, and chemoarchitecture including the connectivity with both cortical and subcortical structures; instead, much less is known about the presence of the endocannabinoid system (ECs) an important neuromodulatory complex in this brain area. In an attempt to better understand the role of the ECs within the Cl circuitry, we undertook an immunohistochemical analysis to describe the distribution of the CB1 and of the endogenous cannabinoids degrading enzymes MGL and FAAH in the pig Cl as well as their relationship with both the catecholaminergic system and with parvalbumin (PV) expressing neurons. CB1, FAAH and MGL were expressed throughout the entire Cl. CB1 was expressed by fibers and puncta, while FAAH and MGL were mainly localized in the neuropil. FAAH also showed a faint cell body localization that colocalized with PV. Tyrosine hydroxylase positive fibers (catecholaminergic system), did not demonstrate the presence of CB1. Taken together, the results described herein provide evidence for an anatomical distribution of a CB1/PV signaling system in the pig Cl suggesting that PV cells may play a role within the ECs.

Retinal ganglion cells in Strigidae raptors: distribution and morphometry / Células ganglionares de la retina en aves rapaces Strigidae: distribución y morfometría

The authors studied the morphometry and the topographical distribution of Retinal Ganglion Cells (RGCs) in four nocturnal raptors of the order of Strigiformes, family of Strigidae: little owl, tawny owl, scops owl, eared owl. In order to recognize specialized retinal vision areas (fovea and visual streak), the number of RGCs/mm2 and the soma size in the four retinal fields (dorsal, ventral, temporal and nasal) by the histological analysis of retinal radial sections were recorded. A temporal fovea was identified in little owl, tawny owl and eared owl while in scops owl this visual area was localized near the fundus oculi. A radial visual streak ventrally directed was pointed out in the retinas of the four raptors with different shape according to its width. The Authors linked the obtained data with the predatory behavior of nocturnal raptors in their habitat.

Se estudió la morfometría y la distribución topográfica de las células ganglionares de la retina (CGR) en cuatro aves rapaces nocturnas del orden de los Strigiformes, familia Strigidae: búho pequeño, mochuelo, autillo, y cárabo. Con el objetivo de definir las áreas de visión retiniana especializadas (fóvea y campo visual), se registró el número de CGRs/mm2 y el tamaño del soma en los cuatro campos retinianos (dorsal, ventral, temporal y nasal), mediante análisis histológico de las secciones radiales de la retina. Se identificó una fóvea temporal en mochuelo, búho leonado y búho pequeño, mientras que en el búho real, esta área visual se localizó cerca del fondo de ojo. Se observó un campo radial visual dirigido ventralmente en las retinas de las cuatro aves rapaces, con diferentes formas según su extensión. Se relacionaron los datos obtenidos con el comportamiento predatorio de aves rapaces nocturnas en su hábitat.

Number and topographical distribution of retinal ganglion cells in diurnal and nocturnal raptors / Número y distribución topográfica de células ganglionares de la retina en rapaces diurnas y nocturnas

Retinal Ganglion Cells (RGCs) in two diurnal raptors, the buzzard (Buteo buteo) and kestrel (Falco tinnunculus), and in two nocturnal raptors, the little owl (Athene noctua) and tawny owl (Strix aluco), were studied in order to highlight their topographical distribution, taking into account the soma size. Cell density (RGCs/mm2) and median soma area from histological radial sections in four retinal fields (dorsal, ventral, temporal and nasal) were recorded in order to identify specialized retinal vision areas. The results showed a different RGCs distribution between diurnal and nocturnal raptors related to the location of the foveas and shape of the horizontal streak, confirming the bibliographic data. In diurnal raptors, a higher cell density was found in the temporal and central retinal fields as revealed by the presence of a temporal and a central fovea which showed a "horizontal streak". In nocturnal raptors the cell density was higher in the peripheral temporal field likely due to a temporal fovea. A peak in cell density observed in the ventral field, especially in the retina of little owls, might be linked to a more "radial visual streak". Comparing the data obtained we highlighted that the morphology and the number of RGCs are closely linked to the habitat and to the type of predation.

Se estudiaron las células ganglionares de la retina (RGC) en dos aves rapaces diurnas, el ratonero (Buteo buteo) y el cernícalo (Falco tinnunculus), y en dos aves rapaces nocturnas, el mochuelo (Athene noctua) y el búho leonado (Strix aluco), buscando resaltar su distribución topográfica, teniendo en cuenta el tamaño del soma. Se registraron la densidad celular (RGCs/mm2) y la media del área del soma de las secciones histológicas radiales en cuatro campos retinianos (dorsal, ventral, temporal y nasal) para identificar áreas de visión retinianas especializadas. Los resultados mostraron una distribución diferente de RGCs entre las rapaces diurnas y nocturnas relacionadas con la ubicación de las fóveas y la forma de la línea horizontal, lo que confirma los datos bibliográficos. En las aves rapaces diurnas, se encontró una densidad celular más alta en los campos de retina temporales y centrales como lo revela la presencia de una fóvea temporal y central que mostraba una "veta horizontal". En aves rapaces nocturnas, la densidad celular fue mayor en el campo temporal periférico probablemente debido a una fóvea temporal. Un aumento en la densidad celular, observado en el campo ventral, especialmente en la retina de los pequeños buhos, podría estar relacionado con una "línea visual radial". Comparando los datos obtenidos, resaltamos que la morfología y el número de RGC están estrechamente relacionados con el hábitat y el tipo de depredación.

Ultramicronized palmitoylethanolamide counteracts the effects of compound 48/80 in a canine skin organ culture model.

BACKGROUND: Ultramicronized palmitoylethanolamide (PEA-um) has been reported to reduce pruritus and skin lesions in dogs with moderate atopic dermatitis and pruritus. HYPOTHESIS/OBJECTIVES: A canine ex vivo skin model was used to investigate the ability of PEA-um to counteract changes induced by compound 48/80, a well-known secretagogue that causes mast cell degranulation. ANIMALS: Normal skin was obtained from three donor dogs subjected to surgery for reasons unrelated to the study. METHODS: Cultured skin biopsy samples in triplicate were treated with 10 and 100 µg/mL compound 48/80, without or with 30 µM PEA-um. Mast cell (MC) degranulation, histamine release into the culture medium, local microvascular dilatation, epidermal thickness, keratinocyte proliferation and epidermal differentiation markers were evaluated. RESULTS: Exposure of the skin organ culture to PEA-um 24 h before and 72 h concomitantly to compound 48/80 resulted in a significant decrease of degranulating MCs. PEA-um also reduced the histamine content in the culture medium by half, although the effect did not reach statistical significance. PEA-um significantly counteracted vasodilation induced by 100 µg/mL compound 48/80. Finally, PEA-um alone did not induce changes in epidermal thickness, differentiation markers, keratinocyte proliferation, MC density and/or degranulation. CONCLUSIONS AND CLINICAL IMPORTANCE: Collectively, these results support the protective action PEA-um on the skin of dogs undergoing allergic changes.

Histological Characteristics, Fatty Acid Composition of Lipid Fractions, and Cholesterol Content of Semimembranosus and Triceps Brachii Muscles in Maremmana and Limousine Bovine Breeds.

This study examined the histological properties of Semimembranosus and Triceps brachii muscle in two different bovine breeds, Maremmana (Ma) (an autochthonous breed from Tuscany, Italy) and Limousine (Lm). The animals were grazed in two adjoining pastures, received the same feed supplementation, and were weighed monthly. The experimental period lasted from weaning (6 months old) to slaughter (19 months old). Muscle samples were collected immediately after slaughter, before carcass cooling. Regarding the histological properties, the number of muscle fibers (TNF), mean sarcolemma perimeter (MSP), cross-sectional area, and total sarcolemma perimeter (TSP) were determined. Samples were also analyzed for proximate composition, fatty acid profile of total lipids (TLs), phospholipids (PLs), and neutral lipids (NLs), and for total cholesterol content. Breed was a significant variation factor for the performance parameter and histological muscle fiber properties. Interestingly, despite that Ma being a less extensively genetically improved breed than Lm, it showed higher weight at slaughter (+18%) and daily weight gain (+19%). Ma also showed smaller muscle fibers than Lm and, consequently, the TSP was higher. This difference affected the lipid fraction distribution (Lm was higher in PLs and lower in NLs than Ma) and, consequently, the fatty acid composition of TLs (Lm was high in polyunsaturated fatty acids, while Ma was high in monounsaturated fatty acids). The results of this experiment highlight the importance of environmental and management conditions on the full expression of genotypic potential.

Spatial distribution of cannabinoid receptor 1 and fatty acid amide hydrolase in the cat ovary and oviduct.

Involvement of the endocannabinoid system in female reproduction has been extensively described in humans with the cognate receptors and ligands being found in the ovaries and genital tract. In human, an imbalance of the endocannabinoid system is linked with both ectopic pregnancy and infertility. In bovine species anandamide levels regulate aspects of sperm-oviduct interaction. Here we report the immunohistochemical distribution of cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) in cat ovary and oviduct, using paraffin-embedded tissue samples and commercially available antibodies. We found a differential expression of both CB1R and FAAH during different stages of ovarian function and in the oviduct. CB1R was detected only in tertiary follicle granulosa cells while more immature follicles were negative. FAAH was instead found in ovarian pre-antral follicles, the oocyte cytoplasm, and in granulosa cells of primary, secondary and tertiary follicles. Secondary and tertiary follicles were also FAAH immunoreactive. Luteal cells were immunopositive for both CB1R and FAAH. Because CBR1 in oviduct was found only in ciliated cells, it might represent a specific marker at least in cats. In contrast, FAAH immunoreactivity was observed in both ciliated and non-ciliated cells. The present study may thus serve as the starting point for further investigations on the role of the endocannabinoid system in cat reproduction. Additional work will be needed to assess whether the morphological distribution of CB1R and FAAH changes in different conditions such as pre-pubertal age, follicular phase of the sexual cycle and pregnancy.

Morphological study of the iris musculature in diurnal and nocturnal raptors / Estudio morfológico de la musculatura del diafragma en aves rapaces diurnas y nocturnas

In literature it is established that the iris musculature consists of striate muscle fibers in birds while in mammals it consists of smooth muscles. Some authors report the presence of smooth muscle tissue also in the iris of some species of birds. In the present study we report on the iris muscle tissues (type of tissue, direction and mean diameter of muscle fibers or cells) in five species of Accipitriformes (diurnal raptors) and four species of Strigiformes (nocturnal raptors) because they show different way of life depending of their predatory behavior. This morphological study was carried out from raptors died or euthanized at the Wild Life Rescue Centre of Sea and Water birds in Livorno (Italy). From histological examination of iris serial radial sections we find both striated and smooth musculature even if with marked differences among analyzed species, not directly correlated with diurnal or nocturnal lifestyle. Striated fibers are always present, mainly with cross direction, throughout the iris stroma, while the histological differences concern the smooth cells. Indeed, harrier and sparrow hawk (Accipitriformes) and great horned owl and little owl (Strigiformes) show a compact layer of cross smooth muscle cells throughout the iris stroma. In the other species analyzed smooth muscle cells are slightly detectable as scattered or not detectable. Since the cross smooth muscle tissue allows to maintain a myotic state for extended periods of time, our results might be correlated more to the predatory behavior than the taxonomic order.

En la literatura, se establece que la musculatura del iris se compone de fibras musculares estriadas en las aves, mientras que en los mamíferos, la forman los músculos lisos. Algunos autores informan también de la presencia de tejido muscular liso en el iris de algunas especies de aves. El presente estudio informa sobre los tejidos musculares del iris (tipo de tejido, la dirección y diámetro de las fibras musculares o células) en cinco especies de Accipitriformes y cuatro especies de Strigiformes que muestran diferentes hábitos en función de su comportamiento depredador. Este estudio morfológico se realizó en aves rapaces que murieron o fueron eutanasiadas en el Centro de Vida Salvaje de Rescate de Aves Marinas y Acuáticas de Livorno (Italia). El examen histológico de secciones seriadas radiales del iris mostró la presencia tanto de musculatura estriada como lisa, aunque con marcadas diferencias entre las especies analizadas, pero sin correlación directa con el estilo de vida diurna o nocturna. Las fibras estriadas estuvieron siempre presentes, principalmente en dirección transversal a lo largo del estroma del iris, mientras que las diferencias histológicas fueron de las células lisas. Tanto el aguilucho y el gavilán (Accipitriformes) como el buho real y el mochuelo (Strigiformes) mostraron una capa compacta de células musculares lisas transversales en todo el estroma del iris. En las otras especies analizadas, las células musculares lisas fueron ligeramente detectables de manera dispersa o no detectables. Dado que el tejido del músculo liso transversal permite mantener un estado miótico durante largos períodos de tiempo, nuestros resultados podrían estar más correlacionados con el comportamiento depredador, y no con el orden taxonómico.

Establishment of a 2-week canine skin organ culture model and its pharmacological modulation by epidermal growth factor and dexamethasone.

Although canine skin models are already available as either monocellular or organotypic cultures, they only partly recapitulate normal skin morphological features and function. The objective of this study was to establish a canine serum-free skin organ culture model and verify whether dexamethasone could rescue epidermal growth factor-induced changes. The study of morphological changes as a response to pharmacological substances may indeed help to investigate skin physiology and pathology. Normal skin was obtained from five client-owned dogs subjected to surgical procedures unrelated to dermatological conditions. Two experimental designs were performed: (i) two-week viability of the skin culture; (ii) dexamethasone (DMS) inhibition of epidermal growth factor (EGF)-induced effects. Serum-free submerged organ cultures were established in Williams' E medium supplemented with penicillin-streptomycin, insulin, hydrocortisone and l-glutamine. General morphological features of skin anatomical structures were well maintained up to day 14, scattered pyknotic nuclei were visible in the epidermis from day 7. Normal keratinocyte differentiation was confirmed by cytokeratin (K) 10, K14 and loricrin immunostaining. Epidermal thickness did not decrease throughout the study. A decrease in keratinocyte proliferation was observed at day 7 and 14. Treatment with EGF induced both keratinocyte proliferation and thickening of the epidermis; both responses were counteracted by DMS. Treatment with EGF increased the length of epithelial tongues at the edge of the skin explants; this effect was further enhanced by DMS supplementation. Our findings demonstrate the potential use of a full-thickness canine skin organ culture model for the study of skin physiology and pharmacological response to exogenous compounds, especially in the field of re-epithelialisation and keratinization disorders.

Immunohistochemical distribution of the cannabinoid receptor 1 and fatty acid amide hydrolase in the dog claustrum.

Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum.

Preferential epithelial expression of type-1 cannabinoid receptor (CB1R) in the developing canine embryo.

The use of cannabinoid receptor agonists is gaining a strong interest both in human and veterinary medicine. The potential use of cannabimimetic compounds in companion animals was reviewed in 2007 for their role in tissue inflammation and pain. A better knowledge of type-1 cannabinoid receptor (CB1R) expression on the target population may help in risk management in order to prevent unwanted side effects. We used 30-days old canine embryos to describe the distribution of CB1R by means of immunohistochemistry with a commercially available antibody.CB1R immunoreactivity was mainly epithelial and included most structures of central and peripheral nervous system, inner ear, olfactory epithelium and related structures, eye and thyroid. Further investigative research on the role of the endocannabinoid system in the developmental biology field is needed, however, we show that in the canine species we must consider pregnancy as risk condition for developmental abnormalities that may arise upon the use of CB1R receptor agonists.

Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum.

The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyze the localization and morphology of neurons expressing parvalbumin (PV), calretinin (CR), NPY, and somatostatin (SOM) in the claustrum of man (# 5), chimpanzee (# 1) and crab-eating monkey (# 3). Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir) neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY), was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex.

Topical formulations containing finasteride. Part II: determination of finasteride penetration into hair follicles using the differential stripping technique.

The differential stripping technique consists of a tape-stripping phase followed by a cyanoacrylate biopsy. This technique not only allows the quantification of drug retained in the stratum corneum (SC) and in the hair follicles but also differentiates transepidermal from transfollicular penetration. Our study aimed at both validating the differential stripping procedure on hairless rat skin and assessing the role of the hair follicle in the cutaneous penetration of finasteride (FNS) after application of two experimental formulations for 6 or 24 h: P-08-016, a hydroxypropyl chitosan (HPCH)-based formulation and P-10-008, an anhydrous formulation devoid of HPCH. Microscopic and histological evaluation showed that after 15 tape strips both the SC and the viable epidermis were completely removed. A subsequent cyanoacrylate skin surface biopsy led to the removal of the infundibula content. The largest amounts of FNS were found in the epidermis and in the appendages after application of P-08-016, regardless of the time from application. In contrast, smaller and statistically significant amounts of FNS were recovered with P-10-008 6 h after application, compared with that at 24 h. In conclusion, the differential stripping technique allowed determination of the amount of FNS localized in different skin districts, focusing particularly on the follicular contribution.

Topical formulations containing finasteride. Part I: in vitro permeation/penetration study and in vivo pharmacokinetics in hairless rat.

In hair follicle (Hf) cells, the type-2 5-α-reductase enzyme, implicated in androgenetic alopecia, is selectively inhibited by finasteride (FNS). Because an effective topical formulation to deliver FNS to Hf is currently unavailable, this investigation aimed at evaluating in vitro FNS skin permeation and retention through and into hairless rat and human abdominal skin. Four hydroxypropyl chitosan (HPCH)-based formulations (P-08-012, P-08-016, P-08-063, and P-08-064) and one anhydrous formulation without HPCH (P-10-008) were tested. The pharmacokinetics in plasma and skin after application of P-08-016 or P-10-008 on dorsal rat skin with single and repeated doses was investigated. P-08-016 performed the best in driving FNS to the reticular dermis without producing a high transdermal flux. Neither the in vivo single nor the repeated dose experiments produced plasma levels of FNS and no differences were found between formulations concerning skin retention. No increase in the amount of drug retained in the skin was obtained with the repeated dose experiment. In conclusion, the HPCH-based formulation P-08-016 might represent an alternative to systemic therapy for its ability to promote a cutaneous depot of FNS in the region of hair bulbs, minimizing systemic absorption even after repeated treatments.

An autologously generated platelet-rich plasma suturable membrane may enhance peripheral nerve regeneration after neurorraphy in an acute injury model of sciatic nerve neurotmesis.

BACKGROUND: The aim of this study was to investigate the ability of suturable platelet-rich plasma (PRP) membrane to promote peripheral nerve regeneration after neurotmesis and neurorraphy. METHODS: A total of 36 rats were used: 32 animals underwent surgery and were split in two groups. An interim sacrifice was performed at 6 weeks postsurgery and final sacrifice at 12 weeks; four animals did not sustain nerve injury and served as control. Clinical, electromyographic (EMG), gross, and histological changes were assessed. The EMG signal was evaluated for its amplitude and frequency spectrum. Number of regenerating fibers, their diameter, and myelin thickness were histologically analyzed. RESULTS: Both EMG parameters showed a significant (p < 0.05) effect of treatment at 6 and 12 weeks postsurgery. At 6 weeks, the fiber density was statistically different between treated and untreated animals with a higher observed density in treated nerves. No difference in fiber density was observed at 12 weeks postsurgery. The distribution of fiber diameters showed an effect at 12 weeks when only the sections of the nerves sutured with PRP showed fibers with diameters greater than 6 µm. DISCUSSION: Our data show that the application of a PRP fibrin membrane around the neurorraphy improves the nerve regeneration process in a rat sciatic nerve model. The use of PRP as a suturable membrane could perform an action not only as a source of bioactive proteins but also as a nerve guide to hold the scar reaction and thus improve axonal regeneration.

Topography of Gng2- and NetrinG2-expression suggests an insular origin of the human claustrum.

The claustrum has been described in the forebrain of all mammals studied so far. It has been suggested that the claustrum plays a role in the integration of multisensory information: however, its detailed structure and function remain enigmatic. The human claustrum is a thin, irregular, sheet of grey matter located between the inner surface of the insular cortex and the outer surface of the putamen. Recently, the G-protein gamma2 subunit (Gng2) was proposed as a specific claustrum marker in the rat, and used to better delineate its anatomical boundaries and connections. Additional claustral markers proposed in mammals include Netrin-G2 in the monkey and latexin in the cat. Here we report the expression and distribution of Gng2 and Netrin-G2 in human post-mortem samples of the claustrum and adjacent structures. Gng2 immunoreactivity was detected in the neuropil of the claustrum and of the insular cortex but not in the putamen. A faint labelling was present also in the external and extreme capsules. Double-labelling experiments indicate that Gng2 is also expressed in glial cells. Netrin-G2 labelling was seen in neuronal cell bodies throughout the claustrum and the insular cortex but not in the medially adjacent putamen. No latexin immunoreactive element was detected in the claustrum or adjacent structures. Our results confirm that both the Gng2 and the Netrin-G2 proteins show an affinity to the claustrum and related formations also in the human brain. The presence of Gng2 and Netrin-G2 immunoreactive elements in the insular cortex, but not in the putamen, suggests a possible common ontogeny of the claustrum and insula.