The isoproterenol-induced myocardial fibrosis: A biochemical and histological investigation.

The isoproterenol (ISO)-induced myocardial fibrosis is considered a reliable and repeatable experimental model characterized by a relatively low mortality rate. Although is well-known that ISO stimulates the ß1 adrenergic receptors at the myocardial level, a high degree of heterogeneity emerges around the doses and duration of the treatment generating unclear results. Therefore, we propose to gain insights into the progression of ISO-induced myocardial fibrosis, in order to critically analyze and optimize the experimental model. Male Wistar rats (12-14-week-old) were submitted to subcutaneous injection of ISO, in particular, two doses were selected: the commonly used dose of 5 mg/kg and a lower dose of 1 mg/kg, administered for 3 and 6 days. Biochemical and histological examinations were conducted either immediately after the last administration or after a recovering period of 7 or 14 days from the initial administration. Noteworthy, from our investigation emerged that even the lower dose of ISO was able to induce the maximal biochemical and histological alterations, suggesting that lower doses should be considered to control the progression of the damage more precisely and to identify a prodromic phase in which intervention with pharmacological or nutraceutical tools can be effectively attempted.

Three-dimensional morphometry of kidney in New Zealand rabbit using unbiased design-based stereology.

The rabbit is widely used as a laboratory animal in experimental models of kidney diseases. This species is also important from a veterinary perspective as a companion animal. Stereology has been accepted as an accurate approach to kidney morphometry. The objective of the present project was to provide normal quantitative stereological parameters for adult rabbit kidneys. The left kidneys of five adult male New Zealand rabbits were used. Isotropic sections were obtained using the orientation method. Total kidney volume was calculated by the Cavalieri principle. The volume fraction of the renal structures was estimated using the point counting system. The lengths of the proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) were calculated using counting frames. The total glomerular number was accounted for using the physical/fractionator technique. The mean glomerular volume was obtained by dividing the total volume of glomeruli by their total number. The total volume of rabbit kidneys calculated was 10.39 ± 1.98 cm3. The fractional volume of the kidney cortex and medulla accounted for 57.79 ± 0.65% and 42.2 ± 0.65%, respectively. The total glomerular volume was 2.18 ± 0.32% of the whole kidney. The total number of glomeruli in the rabbit kidney was estimated as 204.68 ± 12 × 103. The mean glomerular volume measured 1.07 ± 0.12 × 106 µm3. The total length of PCT and DCT was 2.96 ± 0.29 km and 1.38 ± 0.24 km, respectively. These findings can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals by comparing with available data on humans and other species. RESEARCH HIGHLIGHTS: Three-dimensional morphometry of adult rabbit kidney structures was analyzed using quantitative stereology. Total volume of kidney, fractional volume of cortex and medulla, length of renal tubules and number of nephrons were estimated. These three-dimensional morphometrical data can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals.

Improved Cardiovascular Effects of a Novel Pomegranate Byproduct Extract Obtained through Hydrodynamic Cavitation.

The healthy properties of pomegranate fruit, a highly consumed food, have been known for a long time. However, the pomegranate supply chain is still rather inefficient, with the non-edible fraction, whose weight is roughly half the total and is endowed with plenty of valuable bioactive compounds, either disposed of or underutilized. A novel extract obtained from non-edible byproducts (called PPE), using hydrodynamic cavitation, a green, efficient, and scalable technique, was investigated for its cardiovascular effects in vivo. PPE showed efficacy in an acute phenylephrine (PE)-induced hypertensive rat model, similar to the extract of whole fruit (PFE) obtained using the same extractive technique, along with good intestinal bioaccessibility after oral administration. Finally, when chronically administered for 6 weeks to spontaneously hypertensive rats, PPE was shown to significantly contain the increase in systolic blood pressure, comparable to the reference drug Captopril, and at a dose remarkably lower than the reported effective dose of ellagic acid. The extract from the non-edible fraction of the pomegranate fruit also showed good anti-inflammation and anti-fibrotic effects. The findings of this study, along with the extraction technique, could contribute to enhancing the value of the pomegranate supply chain, relieve the related environmental burden, and potentially improve public health.

Swine Pudendal Nerve as a Model for Neuromodulation Studies to Restore Lower Urinary Tract Dysfunction.

Lower urinary tract dysfunction, such as incontinence or urinary retention, is one of the leading consequences of neurological diseases. This significantly impacts the quality of life for those affected, with implications extending not only to humans but also to clinical veterinary care. Having motor and sensory fibers, the pudendal nerve is an optimal candidate for neuromodulation therapies using bidirectional intraneural prostheses, paving the way towards the restoration of a more physiological urination cycle: bladder state can be detected from recorded neural signals, then an electrical current can be injected to the nerve based on the real-time need of the bladder. To develop such prostheses and investigate this novel approach, animal studies are still required since the morphology of the target nerve is fundamental to optimizing the prosthesis design. This study aims to describe the porcine pudendal nerve as a model for neuromodulation studies aiming at restoring lower urinary tract dysfunction. Five male farm pigs were involved in the study. First, a surgical procedure to access the porcine pudendal nerve without muscle resection was developed. Then, an intraneural interface was implanted to confirm the presence of fibers innervating the external urethral sphincter by measuring its electromyographic activity. Finally, the morphophysiology of the porcine pudendal nerve at the level of surgical exposure was described by using histological and immunohistochemical characterization. This analysis confirmed the fasciculate nature of the nerve and the presence of mixed fibers with a spatial and functional organization. These achievements pave the way for further pudendal neuromodulation studies by using a clinically relevant animal model with the potential for translating the findings into clinical applications.

Three-dimensional morphometry of the testis in dog using design-unbiased stereology.

Testis is considered the main organ of the male reproductive system. Dogs are used as a suitable experimental model of testicular diseases in humans. From the veterinary aspect, several disorders have been reported to affect the testis in dogs. Thus, the objective of the present study was to investigate the morphometrical features of the dog testis using design-based stereology. The testes of six male dogs were used. Isotropic, uniform random sections were obtained and processed for light microscopy. Testicular total volume and the fractional volume of the seminiferous tubules, interstitial tissue and germinal epithelium were measured using the Cavalieri's estimator and the point counting system. Germinal epithelial surface area was estimated using test lines, and total length of seminiferous tubules was analysed using the counting frames. The total volume of testis was calculated 13.64 ± 1.94 cm3 . The relative volume fractions of the seminiferous tubules, interstitial tissue and germinal layer expressed as a percentage of total testicular volume were found to be 75.87 ± 6.11%, 23.68 ± 5.15% and 64.15 ± 4.82%, respectively. The surface area of the germinal layer was 915.25 ± 150.48 cm2 . The thickness of germinal layer was estimated to be 96.18 ± 10.72 µm. The total length of seminiferous tubules measured 290.8 ± 35.86 m. No statistical difference in investigated parameters was found between the left and right testes (p > 0.05). Our data might contribute to the male reproductive knowledge, help develop experimental studies in this field and possibly lead to advancement in the diagnosis and treatment of testicular diseases in the dog.

Glial cells are affected more than interneurons by the loss of Engrailed 2 gene in the mouse cerebellum.

Glial cells play a pivotal role in the inflammatory processes, which are common features of several neurodevelopmental and neurodegenerative disorders. Their major role in modulating neuroinflammation underscores their significance in these conditions. Engrailed-2 knockout mice (En2-/- ) are considered a valuable model for autism spectrum disorder (ASD) due to their distinctive neuroanatomical and behavioral traits. Given the higher prevalence of ASD in males, our objective was to investigate glial and interneuron alterations in the cerebellum of En2-/- mice compared with wild-type (WT) mice in both sexes. We employed immunohistochemical analysis to assess cell density for all cell types studied and analyzed the area (A) and shape factor (SF) of microglia cell bodies. Our findings revealed the following: (a) In WT mice, the density of microglia and astrocytes was higher in females than in males, while interneuron density was lower in females. Notably, in En2-mutant mice, these differences between males and females were not present. (b) In both male and female En2-/- mice, astrocyte density exceeded that in WT mice, with microglia density being greater only in females. (c) In WT females, microglia cell bodies exhibited a larger area and a lower shape factor compared to WT males. Remarkably, the En2 mutation did not appear to influence these sex-related differences. (d) In both male and female En2-/- mice, we observed a consistent pattern: microglia cell bodies displayed a larger area and a smaller shape factor. Given the ongoing debate surrounding the roles of glia and sex-related factors in ASD, our observations provide valuable insights into understanding how an ASD-associated gene En2 affects specific cell types in the cerebellum.

Clinical, histopathological, and molecular characterization of canine pigmented viral plaques.

Canine pigmented viral plaques (PVPs) are proliferative epidermal lesions caused by canine papillomaviruses (CPVs). Although the lesions are benign, neoplastic transformation has been reported. Cases reported in the literature are few and mainly focused on genome sequencing. The aim of this study was to collect data on the epidemiology, clinicopathological features, and genotyping of PVPs. Fifty-five canine PVPs were retrospectively retrieved and histologically evaluated. Follow-up was available for 33 cases. The median age was 6.5 years and pugs were the most represented breed (25%). There were 4 clinical presentations: a single lesion (24%), multiple lesions (75%) in one (41%) or different sites (34%), and generalized lesions all over the body (24%). The abdomen and axillae were the most common sites. In single lesions, no recurrence was observed after conventional surgery, whereas different medical treatments reported for multiple lesions were not successful. Spontaneous regression was reported in 3 cases. Neoplasia in contiguity with PVPs was seen in 5 of 55 lesions (9%), and 1 dog was euthanized due to invasive squamous cell carcinoma (SCC). The most useful histopathological features for diagnosis were scalloped profile, epidermal spikes, hypergranulosis, and hyperpigmentation. L1 immunolabeling was present in 14 of 16 cases (87%). Sequencing revealed that 10 of 16 cases were associated with CPV-9 (71%), 2 cases were associated with CPV-4 (14%), and 2 cases were associated with CPV-8 (14%). In conclusion, this represents a large cohort study on canine PVPs reporting data on clinicopathological features, therapy, outcome, and the type of CPV involved for the first time in Italy.

Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum.

The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

Localization and genotyping of canine papillomavirus in canine inverted papillomas.

Numerous canine papillomaviruses (CPVs) have been identified (CPV1-23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species.

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.

Gross and histological findings in the canine placenta and amnion at term: What's normal, abnormal or pathological?

The canine placenta is an underexamined organ. Placental abnormalities can affect foetus development and may be responsible for a low weight of the infant at birth; however, knowledge on their clinical significance in the canine species is limited. We aimed to describe macroscopic and microscopic findings in the canine placenta and amnion at term in clinically uncomplicated pregnancies and to evaluate their relationship with birth weight of healthy puppies. During natural delivery or C-section, the birth weight of 82 puppies was recorded, 72 placentas and 66 amnions were recovered. The foetal and maternal surfaces of the placental girdle, marginal haematoma and amnion were evaluated. Each gross finding was recorded, morphometrically assessed and sampled for histological diagnosis. Furthermore, specimens of placenta and amnion were collected from representative areas and microscopic deviations from normal structure were evaluated in haematoxylin and eosin sections. Gross examination revealed 'abnormalities' in the 75.4% of the collected placentas. Necrosis was the gross change most commonly observed in the placental girdle (72.5%). Congestion (17.4%) and clotted blood/fibrinoid material (2.9%) were also observed. No gross changes of either the marginal haematoma or the amnion were recorded. Histologically, placental girdle showed necrosis (62.3%), mineralization (52.2%), congestion (36.2%) and neutrophilic infiltration (27.5%). Marginal haematoma exhibited mineralization (11.6%) and neutrophils (29%), while necrotic foci were rarely observed (4.3%). In the amnion, the most frequent alteration observed was hypertrophy of the epithelium (35.9%) followed by oedema (31.2%), mineralized foci (28.1%), fibrosis (23.4%), congestion (15.6%) and more rarely neutrophils (12.5%). Puppies' birth weight was not statistically affected by either gross or histological abnormalities. Our study revealed that macroscopic and microscopic 'abnormalities' of the placenta and amnion may be common in uncomplicated pregnancies at term; however, no implications on puppies' birth weight were observed. Deviations from 'normal' morphology of canine foetal adnexa warrant further investigation to assess their clinical implications if present.

Age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs.

There are age-related changes in testicular anatomy and physiology whereby there are modifications of sperm production and reproductive hormone functions. Effects of age on testicular microanatomy are well documented in humans, while there is limited understanding of these changes in dogs. The aim of this study was to evaluate age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs. Dogs (n = 32) were divided into four age groups: peripubertal (n = eight), relatively younger (n = seven), reproductively mature (n = seven) and relatively older (n = ten). Picrosirius Red stained sections were used for morphometrical analysis of testicular tissues, while the characteristics of seminiferous epithelium were assessed using a modified Johnsen scoring system for haematoxylin and eosin stained sections. Seminiferous epithelium and seminiferous tubule area increased from peripuberty to reproductive maturity, indicating there were changes during sexual maturation and subsequently there were decreases with further aging. There was a similar age-related trend for changes in seminiferous epithelium height with values being greatest in reproductively mature dogs; while there were no age-related differences in tubular diameter. Collagen content in the testicular interstitium gradually decreased from peripuberty to the age when dogs were reproductively mature and there were subsequent increases in relatively older dogs, thus, there was an association between the extent of testicular fibrosis and senescence. There was a decrease in spermatogenetic functions from relatively younger to older ages. Further investigations are warranted to establish mechanisms responsible for age-related changes of testicular morphology and related clinical implications.

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.

Relationship between placental characteristics and puppies' birth weight in toy and small sized dog breeds.

Puppy neonatal mortality may be related to low birth weight, which has been shown in humans to be linked to placental factors. The relation between the newborn puppy and the placental characteristics has been poorly investigated in dogs. Twenty bitches, 9 toy-sized (i.e. < 5 Kg) and 11 small-sized (i.e. 5 to 10 Kg), were included in this study. During natural delivery or c-section, puppies were identified and their order of birth, sex and weight were recorded. Puppy weights were registered at birth and daily until Day 6. Placentas were weighed after removal of extraplacental adnexa, after which a photo was taken and morphometrically assessed. The Total Placental Area (TPA) and the Transfer Zone Area (TZA) were calculated and their surface expressed in mm2. Immunohistochemistry with monoclonal mouse anti CD31 antibody was used to identify fetal and maternal vessels in the placental labyrinth zone. A vascularization index (VI) was determined for each placenta and the Total Vascular Area (TVA) was estimated. Puppies' birth weight correlated positively with placental weight (P < 0.001, r = 0.689). A positive correlation was found between the puppies' birth weight and TPA (P < 0.001, r = 0.786), TZA (P < 0.001, r = 0.772), and TVA (P < 0.001, r = 0.482). A positive correlation was also found between placental weight and TPA (P < 0.001, r = 0.661), TZA (P < 0.001, r = 0.583), and TVA (P < 0.001, r = 0.333). In the small-sized breeds, the placentas of low-weight puppies were lighter and had a smaller TZA and TVA (P < 0.05). The VI was higher in the placentas of the toy-sized compared to small-sized bitches (P < 0.01). No effect of parity, litter size, or sex of the puppy was observed on birth weight or growth rates on Day 6. The growth pattern of low-weight puppies did not differ from that of the other puppies during the first 6 days of life. As in humans, placental weight, the extension of the transfer zone and placental total vascular area correlates closely with the puppies' birth weight in normal pregnancies. Our data could represent reference values for placental weight, TZA, TVA and VI in toy and small-sized dog breeds.

Normal microscopic anatomy of equine body and limb skin: A morphological and immunohistochemical study.

INTRODUCTION: Information on microscopic anatomy of equine skin is sparse. In horses, limb wounds often become chronic and/or non-healing whereas body wounds heal normally. These dissimilarities in healing patterns might be a product of different phenotypic characteristics of body and limb skin. The objective of this study was to investigate microscopic anatomy, epidermal thickness, keratinocyte proliferation and differentiation as well as the presence of mast cells in normal equine skin of body and limb. MATERIALS AND METHODS: The study involved body and limb skin biopsies from six horses. Histological characteristics of the epidermis were assessed and epithelial thickness measured. Immunohistochemistry was performed to investigate epidermal differentiation patterns of cytokeratin (CK) 10, CK14, CK16, loricrin, and peroxisome proliferator-activated receptor alpha (PPAR-α), epidermal proliferation (Ki-67 immunostaining), and mast cells distribution in the skin. RESULTS: The epidermis was significantly thicker in the limb skin compared to body skin (p<0.01). Epidermal proliferation and CK distribution did not show differences in the two anatomical areas. Loricrin presence was focally found in the spinous layer in four out of six limb skin samples but not in body skin samples. Tryptase positive mast cells were detected in the dermis and their density (cell/mm2) was not different between body and limb. DISCUSSION AND CONCLUSION: Here we report for the first time about the normal distribution of CK10, CK14, CK16, PPAR-α, and loricrin in equine limb and body skin as well as about epidermal proliferation rate and mast cell count. It will be relevant to investigate the distribution of the investigated epithelial differentiation markers and the role of mast cells during equine wound healing and/or other skin diseases.

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.