Andrea Marchegiani

In vivo Biocompatibility of p(HPMAm-lac)-PEG Hydrogels Hybridized with Hyaluronan

The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm‐lac1–2)‐PEG‐p(HPMAm‐lac1–2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross‐linking forming a viscoelastic spherical depot at the injection site. The residence time in vivo (20 days) was found to be shorter than that observed in vitro (32 days), indicating that the injected hydrogel was resorbed not only by chemical hydrolysis but also by cellular metabolism and/or enzymatic activity. Systemic biocompatibility was tested by analysing routine haematological parameters at different time‐points (7, 14 and 21 days after administration) and histology of the main organs, including the haematopoietic system. No statistically significant difference between parameters of the saline‐treated group and those of the hydrogel‐treated group was found. Importantly, a time‐dependent decrease of important pro‐inflammatory cytokines (TREM1 (Triggering Receptor Expressed on Myeloid cells‐1), tumour necrosis factor‐α and interleukin‐1β) in cultured bone marrow cells extracted from hydrogel treated mice was observed, possibly correlated to the anti‐inflammatory effect of hyaluronic acid released in time as hydrogel degraded. Copyright

Ultrasonographic biometry of the eyes of healthy adult donkeys

Sixty-two healthy adult donkeys were included in this study, giving a total of 124 eyes for examination. The weight of the donkeys was estimated and an ultrasonography of the eyes was performed using a curvilinear transducer. Ocular measurements were taken in a horizontal plane and included the following values: globe axial length (GAL), anterior chamber depth (ACD), vitreous body depth (VD), lens diameter (LDi) and lens depth (LDe). The mean and sds for each measurement are reported in mm: GAL 34.22±2.05; ACD 3.01±0.58; VD 20.20±1.63; LDi 17.96±1.66 LDe 11.06±0.71. Gender was not a variability factor for ocular biometry in donkeys, while the weight was directly related to the ultrasonographic ocular values. Lens dimensions represented an exception and further investigation should be carried out to verify a possible correlation with age rather than weight. This is the first paper reporting reference data for ocular biometry in donkeys. The ultrasonographic evaluation of the equine eye is a manageable procedure that is easy to perform and can provide information not always obtainable with direct ocular examination. It allows the imaging of intraocular and retrobulbar structures and the diagnosis of some important disorders involving these areas (Scotty and others 2004, Michau 2005, Dietrich 2007). Ocular ultrasound is also indicated where it is impossible to directly visualise (eg, with an ophthalmoscope) posterior structures of the globe in cases of corneal oedema or ulceration, cataract or ocular masses (Withcomb 2002). Ultrasound can be used to investigate enophthalmos, buphthalmos or exophthalmos in cases of ocular protrusion and suspicion of disparity in globe size (Withcomb 2002). The most common diseases that can be detected or confirmed with ultrasound are corneal diseases, cataract, lens luxation, intraocular cysts or masses, glaucoma and retinal detachment (Reef 1998, Withcomb 2002). Although horses and donkeys can be affected by …

A survey of ocular ultrasound abnormalities in horse: 145 cases

PurposeTo describe the association of clinical and ultrasonographic (US) findings in horses affected by visual impairments, to estimate the most frequent ultrasonographic alteration as well importance and limits of US as a part of ophthalmic evaluation in equine patients.MethodsOne-hundred-forty-five horses referred to the Veterinary Teaching Hospitals of the University of Perugia and Camerino for ocular problems were submitted to ophthalmic examination and ocular ultrasound. The following group of abnormalities was established: corneal alteration, cataract, synechiae, iris prolapse, anterior chamber alterations, vitreous opacities, globe reduction, globe enlargement, retinal detachment, lens subluxation/luxation, lens rupture, intraocular masses, retrobulbar masses. Specificity, sensitivity, positive and negative predictive value and related 95% confidence interval were calculated. Kappa value was estimated for concordance evaluation.ResultsA total of 384 clinical alterations were observed during ophthalmological examination, while 357 were found by ultrasound investigation. A very good agreement was found for lens subluxation/luxation, intraocular masses, iris prolapse, globe enlargement, lens rupture, vitreous opacities and cataract. Moderate agreement was found for retinal detachment, anterior chamber alterations and synechiae. Corneal alteration and globe reduction resulted in a fair and poor agreement, respectively.ConclusionsUltrasonography affords the benefit of providing a complete cross-sectional view of the eyeball, facilitating the identification of ocular diseases in cases of loss of transparency of ocular media. It also represents an indispensable aid whenever anterior opacification precludes the use of ophthalmoscopic or biomicroscopic examination techniques.SommarioObiettiviDescrivere l’associazione tra reperti clinici ed ultrasonografici in cavalli affetti da alterazioni della visione, valutare i più frequenti reperti ultrasonografici e l’importanza ed i limiti dell’ultrasonografia come parte della valutazione oftalmologica nel cavallo.MetodiCentoquarantacinque cavalli riferiti all’ospedale Veterinario Didattico dell’Università di Perugia e di Camerino per patologie oculari sono stati sottoposti a valutazione clinica e a ultrasonografia oculare. Le alterazioni sono state suddivise nelle seguenti categorie: lesioni corneali, cataratta, sinechie, prolasso dell’iride, alterazioni della camera anteriore, opacità vitreali, riduzione dimensioni globo, aumento dimensioni globo, distacco retinico, lussazione/sublussazione del cristallino, rottura della lente, masse intraoculari, masse retrobulbari. Sono stati calcolati specificità, sensibilità, valore predittivo positivo (VPP) e negativo (VPN) con i relativi intervalli di confidenza (95%). La concordanza è stata valutata per mezzo del k-value.RisultatiSono state riscontrate un totale di 384 lesioni all’esame clinico e 357 a quello ultrasonografico. Concordanza eccellente è stata riscontrata per lussazione/sublussazione del cristallino, masse intraoculari, prolasso dell’iride, aumento di volume del globo, rottura della lente, opacità vitreali e cataratta. Concordanza moderata per distacco retinico, alterazioni della camera anteriore e sinechie. Le alterazioni corneali e la riduzione delle dimensioni del globo avevano concordanza rispettivamente pari a modesta e scarsa.ConclusioniL’ultrasonografia ha il vantaggio di fornire una visione completa della sezione dell’occhio facilitando l’identificazione di patologie causanti perdita di trasparenza delle strutture oculari. Rappresenta quindi un ausilio indispensabile nel caso di opacizzazione delle strutture anteriori che impediscono l’esecuzione di un corretto esame oftalmoscopico o biomicroscopico.

Use of Ozone-Based Eye Drops: A Series of Cases in Veterinary and Human Spontaneous Ocular Pathologies

Conjunctivitis, keratoconjunctivitis, and corneal ulcers are common eye disorders frequently diagnosed in both humans and animals, and are currently treated by topical administration of eye drops containing anti-inflammatory and antibacterial agents. The current molecules often lack efficacy because infections in hypoxic tissue contain methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa; thus, new products for the treatment of ocular pain and inflammation are needed. The use of ozone, a molecule stabilized for topical use as an ozonide, could be providential due to its anti-inflammatory and bactericidal activity in certain anterior segment pathologies, in addition to promoting tissue repair properties. Ozonated oils have the same properties as gaseous ozone and are well tolerated by tissues. In the present study the repair and regeneration effect of ozonated oil in liposomes plus hypromellose (Ozodrop®, FB Vision, Ascoli Piceno, Italy) instilled 3–4 times a day in external ocular spontaneous pathologies both in animals and humans are reported.

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs. Rat-transformed EGCs were treated with 10 ng/ml TcdB for 6 h-48 h, or for 48 h, followed by incubation for additional 4 or 11 days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays. TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-β‑galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c‑Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin‑2 and Sirtuin‑3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways. In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.

Bilateral cervical ribs in a mixed breed dog

A 4-year-old intact female, mixed breed dog was presented with a complaint of dyspnea. Clinical examination revealed symptoms related to disease of the upper airways. Radiographic findings were consistent with tracheal collapse associated with anomalies involving the seventh cervical vertebra and the first ribs bilaterally. Radiographs were highly suggestive of cervical ribs; computed tomography and ultrasound examination allowed complete characterization and better localization of the anomalies with relationship to the adjacent muscle and vasculature. Cervical ribs are malformations widely described in human medicine, but only sporadically in dogs. Herein, we discuss etiological, clinical, diagnostic and therapeutic aspects of cervical ribs and possible correlations between the cervical ribs and other anatomical anomalies noted in this dog.

Metabolic effects of Tart Cherries supplementation in an animal model of obesity

Fruits and vegetables contain non-nutritive phytochemicals that may contribute to their health-promoting effects. Anthocyanins are phytochemical flavonoids principally found in fruits and vegetables. Several studies have suggested that anthocyanin-rich plant extracts can modify lipid metabolism in vitro and can reduce hyperlipidemia in vivo. Tart cherries (Prunus cerasus L.) are a rich source of anthocyanins. This study was designed to evaluate the effects of anthocyanin-rich tart cherries extract and seeds powder on Diet-Induced Obesity (DIO) rats, that provide a useful animal model sharing several common features with human obesity. DIO rats were studied for 17 weeks of hypercaloric diet with the supplementation of 0,1 mg/kg of tart cherries seeds powder (DS) and seeds powder plus tart cherries extract, containing 1mg of anthocyanins (DES). DIO rats were compared to the control rats with not fat diet (Chow).To determine the systemic effects of caloric dense expousure we examined food consumption, fat mass content and fasting glycemia, insulin levels, cholesterol and triglycerides. Ultrasonographic (US) and computed tomography (CT) evaluations were performed to detect adipose tissue deposition. In CT, also fat infarction of the liver was investigated followed by histochemical analysis 17 weeks of fat diet, rats increased significantly their body weight in comparison to the control group. Glycaemia and insulin levels were higher in DIO rats. No difference in body weight was found in DS and DES rats compared to age-matched DIO rats. Supplementation of tart cherries in DS and DES induce a decrease of the blood pressure and the glycemia. Furthermore, decreased the serum levels of thiobarbituric reactive substances. The US and CT analysis indicated an increase of deposition of visceral adipose tissue and evidenced a decrease of hepatic attenuation in DIO rats, suggests a moderate hepatic steatosis prevented by tart cherries supplementation in DS and DES rats. The evidence of the CT was confirmed by histological analysis. DIO rats present a distinctive pattern of steatosis with hepatocytic ballooning degeneration at the perivenular areas. The steatosis elements decrease in DS and DES rats. Tart cherries supplementation, although did not reduce the body weight in DIO rats, prevent the development of related risk factors. Further studies are needed to better clarify the benefits of tart cherry supplementation on health and disease prevention.

Obesity-related nervous system injury: preliminary evidences in diet induced obesity (DIO) rats

Increased food intake, reduced physical activity and altered metabolic processes are the variables that affect energy balance inducing obesity. Obesity is now considered an increasingly medical challenge. Actually, the prevalence of obesity has increased dramatically worldwide over the last decades and has now reached epidemic proportions. On the other hand, obesity is associated with the development of chronic diseases such as cerebrovascular disease promoting the cognitive decline. Caloric-dense diet induced obesity (DIO), provides a useful animal model sharing several common features with human obesity. DIO rats of 7 weeks of age are expose to high fat (45 %) diet ad libitum and after 5 weeks the obese phenotype starts to be develop. To clarify the possible relationships between obesity and nervous system changes, DIO rats were studied after 5 weeks and 17 weeks of hypercaloric diet compared to the control rats with not fat diet (Chow). Memory performance were measured using different cognitive tests. Moreover, ultrasonographic (US) and computed tomography (CT) evaluations were performed to detect adipose tissue changes. Magnetic resonance imaging (MRI) to highlight brain morphological alterations was used. Morphological changes of brain areas (frontal cortex, hippocampus) were evaluated by immunohistochemical analysis. The results confirmed the developed of obesity after 5 weeks of fat diet. At long-term (17 weeks) high fat diet exposure, rats increased significantly their body weight in comparison to the control group and the youngest DIO rats. The US and CT analysis indicated an increase of deposition of both visceral and subcutaneous adipose tissue and evidences a decrease of hepatic attenuation in the older DIO rats.MRI images did not show vascular and morphological alterations in brain. Instead, immuhistochemical and immunochemical analysis, revealed an increase expression of glial-fibrillary acidic protein (GFAP) in the older DIO rats compared to the age- matched Chow rats both in frontal cortex and in hippocampus. DIO rats showed a reduction of retention latency time in the emotional learning task. These preliminary findings indicate that the development of obesity, does not determined gross anatomy alteration in brain, but the occurrence of injury characterized by astrogliosis. The identification of neurodegenerative changes in DIO may represent the first insight to better characterize the neuronal involvement in obesity.