The Impact of Spray Cryotherapy on Lesion-Induced Osteitis in a New Murine Experimental Model.

Background and Objectives: Endoscopic sinus surgery is considered the gold management strategy for difficult-to-treat chronic rhinosinusitis. The inflammatory bony process is incriminated as being involved in the unfavorable evolution and recurrence of the disease. Osteitis is significantly increased in patients that have been previously submitted to surgery, and it is more often present in patients with extended radiological disease and in patients undergoing revision surgery. The aim of the research is to demonstrate the presence of inflammations and neo-osteogenesis associated with nasal mucosal surgical injury and the correlation between their severity and to evaluate the efficacy of low-pressure spray cryotherapy in reducing inflammation and bone remodeling. Materials and Methods: The experimental murine model was conducted over a period of 80 days and included a total of 60 adult female Wistar rats, with three periods of withdrawal of 20 individuals each from the experiment. After inducing a bilateral mechanical injury by brushing, low-pressure spray cryotherapy application was performed unilaterally, and tissue samples were prepared specifically for histological analysis. The scores for inflammation and osteitis were compared over time and between the two nasal fossae. Results: Osteitis and inflammation were induced by a simple mucosal brushing lesion, similar to surgical injury. We identified the presence of inflammation in 95% of the specimens, and it was present over time. Moreover, criteria for bone remodeling were clearly highlighted in a percentage of 72% of the specimens. There was a direct correlation between the severity of inflammation and neo-osteogenesis, with a statistical significance of p = 0.050. Low-pressure spray cryotherapy was safe and effective in reducing inflammation (p = 0.020) and osteitis (p = 0.000) with a safety profile. Conclusions: Low-pressure cryotherapy reduces the severity of mucosal inflammation and osteitis in lesion-induced neo-osteogenesis.

Meningioma with rhabdoid features: Pathologic findings in dogs.

Rhabdoid meningioma is a rare type of meningeal neoplasm in humans. This study reports the clinical, pathological, and ultrastructural features of 4 cases of canine meningioma with rhabdoid features. The cases were female and 8 to 12 years of age. Biopsies from complete surgical resections were examined for all cases. The whole brain with tumor recurrence was collected at necropsy in 2 dogs. Histologically, the tumors consisted of discohesive sheets of oval-polygonal cells with abundant eosinophilic cytoplasm and occasional paranuclear hyaline-like inclusions. Cells were intensely immunopositive for vimentin, negative for melan A and S100 protein in all cases, and showed variable immunolabeling for cytokeratin in 2 cases. Focal glial fibrillary acidic protein (GFAP)-immunopositive cells were present in 1 case. Ultrastructurally, the rhabdoid cells in case 1 contained prominent cytoplasmic whorls of intermediate filaments, recapitulating the ultrastructural features of rhabdoid meningioma in humans. In cases 2 and 3, the meningioma cells contained interdigitating cell processes folded in a maze-like fashion resembling rhabdoid-like meningioma in humans. In case 4, the voluminous cytoplasm contained many round-to-flattened mitochondria admixed with rough endoplasmic reticulum, indicating a predominant oncocytic differentiation and not the rhabdoid differentiation suggested by light microscopy. Thus, rhabdoid morphology occurs in different types of meningiomas, and ultrastructural findings are essential for a correct diagnosis.

Cancer spectrum in TP53-deficient golden Syrian hamsters: A new model for Li-Fraumeni syndrome.

BACKGROUND: The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. Humans who inherit mutant TP53 alleles develop a wide range of early onset cancers, a disorder called Li-Fraumeni Syndrome (LFS). Trp53-deficient mice recapitulate most but not all of the cancer phenotypes observed in TP53-deficient human cancers, indicating that new animal models may complement current mouse models and better inform on human disease development. MATERIALS AND METHODS: The recent application of CRISPR/Cas9 genetic engineering technology has permitted the emergence of golden Syrian hamsters as genetic models for wide range of diseases, including cancer. Here, the first cancer phenotype of TP53 knockout golden Syrian hamsters is described. RESULTS: Hamsters that are homozygous for TP53 mutations become moribund on average ~ 139 days of age, while hamsters that are heterozygous become moribund at ~ 286 days. TP53 homozygous knockout hamsters develop a wide range of cancers, often synchronous and metastatic to multiple tissues, including lymphomas, several sarcomas, especially hemangiosarcomas, myeloid leukemias and several carcinomas. TP53 heterozygous mutants develop a more restricted tumor spectrum, primarily lymphomas. CONCLUSIONS: Overall, hamsters may provide insights into how TP53 deficiency leads to cancer in humans and can become a new model to test novel therapies.

A novel gene editing system to treat both Tay-Sachs and Sandhoff diseases.

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme ß-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex ß subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p < 0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p < 0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay-Sachs and Sandhoff patients.

Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.

Deficiencies in the lysosomal hydrolase ß-galactosidase (ß-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse ß-gal encoding gene, Glb1, was targeted to generate both models of ß-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of ß-gal (ß-gal-/-). Glb1W274L mice showed a significant reduction in ß-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, ß-gal-/- mice were devoid of ß-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). ß-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of ß-galactosidase deficiency with residual enzyme activity has been developed.

Naturally Occurring Canine Glioma as a Model for Novel Therapeutics.

BACKGROUND: Current animal models of glioma are limited to small animal models, which are less predictive of treatment of human disease. Canines often develop gliomas de novo, but the natural history of the disease is not well described. OBJECTIVE: We provide data for naturally occurring canine gliomas; evaluating medical and surgical therapies. METHODS: We reviewed medical records of pet dogs with a presumptive diagnosis of glioma from MRI imaging that underwent surgery as part of the Canine Brain Tumor Clinical Trials Program. Breed, age, sex, median progression-free, and overall survival times and cause of death were recorded for multivariate analysis. RESULTS: Ninety five dogs (56 male; mean age = 8.3 years) were included, but nine were excluded as final pathology was non-neoplastic. Gross total resection was reported in 81 cases based on postoperative MRI. Seventy had high-grade tumors (grade III or IV). Eighty three dogs presented with seizures, being the most common presenting clinical sign. Median survival after surgery was 723 days (95% CI 343-1103) for grade II tumors, 301 days (197-404) for grade III and 200 days (126-274) for grade IV (p = .009 Kaplan-Meier survival analysis; Log Rank test). Age (cox regression, p = .14) or sex (Kaplan-Meier test, p = .22) did not predict survival. CONCLUSIONS: This study establishes normative data for a model exploiting dogs with naturally occurring glioma, which can be used to test novel therapies prior to translation to human trials. Further work will focus on the effects of different therapies, including chemotherapy, radiation therapy, and immunotherapy.

A novel cancer syndrome caused by KCNQ1-deficiency in the golden Syrian hamster.

BACKGROUND: The golden Syrian hamster is an emerging model organism. To optimize its use, our group has made the first genetically engineered hamsters. One of the first genes that we investigated is KCNQ1 which encodes for the KCNQ1 potassium channel and also has been implicated as a tumor suppressor gene. MATERIALS AND METHODS: We generated KCNQ1 knockout (KO) hamsters by CRISPR/Cas9-mediated gene targeting and investigated the effects of KCNQ1-deficiency on tumorigenesis. RESULTS: By 70 days of age seven of the eight homozygous KCNQ1 KOs used in this study began showing signs of distress, and on necropsy six of the seven ill hamsters had visible cancers, including T-cell lymphomas, plasma cell tumors, hemangiosarcomas, and suspect myeloid leukemias. CONCLUSIONS: None of the hamsters in our colony that were wild-type or heterozygous for KCNQ1 mutations developed cancers indicating that the cancer phenotype is linked to KCNQ1-deficiency. This study is also the first evidence linking KCNQ1-deficiency to blood cancers.

Congenital diaphragmatic hernia with concurrent aplasia of the pericardium in a foal.

BACKGROUND: In veterinary medicine congenital abnormalities of the diaphragm and pericardium are rare, idiopathic malformations, being reported mainly in dogs. This report documents an unusual case of developmental defects in a foal consisting of diaphragmatic hernia concurrent with pericardial aplasia. CASE PRESENTATION: Following a normal delivery, a full term, female Friesian stillborn foal with the placenta was presented for necropsy. External morphological examination indicated a normally developed foal. At necropsy, a large oval defect (approximately 20 × 15 cm in size) was observed in the left-dorsal side of the diaphragm (left lumbocostal triangle). This defect allowed the intestinal loops, spleen and partially the liver to translocate into the thorax. The loops of the left ascending colon, including the pelvic flexure and partially the small intestine covered the cranial and dorsal posterior parts of the heart due to the complete absence of the left pericardium. The remaining pericardium presented as a white, semi-transparent strip, partially covering the right side of the heart. The left lung and the main bronchus were severely hypoplastic to approximately one-fifth the size of their right homologue. The intermediate part of the liver, containing mainly the enlarged quadrate lobe was translocated in the thorax, severely enlarged and showed marked fibrosis. Histologically in the herniated lobes we diagnosed hepatic chronic passive congestion, telangiectasia and medial hypertrophy of blood vessels. CONCLUSION: Concomitant malformation involving diaphragmatic hernia and pericardial aplasia in horses have not been previously reported. Moreover, this is the first case describing pericardial aplasia in horse.

Case report: The first description of a thyroglossal duct cyst in a hen (Gallus gallus domesticus).

Thyroglossal duct cyst represents a congenital anomaly of the cervical region, rarely documented in animals. Although previously reported in dogs, cats, horses, goats, pigs, and calves, never in birds. This report describes a rare case of thyroglossal duct cyst in a hen. A necropsy of a Transylvanian Naked Neck hen carried following diphtheroid mucocutaneous lesions. The necropsy revealed a large, cyst-like structure measuring 0.5 cm at the level of the caudal edge of the left thyroid gland. Histologically, the cystic mass, bordered by 1-2 lines of well-differentiated ciliated cuboidal cells, presented nuclear immunoreactivity for Thyroid transcription factor 1. To the best of the authors' knowledge, there are no previous records of thyroglossal duct cysts in avians. Moreover, this is the first case describing a thyroglossal duct cyst in a hen.

Hepatic changes following a high-fat diet: effects of Cornus mas and gold nanoparticles phytoreduced with Cornus mas on oxidative stress, inflammation, and histological damage.

Background and aims: High fat diet (HFD) can lead to liver injury, through oxidative stress and inflammation. The use of natural compounds with antioxidant and anti-inflammatory properties can have a protective potential. We aimed to investigate the effects of Cornus mas (CM) and gold nanoparticles phytoreduced with CM (GNPsCM) on hepatic alterations induced by HFD in rats. Methods: Female Sprague Dawley rats were randomly divided into four groups: control, HFD, HFD +CM and HFD + GNPsCM. The high fat diet was administered for 32 weeks and CM and GNPsCM were administered for 4 weeks after the HFD period. The high fat diet induced oxidative stress in liver, with lipid peroxidation and decreased antioxidant capacity, inflammation and minimal histological alterations. Results: The administration of CM and GNPsCM reduced lipid peroxidation produced by HFD and increased antioxidant potential in liver homogenates, while increasing inflammatory markers. Histological alterations were slightly improved by the intervention of compounds, and hyaluronic acid content of the liver without statistical significance as compared to HFD group. Conclusion: These findings support the potential of these treatments in addressing liver oxidative stress, mitigating liver damage induced by a high-fat diet. This investigation sheds light on the oxidative stress dynamics and histological alterations associated with high-fat diet-induced liver injury, contributing to our understanding of potential therapeutic interventions.

Neoadjuvant systemic oncolytic vesicular stomatitis virus is safe and may enhance long-term survivorship in dogs with naturally occurring osteosarcoma.

Osteosarcoma is a devastating bone cancer that disproportionally afflicts children, adolescents, and young adults. Standard therapy includes surgical tumor resection combined with multiagent chemotherapy, but many patients still suffer from metastatic disease progression. Neoadjuvant systemic oncolytic virus (OV) therapy has the potential to improve clinical outcomes by targeting primary and metastatic tumor sites and inducing durable antitumor immune responses. Here we describe the first evaluation of neoadjuvant systemic therapy with a clinical-stage recombinant oncolytic vesicular stomatitis virus (VSV), VSV-IFNß-NIS, in naturally occurring cancer, specifically appendicular osteosarcoma in companion dogs. Canine osteosarcoma has a similar natural disease history as its human counterpart. VSV-IFNß-NIS was administered prior to standard of care surgical resection, permitting microscopic and genomic analysis of tumors. Treatment was well-tolerated and a "tail" of long-term survivors (∼35%) was apparent in the VSV-treated group, a greater proportion than observed in two contemporary control cohorts. An increase in tumor inflammation was observed in VSV-treated tumors and RNA-seq analysis showed that all the long-term responders had increased expression of a T cell anchored immune gene cluster. We conclude that neoadjuvant VSV-IFNß-NIS is safe and may increase long-term survivorship in dogs with naturally occurring osteosarcoma, particularly those that exhibit pre-existing antitumor immunity.

Neoadjuvant systemic oncolytic vesicular stomatitis virus is safe and may enhance long-term survivorship in dogs with naturally occurring osteosarcoma.

Osteosarcoma is a devastating bone cancer that disproportionally afflicts children, adolescents, and young adults. Standard therapy includes surgical tumor resection combined with multiagent chemotherapy, but many patients still suffer from metastatic disease progression. Neoadjuvant systemic oncolytic virus (OV) therapy has the potential to improve clinical outcomes by targeting primary and metastatic tumor sites and inducing durable antitumor immune responses. Here we described the first evaluation of neoadjuvant systemic therapy with a clinical-stage recombinant oncolytic Vesicular stomatitis virus (VSV), VSV-IFNß-NIS, in naturally occurring cancer, specifically appendicular osteosarcoma in companion dogs. Canine osteosarcoma has a similar natural disease history as its human counterpart. VSV-IFNß-NIS was administered prior to standard of care surgical resection, permitting microscopic and genomic analysis of tumors. Treatment was well-tolerated and a 'tail' of long-term survivors (~35%) was apparent in the VSV-treated group, a greater proportion than observed in two contemporary control cohorts. An increase in tumor inflammation was observed in VSV-treated tumors and RNAseq analysis showed that all the long-term responders had increased expression of a T-cell anchored immune gene cluster. We conclude that neoadjuvant VSV-IFNß-NIS is safe and may increase long-term survivorship in dogs with naturally occurring osteosarcoma, particularly those that exhibit pre-existing antitumor immunity.

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases.

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases. Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Helicobacter Pylori-Induced Gastric Infections: From Pathogenesis to Novel Therapeutic Approaches Using Silver Nanoparticles.

Helicobacter pylori is the first formally recognized bacterial carcinogen and the most important single digestive pathogen responsible for the induction of gastroduodenal diseases such as gastritis, peptic ulcer, and, finally, gastric neoplasia. The recently reported high rates of antimicrobial drug resistance hamper the current therapies of H. pylori, with therapeutic failure reaching up to 40% of patients. In this context, new treatment options and strategies are urgently needed, but the successful development of these new therapeutic tools is conditioned by the understanding of the high adaptability of H. pylori to the gastric acidic environment and the complex pathogenic mechanism. Due to several advantages, including good antibacterial efficiency, possible targeted delivery, and long tissular persistence, silver nanoparticles (AgNPs) offer the opportunity of exploring new strategies to improve the H. pylori therapy. A new paradigm in the therapy of H. pylori gastric infections using AgNPs has the potential to overcome the current medical limitations imposed by the H. pylori drug resistance, which is reported for most of the current organic antibiotics employed in the classical therapies. This manuscript provides an extensive overview of the pathology of H. pylori-induced gastritis, gastric cancer, and extradigestive diseases and highlights the possible benefits and limitations of employing AgNPs in the therapeutic strategies against H. pylori infections.

ssDNA nanotubes for selective targeting of glioblastoma and delivery of doxorubicin for enhanced survival.

Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.

Silver Nanoparticles for the Therapy of Tuberculosis.

Rapid emergence of aggressive, multidrug-resistant Mycobacteria strain represents the main cause of the current antimycobacterial-drug crisis and status of tuberculosis (TB) as a major global health problem. The relatively low-output of newly approved antibiotics contributes to the current orientation of research towards alternative antibacterial molecules such as advanced materials. Nanotechnology and nanoparticle research offers several exciting new-concepts and strategies which may prove to be valuable tools in improving the TB therapy. A new paradigm in antituberculous therapy using silver nanoparticles has the potential to overcome the medical limitations imposed in TB treatment by the drug resistance which is commonly reported for most of the current organic antibiotics. There is no doubt that AgNPs are promising future therapeutics for the medication of mycobacterial-induced diseases but the viability of this complementary strategy depends on overcoming several critical therapeutic issues as, poor delivery, variable intramacrophagic antimycobacterial efficiency, and residual toxicity. In this paper, we provide an overview of the pathology of mycobacterial-induced diseases, andhighlight the advantages and limitations of silver nanoparticles (AgNPs) in TB treatment.

Precision Targeted Ablation of Fine Neurovascular Structures In Vivo Using Dual-mode Ultrasound Arrays.

Carotid bodies (CBs) are chemoreceptors that monitor and register changes in the blood, including the levels of oxygen, carbon dioxide, and pH, and regulate breathing. Enhanced activity of CBs was shown to correlate with a significant elevation in the blood pressure of patients with hypertension. CB removal or denervation were previously shown to reduce hypertension. Here we demonstrate the feasibility of a dual-mode ultrasound array (DMUA) system to safely ablate the CB in vivo in a spontaneously hypertensive rat (SHR) model of hypertension. DMUA imaging was used for guiding and monitoring focused ultrasound (FUS) energy delivered to the target region. In particular, 3D imaging was used to identify the carotid bifurcation for targeting the CBs. Intermittent, high frame rate imaging during image-guided FUS (IgFUS) delivery was used for monitoring the lesion formation. DMUA imaging provided feedback for closed-loop control (CLC) of the lesion formation process to avoid overexposure. The procedure was tolerated well in over 100 SHR and normotensive rats that received unilateral and bilateral treatments. The measured mean arterial pressure (MAP) exhibited measurable deviation from baseline 2-4 weeks post IgFUS treatment. The results suggest that the direct unilateral FUS treatment of the CB might be sufficient to reduce the blood pressure in hypertensive rats and justify further investigation in large animals and eventually in human patients.

Chondroprotective effects of pulsed shortwave therapy in rabbits with experimental osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) represents a public health challenge since the pathogenic treatment, able to induce cartilage regeneration, still remains unknown. Ageing of the population and increasing OA prevalence have led to a lot of research, aiming to identify treatments acting on chondrocytes that play a determinant role in cartilage degeneration÷regeneration balance. Pulsed shortwave therapy (with the classical application form - Diapulse) is a physiotherapy method with anabolic effects demonstrated on nervous, conjunctive and vascular tissues, but its effects on OA cartilage are not known. AIM: Our aim was to demonstrate the effects of Diapulse on the cartilage in experimental induced OA. MATERIALS AND METHODS: Experimental OA was induced in 10 mature female rabbits by anterior cruciate ligament transection (ACLT). Ten weeks after ACLT, rabbits were randomized in a treatment group and a control group. Treatment group was exposed to Diapulse at a frequency of 27.12 MHz, pulse length of 65 µs, pulse frequency of 300 pulses÷s (300 Hz) for 10 minutes÷day. Control group was exposed to sham therapy. After treatment, rabbits were sacrificed and the cartilage was evaluated by histopathological examinations with Hematoxylin-Eosin (HE) staining and transmission electron microscopy (TEM). RESULTS: OA characteristic changes were found in both groups. In the treatment group, we found that Diapulse influenced the degenerative process in the OA cartilage by improving the chondrocyte viability and the capacity to maintain cellular matrix integrity and structure. CONCLUSIONS: Diapulse can be considered a disease modifying therapeutic procedure and could be a reliable option for treatment of OA patients.

Cell proliferation and apoptosis in urinary bladder urothelium of rats following ovariectomy and chronic estrogen replacement therapy.

AIMS: In this study we followed the effect of menopause and estrogenic replacement therapy on the proliferative and apoptotic activity of the bladder urothelial cells. METHODS: The experimental model of menopause was reproduced using a standard protocol of bilateral ovariectomy in rats, estrogen replacement therapy being achieved by systemic administration of hexestrol diacetate for six weeks. Proliferative and apoptotic activity was monitored by quantifying the urothelium imunoexpression for PCNA antigen as a marker of S phase of the cell cycle and Cleaved Caspase 3 for monitoring apoptotic activity. RESULTS: Following ovariectomy, the main changes were urothelial atrophy associated with intensification of the apoptotic activity at these level. Estrogen therapy managed to improve the urothelium activity by reducing the Apoptotic Index and by increasing urothelial proliferative activity. CONCLUSIONS: The results show the important role of estrogens in maintaining urothelial activities, highlighting their potential use in the treatment of urothelium atrophic and degenerative processes associated with menopause.

Contrast enhanced ultrasonography (CEUS) in the characterization of tumor microcirculation. Validation of the procedure in the animal experimental model.

AIM: The aims of this study are the development of a contrast enhanced ultrasonography (CEUS) protocol for rats' evaluation and the assessment of the potential benefits of CEUS in Walker 256 tumor rats. MATERIAL AND METHOD: In the study were used 36 albino Wistar rats grafted subcutaneously with Walker 256 tumor. The implementation of the ultrasound (US) guided injection technique (30 subjects - group A) was performed between 4 to 8 weeks after implantation. The contrast agent (CA) - Sono Vue (Bracco) - was administered either into the lateral vein of the tail or directly into the heart under US guidance. The US validation, focusing on CEUS (6 subjects - group B) was realized at 4 to 6 weeks after implantation. The US procedures aimed to obtain morphological (2D), vascular (color Doppler and pulsed Doppler) and angiospecific functional data (CEUS). The Vevo 2100 equipment was used for US and Time Intensity Curves (TIC) were analyzed with Sonoliver (TomTec). The tumor specimens which were resected after the last study underwent a pathology exam. RESULTS: A number of 23 successfully CEUS explorations were performed in 17 subjects (11 in group A and 6 in group B). Nineteen rats could not be evaluated (in 8 cases the tumor was not viable; 4 subjects died during CA administration; in 4 cases the administration line could not be obtained). In group B, CEUS was performed in 6 subjects at 4 weeks after implantation and in 5 subjects at 6 weeks. The statistical analysis of the TIC parameters identified significant differences between the Time to Peak, mean Transit Time and Rise Time parameters of the muscles and those of the tumor. CONCLUSIONS: CEUS was easily implemented on the studied tumor model and is adequate for the evaluation of tumor vascularity. US guided intracardiac administration of the CA is an easy and reproducible procedure. If the examination is performed at defined time intervals it detects the alterations within the tumor circulatory bed.