Animal Facilities Research Articles

Risk factors for third-generation cephalosporin-resistant and extended-spectrum β-lactamase-producing Escherichia coli carriage in domestic animals of semirural parishes east of Quito, Ecuador.

Extended-spectrum β-lactamase (ESBL)-producing and other antimicrobial resistant (AR) Escherichia coli threaten human and animal health worldwide. This study examined risk factors for domestic animal colonization with ceftriaxone-resistant (CR) and ESBL-producing E. coli in semirural parishes east of Quito, Ecuador, where small-scale food animal production is common. Survey data regarding household characteristics, animal care, and antimicrobial use were collected from 304 households over three sampling cycles, and 1195 environmental animal fecal samples were assessed for E. coli presence and antimicrobial susceptibility. Multivariable regression analyses were used to assess potential risk factors for CR and ESBL-producing E. coli carriage. Overall, CR and ESBL-producing E. coli were detected in 56% and 10% of all fecal samples, respectively. The odds of CR E. coli carriage were greater among dogs at households that lived within a 5 km radius of more than 5 commercial food animal facilities (OR 1.72, 95% CI 1.15-2.58) and lower among dogs living at households that used antimicrobials for their animal(s) based on veterinary/pharmacy recommendation (OR 0.18, 95% CI 0.04-0.96). Increased odds of canine ESBL-producing E. coli carriage were associated with recent antimicrobial use in any household animal (OR 2.69, 95% CI 1.02-7.10) and purchase of antimicrobials from pet food stores (OR 6.83, 95% CI 1.32-35.35). Food animals at households that owned more than 3 species (OR 0.64, 95% CI 0.42-0.97), that used antimicrobials for growth promotion (OR 0.41, 95% CI 0.19-0.89), and that obtained antimicrobials from pet food stores (OR 0.47, 95% CI 0.25-0.89) had decreased odds of CR E. coli carriage, while food animals at households with more than 5 people (OR 2.22, 95% CI 1.23-3.99) and located within 1 km of a commercial food animal facility (OR 2.57, 95% CI 1.08-6.12) had increased odds of ESBL-producing E. coli carriage. Together, these results highlight the complexity of antimicrobial resistance among domestic animals in this setting.

Spreading of SARS-CoV-2 from hamsters to humans

Spreading of SARS-CoV-2 from hamsters to humans

Pro-mutagenic effects of the gut microbiota in a Lynch syndrome mouse model

ABSTRACT The gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in the context of Lynch syndrome. In a mouse model for Lynch syndrome, we found a nearly complete loss of intestinal tumor development when animals were transferred from a conventional “open” animal facility to specific-pathogen-free (SPF) conditions. Using 16S sequencing we detected large changes in microbiota composition between the two facilities. Transcriptomic analyses of tumor-free intestinal tissues showed signs of strong intestinal inflammation in conventional mice. Whole exome sequencing of tumors developing in Msh2-Lynch mice revealed a much lower mutational load in the single SPF tumor than in tumors developing in conventional mice, suggesting reduced epithelial proliferation in SPF mice. Fecal microbiota transplantations with conventional feces altered the immune landscape and gut homeostasis, illustrated by increased gut length and elevated epithelial proliferation and migration. This was associated with drastic changes in microbiota composition, in particular increased relative abundances of different mucus-degrading taxa such as Desulfovibrio and Akkermansia, and increased bacterial-epithelial contact. Strikingly, transplantation of conventional microbiota increased microsatellite instability in untransformed intestinal epithelium of Msh2-Lynch mice, indicating that the composition of the microbiota influences the rate of mutagenesis in MSH2-deficient crypts.

Why serology just is not enough: Strategic parvovirus risk assessment using a novel qPCR assay.

Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

Temporal trends in intake category data for animal shelter and rescue organizations in Colorado from 2008 to 2018.

To investigate trends in animal shelter and rescue organization intake for dogs and cats in Colorado from 2008 to 2018. 482 animal shelters and rescue organizations that reported annual intake data to the State of Colorado Department of Agriculture for 1,086,630 dogs and 702,333 cats. Total intake, intake for each of 5 Pet Animal Care and Facilities Act categories (stray, owner surrender, intrastate transfer, interstate transfer, or other), and community-based intake (total intake after exclusion of transfers) of dogs and cats were assessed in total and for each organization type (shelter or rescue organization). The number taken in per year, number taken in/1,000 capita (human residents)/y, and number in each intake category as a percentage of total intake for the same species per year were analyzed with linear regression models. Trend lines indicated that total dog intake increased over the study period, but there was no change when these data were adjusted for the human population. Cat intake decreased over time according to both of these measures. Total community-based intake decreased, whereas total intake by interstate transfer from other organizations increased for both species during the study period. Increased transfer of dogs and cats across state lines into regions with low community-based shelter intake suggested that regional and national animal disease trends could potentially impact disease profiles for recipient areas. Findings supported efforts toward collecting animal shelter and rescue organization intake and outcome data across larger systems.

Practice and Reflection on Socialized Service of Laboratory Animal Facility Maintenance in Colleges and Universities

Practice and Reflection on Socialized Service of Laboratory Animal Facility Maintenance in Colleges and Universities

Impact of colistin and colistin-loaded on alginate nanoparticles on pigs infected with a colistin-resistant enterotoxigenic Escherichia coli strain

Colistin is frequently used for the control of post-weaning diarrhoea in pigs. Colistin resistance caused by plasmidic genes is a public health issue. We evaluated, in experimental animal facilities, whether free colistin or colistin-loaded on alginate nanoparticles (colistin/Alg NPs) could select a colistin-resistant Enterotoxigenic Escherichia coli. The Alg NPs were produced by a simple top-down approach through ball milling of sodium alginate polymer precursor, and colistin loading was achieved through physical adsorption. Colistin loading on Alg NPs was confirmed using various tools such Fourier transform infrared spectroscopy and dynamic light scattering measurements. Thirty-four piglets were orally inoculated or not with a mcr-1-positive, rifampicin-resistant enterotoxigenic E. coli strain, and the inoculated pigs were either treated or not during five days with commercial colistin (100,000 IU/kg) or colistin/Alg NPs (40,415 IU/kg). Clinical signs were recorded. Fecal and post-mortem samples were analyzed by culture. The result clearly indicated that colistin/Alg NPs had a slightly better therapeutic effect. Both treatments led to a transitory decrease of the total E. coli fecal population with a majority of colistin-resistant E. coli isolates during treatment, but the dominant E. coli population was found susceptible at the end of the trial. Further studies are needed to evaluate, in diverse experimental or field conditions, the therapeutic efficacy of colistin/Alg NPs for post-weaning diarrhoea.

Intermittent fasting enhances hippocampal NPY expression to promote neurogenesis after traumatic brain injury

ObjectivesInterventions for preventing cognitive dysfunction after traumatic brain injury (TBI) are limited. Given that adult hippocampal neurogenesis after brain injury contributes to cognitive recovery, and hippocampal neurogenesis is potentially affected by nutritional factors, the aim of this study was to examine whether fasting could promote hippocampal neurogenesis and thus ameliorate the cognitive defects after TBI. MethodsThe present study used 8- to 10-wk-old C57 BL/6 N mice weighing 23 g, half males and half females. The mice were randomly assigned to each group, with 10 to 18 mice per group. All mice were housed in an approved animal facility with a 12-h light/dark cycle. In the metabolic study (food intake, body weight, blood glucose, triacylglycerol, total cholesterol, and β-hydroxybutyric acid ), 54 mice (male:female = 1:1) were randomized to the ad libitum (AL) group (n = 18) and the intermittent fasting (IF) group (n = 36). In the neurogenesis study, 45 mice (male:female = 1:1) were randomized to AL (n = 18), IF (n = 9), IF + scramble (n = 9), and the IF + neuropeptide Y (NPY)_siRNA (n = 9) groups. In the Morris water maze test, 48 mice (male:female = 1:1) were randomized to AL (n = 12), IF (n = 12), IF + scramble (n = 12), and the IF + NPY_siRNA (n = 12) groups. ResultsWe showed that a 1-mo-long IF regimen enhanced the proliferation of neural stem cells in the subgranular zone of the hippocampus 3 d after TBI, in addition to improving the cognitive performance in the Morris water maze test. Furthermore, an increase in the hippocampal NPY expression was detected in the IF group after the injury, compared with the mice fed AL, and local knockdown of NPY in vivo attenuated the effects of IF on TBI. ConclusionsThese findings suggest that IF promotes hippocampal neurogenesis after TBI by a mechanism that involves enhancement of NPY expression, to alleviate cognitive dysfunction caused by injury.

Development of a Universal Scale for Validating the Process of Sanitization of Equipment, Materials and Surfaces in the Animal Facility

Background: Monitoring of sanitization of cage equipment is an essential function of any laboratory animal facilities, seeking to ensure the animal health and welfare. The purpose of the current study was to develop universal scale for monitoring sanitization through detection of residual ATP for the most effective process of sanitizing components of rodents micro- and macro-environment. Methods: Sterile pens and swabs for the HY-LiTE® Luminometer Instrument were used to measure ATP concentration (RLU) on the cleaned surface samples. We have examined the elements of the microenvironment (rodent and rabbit cage, cage wire meshes, feed separating pieces, water bottles with tips, houses, tunnels, IVC cage frames, cage tops and cage wire meshes). The assessed swab area on each of the surfaces was 10×10 cm. Result: We observed, that combined washing (cleaned manually and with automatic universal washers with detergent) gives the lowest RLU values. Monitoring of the quality of sanitization of equipment and surfacescan be carried out without the use of microbiological tests. Use of pre-washing allows increasing the service life of caging equipment. We have developed an assessment scale to monitor sanitizing, which can be recommended to scientific and breeding animal facilities for monitoring sanitization cage equipments.

Effects of Cage Position and Light Transmission on Home Cage Activity and Circadian Entrainment in Mice.

Light is known to exert powerful effects on behavior and physiology, including upon the amount and distribution of activity across the day/night cycle. Here we use home cage activity monitoring to measure the effect of differences in home cage light spectrum and intensity on key circadian activity parameters in mice. Due to the relative positioning of any individually ventilated cage (IVC) with regard to the animal facility lighting, notable differences in light intensity occur across the IVC rack. Although all mice were found to be entrained, significant differences in the timing of activity onset and differences in activity levels were found between mice housed in standard versus red filtering cages. Furthermore, by calculating the effective irradiance based upon the known mouse photopigments, a significant relationship between light intensity and key circadian parameters are shown. Perhaps unsurprisingly given the important role of the circadian photopigment melanopsin in circadian entrainment, melanopic illuminance is shown to correlate more strongly with key circadian activity parameters than photopic lux. Collectively, our results suggest that differences in light intensity may reflect an uncharacterized source of variation in laboratory rodent research, with potential consequences for reproducibility. Room design and layout vary within and between facilities, and caging design and lighting location relative to cage position can be highly variable. We suggest that cage position should be factored into experimental design, and wherever possible, experimental lighting conditions should be characterized as a way of accounting for this source of variation.

Antifungal resistance and clinical significance in small animals

In conjunction with the increasing number of immunosuppressive diseases in human and animal populations, fungal diseases have also been rising. Despite the introduction of new antifungal agents in recent years, the incidence of fungal infections continues to increase, and subsequently, the prevalence of resistance to these drugs is remarkably increased, posing significant health concerns. While antifungal drug resistance is of great importance in human medicine, especially against Candida spp., there are few studies about antifungal resistance in veterinary medicine. Indeed, several fungal infections in dogs have been reported, especially in cases of neutropenia associated with the usage of cyclophosphamide or parvoviral enteritis infection. ketoconazole, fluconazole, itraconazole, and terbinafine, the mainly used antifungal drugs in treating dermatomycosis, have been encountered in feline and canine medicine. However, it has been reported that the desired success could not be obtained from the treatments applied in various cases in recent years. This review aimed to emphasize the role of the development of resistance against antifungal agents on treatment failures. In this context, monitoring of antifungal resistance in veterinary clinics and animal facilities by veterinarians and other animal health authorities is recommended

Phenotypic Drift in Lupus-Prone MRL/lpr Mice: Potential Roles of MicroRNAs and Gut Microbiota.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors. We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation.

Early puberty in short-haired Guinea pigs kept in laboratory animal facilities.

Lab animals, such as Guinea pigs (Cavia porcellus), are crucial for scientific development, as they play an important role in the development and quality control chain of vaccines and drugs distributed by the Brazilian public health system. Investigating their biological and physiological parameters is fundamental to raise and keep these animals, so the handling of the facilities that hold them can be updated whenever new information comes up, with the well-being of the animals and alignment with the 3 Rs in mind. In the search for understanding reproductive aspects of Guinea pigs, the present study had the main goal of studying puberty by means of estrous cycle analysis in short-haired Guinea pigs. Guinea pigs have a vaginal occlusive membrane that covers the vaginal orifice. Its rupture takes place gradually and naturally, moments before labor and during estrus. The present study followed 42 females as for the presentation of the vaginal occlusive membrane. Once the membranes ruptured spontaneously, a swab was collected to study vaginal cytology. Membrane rupture was observed in 39 females; six females showed membrane rupture with less than 21 days of age (17 to 21 days). Twenty-three females were characterized as being in estrus due to cytology showing a prevalence of anucleated superficial cells. One of these females was younger than 21 days old. The opening of the vaginal occlusive membrane took place most frequently in intervals between 17 and 18 days, and the membrane remained open between one and three consecutive days. It was possible to follow three cycles of membrane opening on six females. The present study showed the need to adapt handling guidelines for C. porcellus kept in research animal facilities. The early age of puberty imposes the need of separate the female daughters from their fathers at 16 days old.

Limiting dilution assay to quantify the self-renewal potential of cancer stem cells in hepatocellular carcinoma.

Cancer stem cells (CSCs) are a heterogeneous subpopulation of self-renewing cancer cells that sustain tumorigenesis in various types of human malignancies. Due to the diverse phenotypes of CSCs in distinct pathological conditions, it remains challenging to define CSCs by specific cell surface markers. Thus, it is essential to develop experimental protocols to quantify the self-renewal and tumorigenic potential of CSC for therapeutic purposes. To address these technical difficulties, we introduce the limiting dilution assay (LDA), a well-recognized experimental approach that accurately measures the self-renewal capacity and tumorigenicity of CSCs. Using hepatocellular carcinoma (HCC) cells as a model, we describe detailed experimental procedures for CSC culture in three-dimensional soft fibrin gel. Moreover, we provide an updated protocol for assessing CSC self-renewal in vitro and tumorigenicity in vivo in NOD/SCID IL-2Rγ-/- (NSG) mice by LDA. Taken together, these protocols are readily applicable to laboratories with basic cell culture equipment and access to experimental animal facilities, providing a valuable toolbox to dissect mechanisms underlying HCC tumorigenesis and identify CSC-targeting drugs via high-throughput screening.

Pathogenicity of Bordetella bronchiseptica isolated from apparently healthy rabbits in guinea pig, rat, and mouse.

Bordetella bronchiseptica (B. bronchiseptica) is associated with respiratory tract infections in laboratory animals. In our laboratory animal facility, B. bronchiseptica was isolated from 21 of 27 apparently healthy rabbits obtained from a breeding farm contaminated with B. bronchiseptica. Restriction fragment length polymorphism (RFLP) analysis showed that the flagellin genotype of isolates from the laboratory animal facility and breeding farm was type A, which is seen relatively frequently in rabbits in Europe. To examine its pathogenicity, guinea pigs, rats, and mice were inoculated intranasally with a representative strain isolated in the laboratory animal facility. Following inoculation of 107 colony forming unit (cfu), severe inflammation was observed in the lungs of guinea pig and mice, although the inflammation was less severe in rats. The strain was recovered from the trachea and lungs of these species after inoculation with lower dose such as 103 or 104 cfu. These results suggest that the isolated strain causes respiratory tract infection in guinea pigs, rats, and mice, and that its pathogenicity higher in mice than in rats. This study extends our knowledge of interpreting the microbiologic status of laboratory animals, which will contribute to the development of reliable and reproducible animal experiments.

Establishing and Maintaining an Etruscan Shrew Colony.

The Etruscan shrew (Suncus etruscus) is one of the smallest mammals on earth and is used in many fields of research, including physiology, behavioral science and neuroscience. However, establishing and maintaining a breeding colony of this species in the laboratory can be challenging, as it requires specific husbandry conditions that greatly differ from those of more common laboratory species such as mice or rats. Over the past 15 y, we have successfully established a long-term thriving colony of 150 to 200 animals originating from 36 founders. The colony shows longer life expectancy and larger litter sizes than wild conspecifics. Breeding occurs year-round, independent of seasons, and a breeding pair can regularly produce 2 to 6 offspring with an average life expectancy of more than 3 y. The shrews are housed in glass or plastic enclosures on a specific soil-sand-mixture bedding and are provided with hideouts and nesting material consisting of moss, wood, or bark. Due to their high basal metabolic rate, the shrews require food intake greater than their body weight per day, can hunt arthropods as large as themselves, and cannot survive more than a few hours without food. Live feed such as crickets or mealworms is crucial and must be provided daily or, at the very least, every 2 d. Although our husbandry practices have constantly been adapted and refined, shrew husbandry remains challenging, and great care is necessary to meet the specific needs of this species. Here, we describe the establishment of a long-term stable colony of Etruscan shrews in a research animal facility and the specific husbandry requirements for animal wellbeing.

Abstract P009: Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer

Abstract Purpose: Colorectal cancer is the fourth most common cancer in the United States, accounting for more than 50,000 deaths annually1. The standard treatment is 5-fluorouracil (5-FU)-based chemotherapy, but emerging methods of immunotherapy have gained the attention of investigators and clinicians. A specific cytokine-based strategy for immunotherapy is the blockade of CCL2/MCP-1 (monocyte chemoattractant protein-1)2. CCL2 is a chemotactic cytokine that recruits circulating monocytes to the tumor microenvironment2. These monocytes differentiate into tumor-associated macrophages (TAMs), which promote pro-tumor functions including tumor growth, immunosuppression, and angiogenesis. CCL2 blockade in certain mouse models of cancer has been shown to reduce tumor burden but had not been studied in colorectal cancer. In this study, we examine how cytokine-targeted anti-CCL2 immunotherapy affects tumor-associated macrophage recruitment as well as the promotion of angiogenesis as a standalone and in combination with 5-FU. Methods: Nine-week-old Balb/c mice (n=13) were acclimated for a week in the Small Animal Facility at the University of Arkansas. CT26 cells, a murine colon carcinoma cell line, were cultured in RPMI-1640 media, supplemented with 10% bovine serum, 0.2% amphotericin B/gentamicin, and 1% antibiotic antimycotic solution. 1 × 106 CT26 cells were subcutaneously injected into the left flank of Balb/c mice (10 weeks old). The tumors grew to 75 mm3, upon which Balb/c mice were randomly divided into saline control, immunotherapy, chemotherapy, and combination (immunotherapy + chemotherapy) groups. The immunotherapy and chemotherapy groups were intraperitoneally injected with a 28G insulin syringe with anti-CCL2 and 5-FU at a concentration of 5.0 mg/kg/dose and 15mg/kg/dose, respectively. The control group was injected with an equal volume of sterile saline on days while the combination group received a dose of anti-CCL2 and 5-FU. Tumor-associated macrophages were quantified via immunohistochemistry using anti-CD68 and tumor vasculature was quantified using anti-CD31. Correlation plots were also created to determine a relationship between macrophage recruitment and new blood vessel formation. Results: There was a decrease in the total number of tumor-associated macrophages from Day 3 to 7 (33% decrease) when tumors were treated with anti-CCL2 and 5-FU. The area of vasculature in the combination group showed a slight decrease from Day 3 to Day 7 (29% decrease). There was a slight positive correlation between total macrophage recruitment and new blood vessel formation (R2 = 0.3041). Conclusion: Overall, we have demonstrated that anti-CCL2 in combination with 5-FU has modestly slowed tumor growth, reduced the area of vasculature, and decreased the number of recruited tumor-associated macrophages in a murine model of colorectal cancer. Acknowledgments: This material is based on work supported by the National Institutes of Health (1R15CA202662- 01), National Science Foundation CAREER award (CBET 1751554), and the Arkansas Biosciences Institute. Citation Format: Shelby N. Bess, Timothy J. Muldoon. Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P009.

Information management in laboratory animal facilities for non-human primates: what we have been using in Brazil

Information management in laboratory animal facilities for non-human primates: what we have been using in Brazil

Polycystic Kidney in Adult Guinea Pig: A Case Report

Polycystic kidney disease (PKD) is a genetic or acquired disorder characterized by progressive distension of multiple tubular segments, manifested by fluid accumulation, and growth of non-neoplastic epithelial cells. A male, 2.5 years old, Dunkin Hartley guinea pig, which was kept as a retired breeder in a lab animal facility, TANUVAS, was found dead and subjected for necropsy. History of dullness, depression, anorexia and emaciation was reported. Grossly, both the kidneys were enlarged with multiple, variable sized, spherical transparent cysts with clear fluid. The gall bladder was slightly enlarged with thick bile and a few small, black coloured stones. The tissue samples were fixed in 10% neutral buffered formalin and processed through paraffin embedding technique. Histopathological examination of kidney revealed multiple, variable sized cysts mostly in the cortical areas and the cyst wall was lined by flattened epithelium. Partial to complete loss of glomerular tufts, glomerulosclerosis, tubular epithelial cells degeneration and necrosis were also observed. Some cysts were filled with eosinophilic fluid. Interstitial areas revealed the presence of mononuclear cells infiltration. The basement membrane which supported the cyst wall showed mild thickening by special staining like Masson’s trichrome and Periodic Acid Schiff. Liver showed multifocal, periportal fibrosis with mononuclear cells infiltration and bile duct hyperplasia. Lungs revealed pulmonary congestion, emphysema and bronchiolar epithelial cell degeneration with peribronchiolar mononuclear cells infiltration. Presence of multiple cysts with degenerative changes in adjacent tubules and glomeruli of both the kidneys might have lead to the death of the animal.

Study on the Application of PRP Enriched with Hyaluronic Acid to Skin Wounds-Macroscopic Result.

Tissue healing is a complex process of replacing damaged tissue structures, being a dynamic process with spatial and temporal involvement. In practice, there are three types of healing: primary, secondary and tertiary. Since even tertiary healing can cause different problems depending on the individual patient, the medical world has always strived for new, easier, more effective, faster, and low cost-effective methods to cover skin wounds. This experiment aims to find a method that is as efficient and fast as possible, with minimal costs, of native healing of skin wounds and the development of a protocol that is as reliable and safe as possible for the patient. The experiment was carried out within the animal facility of the University of Medicine and Pharmacy of Craiova, with the agreement of our institutional Ethics Committee. The experimental group consisted of 30 individuals (Wistar laboratory rats), clinically healthy, male and female, being divided into 3 sublots of 10 individuals each. Each sublot was sacrificed at different time intervals: 7, 14 and 21 days, respectively. The local evolution of each individual was monitored during the experimental days, as well as their general state of health. Before slaughter, the lesions were examined macroscopically, following their size, whether or not the infection was present and the presence of other self-inflicted lesions later. Macroscopically, a faster wound healing was found where PRP enriched with hyaluronic acid was applied from the 7th day, so we can say that at first glance the "free" healing period in the case of enhanced epithelialization was about 7 days shorten. This fact will be confirmed or refuted following histopathological examinations and immunohistochemistry.