Potential therapeutic role of spermine via Rac1 in osteoporosis: Insights from zebrafish and mice.

Osteoporosis is a prevalent metabolic bone disease. While drug therapy is essential to prevent bone loss in osteoporotic patients, current treatments are limited by side effects and high costs, necessitating the development of more effective and safer targeted therapies. Utilizing a zebrafish ( Danio rerio) larval model of osteoporosis, we explored the influence of the metabolite spermine on bone homeostasis. Results showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone resorption. Spermine not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and cardiotoxicity. Notably, spermine showcased protective attributes in the nervous systems of both zebrafish embryos and larvae. At the molecular level, Rac1 was identified as playing a pivotal role in mediating the anti-osteoporotic effects of spermine, with P53 potentially acting downstream of Rac1. These findings were confirmed using mouse ( Mus musculus) models, in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions, suggesting strong potential as a bone-strengthening agent. This study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development, highlighting pivotal molecular mediators. Given their efficacy and safety, human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents.

Ameliorate effects of resveratrol and l-carnitine on the testicular tissue and sex hormones level in busulfan induced azoospermia rats.

Busulfan (Bus), is an alkylating agent widely used in chemotherapy which has been proven to possess toxic side effects on testicles. This study was carried out to compare the probable treatment effects of resveratrol (Res) or/and l-carnitine (Lca), as strong antioxidants, on the testicular tissue as well as on the level of sex hormones in busulfan-induced azoospermic rat models. A total of 78 adult male rats, were divided into six different experimental groups including: 1) Control; 2) Lca + Res; 3) BUS; 4) Bus + Lca; 5) BUS + Res and 6) Bus + Lca + Res. Busulfan was intraperitoneally administered in a single dose (10 mg/kg b.w), while resveratrol (20 mg/kg b.w/day) and l-carnitine (200 mg/kg b.w/day) were orally administered by gavage during 48 consecutive days to the rats. At the end of the experiment in all groups the level of LH, FSH, and testosterone were biochemically analyzed by ELISA and the testicular tissue evaluated histologically using stereological technique. Results showed that Lca or/and Res, increased the body and testis weight, the volume of the testis, interstitial tissue, germinal epithelium, and seminiferous tubule, the number of the different cells of germinal epithelium and the level of testosterone. On the other hand, Lca, Res and their combination decreased the concentration of LH and FSH compared to the group treated with Bus. In conclusion, these results suggested that l-carnitine or/and resveratrol treatment significantly attenuated busulfan -induced changes of the rat reproductive system led to the recovery of both testis and sperm parameters. However, co-administration of L-ca and Res was more effective than their individual treatment. This combination may alleviate the side effects of alkylating drugs, such as busulfan and may be beneficial for spermatogenesis.

Surveillance for diseases, pathogens, and toxicants of muskrat (Ondatra zibethicus) in Pennsylvania and surrounding regions.

Using diagnostic data and contemporary sampling efforts, we conducted surveillance for a diversity of pathogens, toxicants, and diseases of muskrats (Ondatra zibethicus). Between 1977 and 2019, 26 diagnostic cases were examined from Kansas and throughout the Southeast and Mid-Atlantic, USA. We identified multiple causes of mortality in muskrats, but trauma (8/26), Tyzzer's disease (5/6), and cysticercosis (5/26) were the most common. We also conducted necropsies, during November 2018-January 2019 Pennsylvania muskrat trapping season, on 380 trapper-harvested muskrat carcasses after the pelt was removed. Tissue samples and exudate were tested for presence of or exposure to a suite of pathogens and contaminants. Gastrointestinal tracts were examined for helminths. Intestinal helminths were present in 39.2% of necropsied muskrats, with Hymenolepis spp. (62%) and echinostome spp. (44%) being the most common Molecular testing identified a low prevalence of infection with Clostridium piliforme in the feces and Sarcocystis spp. in the heart. We detected a low seroprevalence to Toxoplasma gondii (1/380). No muskrats were positive for Francisella tularensis or Babesia spp. Cysticercosis was detected in 20% (5/26) of diagnostic cases and 15% (57/380) of our trapper-harvested muskrats. Toxic concentrations of arsenic, cadmium, lead, or mercury were not detected in tested liver samples. Copper, molybdenum, and zinc concentrations were detected at acceptable levels comparative to previous studies. Parasite intensity and abundance were typical of historic reports; however, younger muskrats had higher intensity of infection than older muskrats which is contradictory to what has been previously reported. A diversity of pathogens and contaminants have been reported from muskrats, but the associated disease impacts are poorly understood. Our data are consistent with historic reports and highlight the wide range of parasites, pathogens and contaminants harbored by muskrats in Pennsylvania. The data collected are a critical component in assessing overall muskrat health and serve as a basis for understanding the impacts of disease on recent muskrat population declines.

Mortality Associated with Bushfire Smoke Inhalation in a Captive Population of the Smoky Mouse (Pseudomys fumeus), a Threatened Australian Rodent.

A mortality event of nine threatened smoky mice (Pseudomys fumeus) occurred in January 2020 at a captive breeding facility in southeastern Australia that was affected at the time by hazardous levels of bushfire smoke, despite being more than 20 km from the nearest fire. Pathologic and clinical observations indicated smoke inhalation was the cause of death. All animals had significant pulmonary lesions, notably pulmonary edema and congestion, and moderate amounts of dark brown to black pigmented intracellular and extracellular particles from <0.5-2.5 µm in diameter were observed in the central or hilar region of the lungs of four of six animals examined histologically. Deaths occurred between three and 30 d after exposure to smoke and, for seven animals in outdoor acclimatization enclosures, were associated with very high ambient temperature (>40 C). Similar mortalities did not occur in co-located parrots, suggesting differing species sensitivity to smoke inhalation. Our findings highlight the potential for smoke to be an underdiagnosed cause of mortality in free-ranging wildlife during bushfires and for bushfires to affect wildlife populations outside of burnt areas, including in unburnt refugia. Conservation interventions for wildlife after bushfires should consider and, where possible, mitigate the risk of animals dying due to increased respiratory demand following smoke inhalation injury.

Injection Reactions after Administration of Sustained-release Meloxicam to BALB/cJ, C57BL/6J, and Crl:CD1(ICR) Mice.

The sustained-release formulation of meloxicam (MSR) is a compounded NSAID that may provide pain relief for as long as 72 h after administration. MSR injection-site skin reactions have occurred in several species but have not previously been observed in mice. We investigated the development and progression of localized skin reactions after a single injection of MSR in Crl:CD1(ICR), C57BL/6J, and BALB/cJ mice. Each mouse received a subcutaneous injection of MSR (n = 60), standard-formulation meloxicam (MEL; n = 24) or saline (control; SC; n = 24) and was scored daily according to a 5-point system for erythema and mass characteristics. Mice were euthanized at either 7 or 14 d after injection and underwent postmortem analysis. MSR-treated mice had more erythematous and mass reactions than did MEL and SC mice. Mass lesions developed in 49 MSR mice (82%; 95% CI, 70% to 90%), 5 MEL animals (21%; 95% CI, 7% to 42%), and 1 SC mouse. MSR-treated BALB/cJ developed erythematous lesions less frequently than similarly treated Crl:CD1(ICR) or C57BL/6J. Lesions often were ventrolateral to the injection site. The median times to the appearance of mass and erythematous lesions were 2 d and 3 d, respectively. Histologically, the erythematous and mass reactions correlated with necrotizing to pyogranulomatous injection-site panniculitis. Inflammation severity scores at 7 and 14 d after injection were greater in the MSR-treated group than the other 2 groups. No strain- or sex-associated differences emerged except that inflammation severity scores at day 14 were higher in Crl:CD1(ICR) females than males. The character of the inflammatory response in MSR-treated mice did not differ between 7 and 14 d after injection, indicating that MSR-induced inflammation is slow to resolve. The ventral migration and delayed onset of MSR injection-site reactions could result in their being attributed to another cause or not being identified. Researchers and clinicians should be aware of the potential for slowly resolving injection-site reactions with MSR.

Reactive astrogliosis in the dentate gyrus of mice exposed to active volcanic environments.

Air pollution has been associated with neuroinflammatory processes and is considered a risk factor for the development of neurodegenerative diseases. Volcanic environments are considered a natural source of air pollution. However, the effects of natural source air pollution on the central nervous system (CNS) have not been reported, despite the fact that up to 10% of the world's population lives near a historically active volcano. In order to assess the response of the CNS to such exposure, our study was conducted in the island of Sao Miguel (Azores, Portugal) in two different areas: Furnas, which is volcanically active one, and compared to Rabo de Peixe, a reference site without manifestations of active volcanism using Mus musculus as a bioindicator species. To evaluate the state of the astroglial population in the dentate gyrus in both samples, the number of astrocytes was determined using immunofluorescence methods (anti-GFAP and anti-GS). In addition, the astrocytic branches in that hippocampal area were examined. Our results showed an increase in GFAP+ astrocytes and a reduction in GS+ astrocytes in Furnas-exposed mice compared to animals from Rabo de Peixe. In addition, astrocytes in the dentate gyrus of chronically exposed animals exhibited longer branches compared to those residing at the reference site. Thus, reactive astrogliosis and astrocyte dysfunction are found in mice living in an active volcanic environment.

SUSPECTED HYPERVITAMINOSIS D IN RED-RUMPED AGOUTI ( DASYPROCTA LEPORINA) RECEIVING A COMMERCIAL RODENT DIET.

An 8 yr, intact male red-rumped agouti ( Dasyprocta leporina) was evaluated for weight loss. Examination revealed poor body condition, hypercalcemia, elevated serum 25-hydroxyvitamin D, metastatic calcification of soft tissues, and hyperechoic kidneys. The diet, formulated for laboratory rodents, contained elevated levels of vitamin D3. Histopathology from a female conspecific that died 5 mo prior identified dystrophic mineralization and nephrosclerosis, suggestive of a vitamin D3 toxicity. The male agouti responded well to a dietary reduction in vitamin D3 and calcium. Six months into therapy, progressive renal failure was identified and was further managed with enalapril, phosphorus binders, and dietary manipulation. Suspected vitamin D3 toxicity has been reported in pacas ( Cuniculus paca) and agouti and has been linked to exposure to New World primate diets. In this brief communication, an agouti developed suspected hypervitaminosis D after receiving a commercial rodent diet commonly fed to this species in captivity.

Effects of Intracage Ammonia on Markers of Pulmonary Endothelial Integrity in Mice Housed in Static Microisolation Cages.

Time-weighted exposure limits to ammonia are established for humans; however similar guidelines have not been defined for laboratory rodents. The Guide recommends maintaining air pollutants at concentrations below levels irritating to mucous membranes but does not provide specific values. Numerous studies have examined ammonia and its effects on animal health, yet none have assessed the effects of naturally occurring intracage ammonia on the lower pulmonary tree and pulmonary endothelial and epithelial integrity in mice. We performed several assays commonly used in mouse acute lung-injury studies (bronchoalveolar lavage fluid [BAL] cell counts and protein concentration, excess lung water content [ELW], Evans blue permeability assay [EBA], lung tissue myeloperoxidase assay [MPO], and lung histopathology) to evaluate the effects of exposure to cyclical, naturally occurring ammonia levels on pulmonary integrity and inflammation. C57BL/6 mice were maintained in static microisolation or open-top cages. Cages were changed weekly, and ammonia levels were measured for 6 wk on days 0, 1, 3, 5, and 7 of each cage-change cycle. Ammonia levels in static microisolation cages began to increase on day 3 and peaked at a mean of 141.3 ppm on day 7. Ammonia levels in open-top cages never exceeded 5 ppm. Neither BAL cell counts, protein concentration, ELW, EBA, nor MPO differed significantly between groups. Lung histopathology showed minimal, incidental changes in all mice. Our findings indicate that the ammonia concentrations in the static microisolation cages we used did not alter the integrity of the lower pulmonary tract nor influence key indicators used to assess acute lung injury.

Interstrain Differences in CO2-Induced Pulmonary Hemorrhage in Mice.

Carbon dioxide is the most commonly used gas for euthanasia of rodents. The current AVMA Guidelines recommend slowly filling the container with CO2 (SF) and now indicate that the practice of placing conscious animals in containers prefilled with CO2 (PF) is unacceptable. An investigator noted pulmonary hemorrhage (PH) in BALB/c mice euthanized by SF that was not observed after PF. Here we evaluated whether the air-displacement rate (SF compared with PF) influenced the development of PH or nasal hemorrhage (NH) in 2 commonly used mouse strains. In addition, we investigated the prevalence of PH and NH in mice euthanized by isoflurane overdose (IO). Male and female (age groups, 6 wk and 6 mo) BALB/c and C57BL/6 mice were euthanized by SF or PF. In addition, 6-mo-old BALB/c male mice were euthanized by IO. Lung, nasal turbinates, brain, and reproductive organs were collected for gross and histologic evaluation and scored for degree of hemorrhage (score, 0 to 3). Severity of hemorrhage did not differ according to mouse age or sex. PH in BALB/c mice was more severe after SF than PF, and SF and PF induced more severe PH in BALB/c than in C57BL/6 mice. PH in 6-mo-old male BALB/c mice was more severe after SF than IO. Neither SF, PF, nor IO influenced the prevalence of NH in any group. This study demonstrates that the method of euthanasia may need to be altered depending on the mouse strain used.

Chronic noise exposure and testosterone deficiency -- meta-analysis and meta-regression of experimental studies in rodents.

INTRODUCTION: Chronic psychological distress can cause suppression of the hypothalamic-pituitary-testicular axis and thus lead to male hypogonadism, which is associated with psycho-social dysfunction, chronic diseases, and as a result, considerable economic costs. Conversely, noise is a prototypal environmental stressor of growing importance, already linked to birth outcomes and diabetes. However, its effects on male testosterone levels have been paid little attention. MATERIAL AND METHODS: This paper reports a systematic review and meta-analysis of experimental studies in rodents, which have examined the effect of chronic noise stress on serum testosterone levels. A systematic search in MEDLINE, EMBASE and the Internet yielded seven studies. A quality effects meta-analytical model was applied to compute pooled Hedges's g. Quality effects meta-regression was carried out as well. RESULTS: We found pooled Hedges's g of -2.41 (95% CI: -3.28, -1.54), indicating a very large effect of noise exposure on testosterone. Metaregression confirmed that the overall duration of exposure explained a significant proportion of the variance across individual effect sizes (Q (1) = 3.95, p = 0.047). However, there was considerable inter-study heterogeneity (I2 = 82%) and publication bias (p = 0.016). After inputting two studies previously thought to be missing, the pooled effect dropped to g = -1.53 (95% CI: -3.01, -0.05). CONCLUSIONS: Chronic noise exposure of ≈ 100 dB leads to a significant reduction of serum testosterone in male rodents. Research on humans is highly warranted, especially given the steady trend in Western societies for increasing the burden of both male hypogonadism and noise pollution.

[Background data of spontaneous tumors in F344/DuCrlCrlj rats].

INTRODUCTION: We investigated the 2-year survival rate and incidence of spontaneous tumors in F344/DuCrlCrlj rats used in carcinogenicity studies of chemical substances. Records for animals used in the control groups of carcinogenicity studies which were conducted during the last 10 years were obtained from the database of the Japan Bioassay Research Center (JBRC). Six hundred ninety-nine males and 550 females were used in 14 and 11 inhalation studies, respectively, and 500 animals of each sex were used in 10 male and 10 female oral studies. METHODS: In each study, SPF (specific pathogen free) animals were housed for 2 years (104 weeks) as control groups in the carcinogenicity studies. All animals underwent necropsy and histopathological examination. Each study was conducted in accordance with the Good Laboratory Practice. RESULTS: The incidence of interstitial cell tumors was highest in both inhalation studies and oral studies (inhalation studies 86.1%, oral studies 68.6%). Tumors which had an incidence of 6% or higher were adenoma of the pituitary, C-cell adenoma of the thyroid, and mononuclear cell leukemia (LGL leukemia) of the spleen in male and female rats; fibroma of the subcutaneous tissue, adrenal pheochromocytoma, and islet cell adenoma of the pancreas in male rats; and endometrial stromal polyps and fibroadenoma of the mammary gland in female rats. Tumors other than the above had rare incidence rates. A clear difference in the incidence of spontaneous tumors was not observed between the inhalation and oral studies. The incidences of spontaneous tumors in control groups of previous oral studies are similar to our findings. There are no other reports of the spontaneous tumor incidence in the control groups of inhalation studies using F344/DuCrlCrlj rats. The 2-year survival rate was about 77% in both the inhalation and oral studies, and a gender difference was not observed. The F344/DuCrlCrlj rats used at JBRC had a higher 2-year survival rate than F344/N rats. This difference is possibly due to the low incidence of LGL leukemia in the F344/DuCrlCrlj rat. CONCLUSIONS: The incidences of spontaneous tumors in F344/DuCrlCrlj rats used in control groups of both inhalation and oral studies during the last 10 years at JBRC are similar to each other and similar to those reported in other studies. This is the first report on the incidence of spontaneous tumors in inhalation studies and contributes to the toxicological evaluation of studies using F344/DuCrlCrlj rats.

Comparative assessment of effectiveness of ketoprofen and ketoprofen/beta-cyclodextrin complex in two experimental models of inflammation in rats.

Oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) can lead to adverse effects such as gastrointestinal distress. The complexation of different groups of active substances with ß-cyclodextrin (ß-CD) has drawn considerable interest over recent years. The purpose of this study was to analyze the ketoprofen/ß-cyclodextrin (K/ß-CD) conjugate complex as well as to assess its anti-inflammatory effect after oral administration (doses of 30 mg/m(2) and 15 mg/m(2) of body surface), compared with ketoprofen. The studies were done on two models of experimentally-induced acute inflammation in rats (n = 48, 6/group), by means of intraplantar administration of a 10% aqueous kaolin suspension and intraperitoneal administration of a 1% sodium thioglycolate solution. The dynamics of the acute inflammatory process and the anti-inflammatory effects were monitored using plethysmometric determinations after 3, 6, 9, 12, 24 and 48 h (plantar inflammation), and the absorbance of the exudates (spectrophotometrically read) and nucleated cell counts after 24 h (peritoneal inflammation). The coupling of ketoprofen with ß-CD resulted in increased solubility (100% in 60 min) of the newly-formed product, which further resulted in a higher bioavailability compared with ketoprofen (<40% in 120 min). In both models of experimentally-induced inflammation, the K/ß-CD complex had a higher anti-inflammatory activity than ketoprofen.

Hypervitaminosis D in guinea pigs with α-mannosidosis.

A colony of guinea pigs (n = 9) with α-mannosidosis was fed a pelleted commercial laboratory guinea pig diet. Over 2 mo, all 9 guinea pigs unexpectedly showed anorexia and weight loss (11.7% to 30.0% of baseline weight), and 3 animals demonstrated transient polyuria and polydipsia. Blood chemistry panels in these 3 guinea pigs revealed high-normal total calcium, high-normal phosphate, and high ALP. Urine specific gravity was dilute (1.003, 1.009, 1.013) in the 3 animals tested. Postmortem examination of 7 animals that were euthanized after failing to respond to supportive care revealed renal interstitial fibrosis with tubular mineralization, soft tissue mineralization in multiple organs, hepatic lipidosis, and pneumonia. Analysis of the pelleted diet revealed that it had been formulated with a vitamin D3 content of more than 150 times the normal concentration. Ionized calcium and 25-hydroxyvitamin D values were both high in serum saved from 2 euthanized animals, confirming the diagnosis of hypervitaminosis D. This report discusses the clinical signs, blood chemistry results, and gross and histologic findings of hypervitaminosis D in a colony of guinea pigs. When unexpected signs occur colony-wide, dietary differentials should be investigated at an early time point.

Effects of housing density on nasal pathology of breeding mice housed in individually ventilated cages.

The 2011 edition of the Guide for the Care and Use of Laboratory Animals includes new recommendations for the amount of floor space that should be provided to breeding mice. When pairs or trios of continuously breeding mice are housed in shoebox cages, they may have less than this recommended amount of floor space. High housing densities may adversely affect animal health, for example, by compromising air quality inside the cage. Hence, some institutions are carefully reevaluating the microenvironments of breeding cages. The use of individually ventilated cages (IVCs) to house research mice allows for greater control over the quality of the cage microenvironment. The authors evaluated the microenvironments of shoebox cages in an IVC rack system housing breeding and non-breeding Swiss Webster mice. Ammonia concentrations were significantly higher in cages housing breeding trios with two litters. Histopathologic lesions attributable to inhaled irritants such as ammonia were found in mice housed in breeding pairs and trios. The authors conclude that the microenvironments of cages in an IVC rack system housing breeding pairs and trios may be detrimental to animal health.

Overexpression of PDGFA and its receptor during carcinogenesis of Opisthorchis viverrini-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a crucial health problem in northeastern part of Thailand, which is caused by a combination of Opisthorchis viverrini infection and nitrosamine. A better understanding of its molecular mechanism is an important step to discover and develop the new diagnostics and therapies for CCA. To reveal the involvement of potential genes in the development of CCA, the present study investigated the expression kinetics of platelet-derived growth factor alpha (Pdgfa) and its receptor (Pdgfra) during the tumorigenesis of CCA induced by O. viverrini infection with quantitative RT-PCR, and confirmed the expression with immunohistological staining. The results showed that in the hamster model of opisthorchiasis-associated CCA, the expression of Pdgfa was increased after infection plus N-nitrosodimethylamine (NDMA) administration, reached its peak at 2 months post infection, and remained at the high level until 6 months. Similarly, the expression of Pdgfra was increased time-dependently. The positive immunostaining for PDGFA proteins was observed in the cytoplasm of epithelial tumor cells of hamster CCA. Moreover, the analysis of the expression of these genes in 10 cases of human opisthorchiasis-associated CCA showed that Pdgfa was overexpressed in 80%, and Pdgfra was overexpressed in 40% cases (>3.0 folds, compared with the expressions of adjacent normal tissues). This result suggests that PDGFA is likely involved in the tumorigenesis of opisthorchiasis-associated CCA, and may be a promising candidate biomarker for diagnosis and treatment strategies of CCA.

A therapeutic dose of ketoprofen causes acute gastrointestinal bleeding, erosions, and ulcers in rats.

Perioperative treatment of several rats in our facility with ketoprofen (5 mg/kg SC) resulted in blood loss, peritonitis, and death within a day to a little more than a week after surgery that was not related to the gastrointestinal tract. Published reports have established the 5-mg/kg dose as safe and effective for rats. Because ketoprofen is a nonselective nonsteroidal antiinflammatory drug that can damage the gastrointestinal tract, the putative diagnosis for these morbidities and mortalities was gastrointestinal toxicity caused by ketoprofen (5 mg/kg). We conducted a prospective study evaluating the effect of this therapeutic dose of ketoprofen on the rat gastrointestinal tract within 24 h. Ketoprofen (5 mg/kg SC) was administered to one group of rats that then received gas anesthesia for 30 min and to another group without subsequent anesthesia. A third group was injected with saline followed by 30 min of gas anesthesia. Our primary hypothesis was that noteworthy gastrointestinal bleeding and lesions would occur in both groups treated with ketoprofen but not in rats that received saline and anesthesia. Our results showed marked gastrointestinal bleeding, erosions, and small intestinal ulcers in the ketoprofen-treated rats and minimal damages in the saline-treated group. The combination of ketoprofen and anesthesia resulted in worse clinical signs than did ketoprofen alone. We conclude that a single 5-mg/kg dose of ketoprofen causes acute mucosal damage to the rat small intestine.