Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

BACKGROUND: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction. METHODS: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease. RESULTS: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease. CONCLUSIONS: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo.

Serum Concentrations of a Long-acting Cat Formulation of Transdermal Buprenorphine in C57BL/6 Mice.

Long-acting analgesics such as extended-release buprenorphine are desirable in rodent medicine because they reduce need for administration of additional medication and provide stable drug levels. We measured the serum concentrations of buprenorphine after topical administration of a long-acting transdermal buprenorphine (LAT-bup) solution to female C57BL/6 mice. We hypothesized that LAT-bup dosed topically at 40mg/kg would achieve serum drug concentrations of greater than 1ng/mL, which is considered the therapeutic level for adequate analgesia in rodents. LAT-bup administered at 40mg/kg resulted in serum drug concentrations above 1ng/mL for all mice at time points 2, 4, 24, 48, 72, and 96 h (n = 3/time point), as assessed by liquid chromatography-mass spectrometry. No adverse effects were noted when LAT-bup was dosed at either 30mg/kg or 40mg/kg. We conclude that LAT-bup is easily administered to mice and achieves adequate blood levels for 96 h. Further studies evaluating analgesic efficacy are indicated.

Evaluation of topical oclacitinib and nail trimming as a treatment for murine ulcerative dermatitis in laboratory mice.

Murine ulcerative dermatitis (UD) is a common, multifactorial skin disease of C57BL/6 and C57BL/6-background strains of mice. Many treatment options have been previously reported but have been variably successful and may interfere with specific research studies. Janus kinase (JAK) inhibitors, such as oclacitinib, have been used to treat allergic dermatitis in humans, dogs, and other species. Additionally, topical oclacitinib was shown to improve an induced model of dermatitis in mice. We hypothesized that topical application of oclacitinib in conjunction with hind limb nail trimming would improve UD lesion scores more than our institutional standard treatment regime using meloxicam, topical antibiotic ointment, and nail trimming or nail trimming alone. To test this, mice with naturally occurring UD were recruited to the study and assigned to one of three treatment groups (n = 14/group): nail trim only; nail trim plus meloxicam and topical triple antibiotic ointment; or nail trim plus topical oclacitinib. UD was assessed on days 1, 7, and 14 for all treatment groups, and scored based on a previously published scoring system that quantitatively scored UD lesions based on pruritus, character of the lesion, size of lesion, and location of lesion. Here we found that mean UD scores decreased from day 1 to day 7 and from day 1 to day 14 for all treatment groups. However, there was no significant difference in mean UD score between the treatment groups at any timepoint. These data show that topical oclacitinib and nail trimming together improved UD lesion scores comparably to our institutional standard treatment and nail trimming alone. However, further studies may be warranted to investigate other potential applications of oclacitinib to treat UD.

Effects of Cling Film Draping Material on Body Temperature of Mice During Surgery.

General anesthesia induces many systemic effects, including thermoregulatory impairment and subsequent perioperative hypothermia. Due to the animals' small size, monitoring and maintaining body temperatures in laboratory rodents during anesthesia is important for successful surgical outcomes and prompt anesthetic recovery. Draping materials have the potential to aid in thermal support during surgical anesthesia. In this study, rectal and surface (infrared) temperatures were measured in C57BL/6 mice under isoflurane anesthesia every 5 min for the duration of a 35-min sham surgery. In addition to placement on a circulating water bath, mice (n = 6/group) were draped with commercial cling film (CF; Press'n Seal, Glad, Oakland, CA), a conventional paper drape (PD), or no drape (ND) during surgery. Results demonstrated that CF-draped animals had significantly higher rectal temperatures than nondraped animals. Furthermore, surface temperatures of CF-draped mice were considerably higher than those of both paper-draped and undraped animals. The data indicate that cling film is an effective material to help minimize hypothermia in mice and potentially in other laboratory rodents requiring general anesthesia.

Cause and Treatment of Exophthalmos in Aged Cotton Rats (Sigmodon hispidus).

Aged cotton rats (Sigmodon hispidus) from an established breeding colony displayed signs of spontaneous exophthalmos. Of a total of 118 colony animals that were older than 6 mo of age, 37 (31%) displayed signs of exophthalmos. These rats were clinically healthy and had no other signs of disease. Ophthalmic exams, molecular and microbiologic testing, and histopa- thology were performed to determine the cause of the exophthalmos and to provide appropriate treatment. Environmental monitoring records were also reviewed for vivarium rooms in which the cotton rats were housed. Histopathology findings supported that the exophthalmos in these cotton rats was secondary to retro-orbital thrombosis associated with cardiomyopathy. The exophthalmic eyes were treated by either removal of the affected eye (enucleation) or surgical closure of the eyelids (temporary tarsorraphy). Enucleation of the exophthalmic eye was the best intervention for these aged cotton rats. These findings demonstrate the potential for a high incidence of ocular problems occurring secondary to cardiomyopathy in aged cotton rats. Enucleation as a therapeutic intervention for exophthalmic eyes in aged cotton rats prolongs the morbidity-free time span during which these aged animals can be used experimentally.

Mammary tumor and mastectomy synergistically promote neuroinflammation in a breast cancer survivor model.

Understanding why breast cancer survivors are at an increased risk for cognitive and affective disorders is essential for developing targeted treatment plans and improving quality of life. Microglia priming results in chronic neuroinflammation and can contribute to neuronal degeneration and dysfunction, thereby offering a potential mechanism for altered brain function that persists after tumor removal. This study examined whether mammary tumors alter microglia and augment the inflammatory profile and behavior of mice. To test this, non-metastatic mammary tumor cells (67NR) were injected orthotopically into the mammary glands of BALB/c mice, allowed to grow for 16 days, and then the tumors were removed via mastectomy. Following a 14-day surgical recovery, the mice were challenged with lipopolysaccharide (LPS), and then central and peripheral inflammation, anxiety, and depressive-like behavior were evaluated. Here we show that major central and peripheral inflammatory markers were not altered by tumor growth nor mastectomy surgery alone. However, hippocampal mRNA expression of major proinflammatory cytokines IL-1ß and TNFα was increased in tumor removal animals, persisting past surgical recovery. Nonetheless, the immune and behavioral responses following LPS administration were comparable among groups. In sum, these data demonstrate that the combination of tumor and mastectomy promotes neuroinflammation; however, immune challenge did not elucidate this inflammation as maladaptive for the host.

Evaluation of the Sterility of Press'n Seal Cling Film for Use in Rodent Surgery.

level and improve surgical outcomes. Recently, some institutions have approved the use of Press'n Seal cling film (CF; Glad Products, Oakland, CA) as a practical, cost-effective alternative to sterile drapes for rodent surgeries. The purpose of this study was to evaluate the sterility of CF by using ATP and replicate organism detection and counting (RODAC) plates. We tested 10 boxes of CF at days 0, 14, and 28 after opening the box and compared the results with traditional packaged sterile drapes. Our data indicated that CF ATP bioluminescence remained at or below 10 relative light units for 28 d after opening the box. In addition, RODAC plates had no growth for 70% of CF boxes at day 0, 100% at day 14, and 90% at day 28. The mean growth for the positive plates was 0.024 cfu/cm² sampled after contacting locations on the front and back of the CF. The results of this study support the use of CF as an acceptable alternative to traditional sterile drapes during rodent aseptic surgery.

Effects of light at night on laboratory animals and research outcomes.

Light has substantial influences on the physiology and behavior of most laboratory animals. As such, lighting conditions within animal rooms are potentially significant and often underappreciated variables within experiments. Disruption of the light/dark cycle, primarily by exposing animals to light at night (LAN), disturbs biological rhythms and has widespread physiological consequences because of mechanisms such as melatonin suppression, sympathetic stimulation, and altered circadian clock gene expression. Thus, attention to the lighting environment of laboratory animals and maintaining consistency of a light/dark cycle is imperative for study reproducibility. Light intensity, as well as wavelength, photoperiod, and timing, are all important variables. Although modern rodent facilities are designed to facilitate appropriate light cycling, there are simple ways to modify rooms to prevent extraneous light exposure during the dark period. Attention to lighting conditions of laboratory animals by both researchers and research care staff ensures best practices for maintaining animal welfare, as well as reproducibility of research results. (PsycINFO Database Record

A Role for Hypocretin/Orexin in Metabolic and Sleep Abnormalities in a Mouse Model of Non-metastatic Breast Cancer.

We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.