Lateral Tail Research Articles

Electromyostimulation with blood flow restriction enhances activation of mTOR and MAPK signaling pathways in rat gastrocnemius muscles.

Neuromuscular electrical stimulation (NMES) combined with blood flow restriction (BFR) induces muscle hypertrophy. However, cellular mechanisms underlying the muscle hypertrophy induced by NMES combined with BFR remain unclear. We tested the hypothesis that NMES combined with BFR would enhance the mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling pathways. Age-matched male Wistar rats (6 months old, n = 7 per group) were assigned randomly to control, BFR alone (BFR), NMES alone (NMES), and NMES combined with BFR (NMES/BFR) groups. NMES induced 25 isometric contractions lasting 8 s with 4-s resting periods between contractions in the gastrocnemius muscle. Four sets in total were performed, with 1-min intervals between sets. A latex cuff was placed on the proximal portion of the hind limb and BFR at 200 mm Hg was conducted in 4 sets (each set 5 min) with 1-min rest intervals between sets. Venous blood was collected from the lateral tail vein to determine pH, H+ concentration, and lactate concentration before and immediately after the treatments. Expression levels of proteins related to muscle hypertrophy were determined by Western blot analysis. The application of NMES/BFR promoted muscle fatigue more than NMES alone. NMES/BFR induced greater changes in accumulation of metabolites and increase in gastrocnemius muscle weight. The phosphorylation of mTOR and MAPK signaling-related proteins was also enhanced following NMES/BFR, compared with other conditions. Thus, NMES enhanced the activation of mTOR and MAPK signaling pathways when combined with BFR.

Phage vaccines displaying YGKDVKDLFDYAQE epitope induce protection against systemic candidiasis in mouse model

Candida albicans is a common commensal and opportunistic fungal pathogen in human, which poses threat to human health, especially in immunocompromised patients. Unfortunately, few effective prophylactic and therapeutic strategies were applied to clinic practice. Recently, the peptide YGKDVKDLFDYAQE from Fructose-bisphosphate aldolase 1 (Fba1), as a vaccine, was reported to induce protection effects against systemic candidiasis. Here, we displayed this epitope peptide on the coat proteins (pIII or pVIII) of filamentous phage, and investigated their protective effects against C. albicans infections. Mice were immunized with recombinant phages (designated as phage-3F and phage-8F) or protein (rFba1), then challenged with C. albicans yeast cells via lateral tail vein. Results demonstrated that the recombinant phages as well as rFba1 apparently induced humoral and cellular immune responses, reduced fungal burden and relieved kidney damage in infected mice and significantly improved their survival rates. Briefly, all these findings indicated that the recombinant phages displaying the epitope YGKDVKDLFDYAQE have the potential to be developed into a new vaccine against C. albicans infections.

Comparative pathogenicity analysis of Cryptococcus neoformans RAPD types

Cryptococcus neoformans and Cryptococcus gattii are the major causes of Cryptococcosis, an invasive fungal infection of human and animals. Few data have been reported on the pathophysiology of Pattern I (PI) and Pattern IV (PIV) of C. neoformans with context of their clinical outcome. In this study an extensive comparison of PI and PIV was conducted to provide a more complete and reliable assessment of their biological and clinical behaviour. Representative strains of PI and PIV of C. neoformans were collected from Outreach Programme on Zoonotic Diseases (OPZD) at Department of Veterinary Public Health of West Bengal University of Animal and Fishery Sciences and infective inoculum was prepared by adjusting the number of Cryptococci at 3.25X 105 / ml in phosphate buffer saline (pH 7.4). Mice were challenged with 0.2 ml of inoculum at lateral tail vein and infected mice were examined for histopathological study and survivability study for 21 days. This present study showed that PIV produced more cystic structure in brain (c2 =4.22, P 0.05). PIV was associated with an approximate 4-fold increase in risk of death as compared with PI (P< 0.05, OR= 4.38, 95% CL= 1.32 to 14.50). Our study suggested that Pattern IV produced more severe Cryptococcosis in murine model due to its difference in genotype.

Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia.

Intravenous (IV) administration in mice is predominantly performed via the lateral tail veins. The technique requires adequate training before it can be used safely and routinely. A novel anaesthesia induction chamber has been developed to simplify the treatment and to facilitate IV injection in mice, particularly for untrained personnel. We have assessed the benefits of the chamber in refining IV injection in isoflurane-anaesthetized mice in direct comparison with the common restrainer method on conscious animals. The body weight, nesting behaviour and concentrations of faecal corticosterone metabolites were taken as indicative of distress induced by the various procedures. The results suggest that both methods of tail-vein injection induce similar levels of momentary stress in the animals, revealed by a short-term increase in the levels of stress hormone metabolites in faeces. A temporary reduction of body weight was observed after IV injection under isoflurane anaesthesia but not for conscious mice injected in the common restrainer. We conclude that the severity of tail-vein injection in mice is 'mild' for both methods. There was no evidence that refining the procedure by using isoflurane anaesthesia in the induction chamber was associated with any benefit.

Friday, September 28, 2018 10:30 AM–12:00 PM abstracts: innovation, surface technology and biomechanics: 177. Non invasive, PET imaging with zirconium probes accurately localizes staphylococcus aureus spine implant infections

BACKGROUND CONTEXT Diagnosis and precise localization of spine infections are an elusive pursuit. Despite widespread technical advances, the current standard of magnetic resonance imaging lacks the precision to discern infection versus inflammation due to other causes. Nuclear medicine utilizing the metabolic positron emission tomography (PET) probe18F-fluorodeoxyglucose (18F-FDG) can identify inflammation, but it lacks specificity for bacterial infection. However, adjuncts compatible with PET/CT imaging have the potential to noninvasively increase the specificity for bacteria responsible for spine infections and guide surgeons with regard to the locale and extent of infection. PURPOSE The purpose of this study is to assess the diagnostic potential of utilizing PET/CT and 89-Zirconium (89Zr) labeled bacterial probes in noninvasive, accurate localization of spine implant infections. STUDY DESIGN/SETTING This is a basic science investigation utilizing a mouse model of spine infection. The spine implants are inoculated with bioluminescentStaphylococcus aureusfor longitudinal quantification of bacterial burden, and PET/CT signal intensity is quantified utilizing 18F-FDG and 89Zr-DFO-1D9 mAb intravenous injections. PATIENT SAMPLE Fifteen12-week-oldC57BL/6 wild type mice were utilized. OUTCOME MEASURES In vivobioluminescence and PET/CT signal intensity was quantified utilizing imaging software. Three-dimensional PET/CT images were utilized to localize signal intensities of interest. METHODS In accordance with an established mouse model of spine implant infection, a spinal implant was fixed into the spinous process of the L4 vertebra. Inoculation of the implant and wound bed was performed with bioluminescent S. Aureus. On postoperative day 7, 18F-FDG PET/CT imaging was completed in 11 infected and four sterile control mice. They were also injected intravenously via lateral tail vein with 68.6±1.6 μCi (∼14μgmAb) of89Zr-DFO-1D9 mAb. One week following89Zr-DFO-1D9 injection, mice underwent PET/CT to detect 89Zr-DFO-1D9 mAb signal intensities. RESULTS Bioluminescence imaging revealed signal emitted solely from the surgical bed of infected mice which matched that of historical infected controls – bioluminescence signals remained elevated over the duration of the experiment suggestive of sustained bacterial infection. PET-CT imaging with 18F-FDG probe revealed high signal intensity at the site of the spine implant with lower levels of signal within the gut, bladder, and heart. The 89Zr-DFO-1D9 mAb probe signal in the region of the implant was significantly higher in infected mice compared to sterile controls (2.72e9±1.19e9 versus 6.60E8±3.56E8 total radiant efficiency, respectively;p CONCLUSIONS TheS. aureusspecific89Zr-DFO-1D9 mAb probe was shown by PET/CT imaging to accurately detectinfections in-vivo. This study represents the first in-vivo investigation utilizing PET/CT with intravenous bacterial specific probes which may vastly improve the ability to accurately and noninvasively diagnose and localize spine infections.

English

The prophylactic effect of ethanolic leaf extract of Nauclea latifolia, widely used in herbal malarial treatment, was compared against standard drug Coartem® (Artemether/Lumefantrine) in Plasmodium-infected mice was investigated, and thereafter analysed for organosomatic index, parasitemia, histomorphological and immunohistochemical changes in the hippocampus. Twenty male mice about 6 to 8 weeks weighing 20 to 24 g were alloted into four groups of five mice each. Group 1 served as control received placebo; group 2 received extract 500 mg/kg; group 3 received extract 1000 mg/kg; and group 4 received 5 mg/kg of Artemether/Lumefantrine. Extract and drug administrations were performed for 3 days, and thereafter mice were infected with 106 of Plasmodium berghei parasites, and monitored for 4 days, after which the experiment was terminated. Thick blood smear were prepared from lateral tail vein, then under anesthesia via a cocktail of xylaxine and ketamine, intracardiac perfusion was performed first with phosphate buffered saline to clear systemic blood, and then 4% paraformaldehyde to fixed brain for routine histology and immunohistochemistry. Result indicates organosomatic index was not statistically significant, parasitemia in the treated groups were significantly (p<0.05) decreased compared to control and were corroborated in the photomicrographs of respective blood morphology, however, histologically there was moderate to severe distortion of the hippocampus across the groups, but glial fibrillary acidic protein expression, a marker for neurotoxicity indicated that group 4 had the most immunolabelling intensity compared to other test groups. In conclusion, the prophylactic ethanolic extract of N. latifolia and Artemether/Lumefantrine cleared parasitemia also seen in the blood morphology, and low dose N. latifolia plausibly has better safety and hippocampal toxicity profile with decreased neuronal shrinkage and distortions, with a more down regulated glial fibrillary acid protein than higher extract doses and Artemether/lumefantrine. Key words: Malaria, hippocampus, neuronal perturbation, Artemether/Lumefantrine, Nauclea latifolia.

Establishment and validation of microsampling techniques in wild rodents for ecotoxicological research.

Compounds and products in the biocide and plant protection sector can only be registered after formal risk assessment to ensure safety for users and the environment. In bird and mammal risk assessment, this is routinely done using generic focal species as models, which are of particular exposure risk. Such a species is the common vole (Microtus arvalis) due to its high food intake relative to the low body weight. For wild species, biological samples, data and hence realistic exposure estimations are particularly difficult to obtain. In recent years, advances have been made in the techniques related to serial microsampling of laboratory mice and rats that allow for a reduction in sampling volumes. Similar progress in wild species sampling is missing. This study presents a proof of concept to dose wild rodents with relevant compounds and to draw serial, low volume blood samples suitable for state-of-the art toxicokinetic analyses. For the first time, the jugular vein of common voles was used to administer compounds (two frequently used fungicidal components). This procedure and the following microsampling of blood (2×10μl six times within 24hours) from the lateral tail vein did not affect body weight and mortality of voles. Samples were sufficient to detect dissipation patterns of the compounds from blood in toxicokinetic analysis. These results suggest that microsampling can be well translated from laboratory mice to wild rodent species and help to obtain realistic exposure estimates in wild rodents for ecotoxicological studies as well as to promote the 3R concept in studies with wild rodent species.

Abstract 222: Translocator Protein as Novel Marker for Early Atherosclerosis

Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. Here, we aimed to determine if (i) the TSPO radioligand, 18 F-PBR111, can identify fatty streak formation that precedes atherosclerotic lesion development and (ii) TSPO expression can be used to identify distinct macrophage populations during different stages of lesion progression. Atherosclerosis-prone apoE -/- mice were maintained on a high-fat diet for 3, 9 and 12 weeks. C57BL6 mice maintained on chow diet served as controls. Mice were anesthetized, injected with 18 F-PBR111 (8-18 MBq, 0.1 mL, 0.2 nM) via the lateral tail vein and imaged using an Inveon PET/CT Scanner over 60 min. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, fluorescently labelled anti-TSPO antibody and the macrophage markers F4/80 and CD11b. Tissues were visualized on a Lightsheet Z.1 microscope and analysed with 3D imaging software. A 3-fold increase in the uptake of 18 F-PBR111 was observed by PET/CT in the aortas of atherosclerotic mice relative to controls. Increased 18 F-PBR111 uptake was observed in the aortic arch and thoracic aorta, corresponding with sites of atherosclerotic lesion development. Light sheet microscopy revealed that TSPO expression increased correspondingly to atherosclerosis progression. The expression pattern of macrophage populations in early lesions differed from that in mature plaques. While F4/80-positive macrophages were evident uniformly throughout the aortas, CD11b-positive macrophages were increased in atherosclerotic areas. In conclusion, imaging TSPO expression is a new approach for studying atherosclerotic lesion progression and is associated with specific inflammatory cell infiltration. The TSPO ligand 18 F-PBR111 is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

Effects of aging and gender on micro-rheology of blood in 3 to 18 months old male and female Wistar (Crl:WI) rats.

Age- and gender-related alterations of hemorheological parameters have not been completely elucidated to date. Experiments on older animals may give valuable information on this issue. However, the majority of rheological studies have been performed in young rodents. We aimed to investigate the influence of aging and gender on hemorheological parameters in rats. Coeval male (n=10) and female (n=10) Wistar (Crl:WI) rats were followed-up over 15 months. Blood samples were obtained from the lateral tail vein at 3, 4, 5, 9, 12, 15 and 18 months of age. Hematological parameters, red blood cell deformability (elongation under shear), osmotic gradient deformability and erythrocyte aggregation were tested. Body weight and the estrus cycle (in females) were also examined. Erythrocyte aggregation showed age- and gender-related variations. Red blood cell deformability was greater in females and gradually decreased over the 15-month period in both genders. Erythrocyte aggregation was greater in male rats at most ages, but did not show consistent changes with age. The micro-rheological parameters showed age-related alterations with gender differences. The effect of the estrous cycle cannot be excluded in female rats. The results provide reference data for studies of aging in rats and of the mechanism related to age and gender differences in hemorheology.

Electromyostimulation with Blood Flow Restriction Enhances Activation of mTOR and MAPK Signalling Pathways in Rat Gastrocnemius Muscles

AimNeuromuscular electrical stimulation (NMES) combined with blood flow restriction (BFR) can induce muscle hypertrophy. However, cellular mechanisms underlying the muscle hypertrophy induced by NMES combined with BFR remain unclear. Thus, the purpose of this study was to elucidate the signalling pathways involved in NMES with BFR induced muscle hypertrophy.MethodsAge‐matched male Wistar rats (6 months old, n=7 per group) were assigned randomly into control, BFR alone (BFR), NMES alone (NMES) and NMES combined with BFR (NMES/BFR) groups. NMES induced 25 isometric contractions lasting 8 s with a 4 s resting period between each contraction in the gastrocnemius muscle. Four sets in total were performed, with 1‐min intervals between each set. Latex cuff was placed on the proximal portion of hind limb and BFR was conducted in four sets (each set 5 min) with 1‐min rest intervals in between at 200 mmHg. Venous blood was collected from the lateral tail vein to determine pH, H+, lactic acid concentration before and immediately after the treatments. Expression levels of proteins related to muscle hypertrophy were determined by western blot analysis.ResultsThe application of NMES combined with BFR encouraged muscle fatigue more than NMES alone. NMES/BFR induced greater changes in accumulation of metabolites and increase in the weight of gastrocnemius muscle. The phosphorylation of mTOR and MAPK signalling related proteins also enhanced following NMES combined with BFR, compared with other conditions.ConclusionsWe elucidated that NMES enhanced the activation of mTOR and MAPK signalling pathways when combined with BFR.Support or Funding InformationThis work was supported by Japan Society for Promotion of Science (JSPS) KAKENHI Grant No. 16K16564 (T. Natsume) and Grant No. 17K01765 (T. Yoshihara). Grants from the MEXT‐Supported Program for Strategic Research Foundation at Private Universities also supported this research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intravenous infusion of xenon-containing liposomes generates rapid antidepressant-like effects

AimSimilar to ketamine, xenon gas acts as a glutamatergic N-methyl-d-aspartate receptor antagonist, but devoid of propensity to cause untoward effects. Herein, we loaded xenon gas into a liposomal carrier called xenon-containing liposomes (Xe-liposome) for systemic delivery, and investigated its effect as an antidepressant and also analyzed synaptic biomarkers including brain-derived neurotrophic factor (BDNF), protein kinase B (AKT), mammalian target of rapamycin (mTOR), protein kinase C (PKC) and extracellular signal-regulated kinase-1/2 (ERK1/2) in blood and brain. MethodsXe-liposomes (15 μl/mg) were prepared by a pressurized freeze-thaw method, and injected via the lateral tail vein (0.6 mL/rat) in male Wistar rats. The uncaging of xenon gas from circulating Xe-liposome was facilitated by continuous ultrasound application externally on the neck over the internal common carotid artery. One-hour after Xe-liposome infusion, animals were assessed for depression-like behaviors using a forced swimming test (FST), and spontaneous locomotor activity. Blood, as well as frontal cortex and hippocampal samples were obtained for immunoblotting and/or enzyme-linked immune sorbent assays. ResultsAcute intravenous infusion of Xe-liposome, at 6 mg/kg, showed an increase in swimming time in the FST (p < 0.006), indicating antidepressant-like phenotypes. Higher doses of Xe-liposomes (9 mg/kg) failed to improve swimming duration. This behavioral discrepancy was not associated with locomotion aberrations, as gross activity of rats remained similar for both doses. In biochemical analyses of frontal cortex, protein levels of BDNF increased by 64%, and enhanced phosphorylation of AKT (43%) and mTOR (93%) was observed at the 6 mg/kg dose level of Xe-liposomes, while these biomarkers and phosphorylated PKC and ERK1/2 levels remained unchanged at the higher dose. Moreover, Xe-liposomal treatment did not change the plasma and protein levels of BDNF, and phosphorylated AKT, mTOR, PKC and ERK1/2 hippocampal expressions. ConclusionXe-liposomes mediate a rapid antidepressant-like effect through activation of AKT/mTOR/BDNF signaling pathway.

Pharmacodynamics of a Long-Acting Echinocandin, CD101, in a Neutropenic Invasive-Candidiasis Murine Model Using an Extended-Interval Dosing Design.

Echinocandins are important in the prevention and treatment of invasive candidiasis but limited by current dosing regimens that include daily intravenous administration. The novel echinocandin CD101 has a prolonged half-life of approximately 130 h in humans, making it possible to design once-weekly dosing strategies. The present study examined the pharmacodynamic activity of CD101 using the neutropenic invasive candidiasis mouse model against select Candida albicans (n = 4), C. glabrata (n = 3), and C. parapsilosis (n = 3) strains. The CD101 MIC ranged from 0.03 to 1 mg/liter. Plasma pharmacokinetic measurements were performed using uninfected mice after intraperitoneal administration of 1, 4, 16, and 64 mg/kg. The elimination half-life was prolonged at 28 to 41 h. Neutropenic mice were infected with each strain by lateral tail vein injection, treated with a single dose of CD101, and monitored for 7 days, at which time the organism burden was enumerated from the kidneys. Dose-dependent activity was observed for each organism. The pharmacokinetic/pharmacodynamic (PK/PD) index of the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC index) correlated well with efficacy (R2, 0.74 to 0.93). The median stasis 24-h free-drug AUC/MIC targets were as follows: for C. albicans, 2.92; for C. glabrata, 0.07; and for C. parapsilosis, 2.61. The PK/PD targets for 1-log10 kill endpoint were 2- to 4-fold higher. Interestingly, the aforementioned PK/PD targets of CD101 were numerically lower for all three species than those of other echinocandins. In summary, CD101 is a promising, novel echinocandin with advantageous pharmacokinetic properties and potent in vivo pharmacodynamic activity.

Abstract 180: Restoration of MiRNA MiR-181c-5p Rescue From the Detrimental Effect of Social Isolation in the Mice Subjected to Ischemic Stroke

Introduction: MicroRNAs (miRNAs) are a class of short non-coding RNAs. Dysregulation of a variety of miRNAs have been tied to several neurological disorders including stroke. Recent studies have also found that microRNA profiles can change as a result of social environments. We have previously shown that the social isolation is detrimental to post-stroke recovery. MiR 181c-family of miRNA is one of the evolutionarily conserved and highly expressed miRNA family in the brain. Therefore, in this study we examined the specific role of miR-181c-5p after post-stroke social isolation. Methods: 2-3-month old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, a 60-minute right middle cerebral artery occlusion surgery (MCAO) was performed and mice were randomly assigned to one of two housing conditions, isolation or continued pair-housing (ST-ISO or ST-PH). In the treatment study group, ST-ISO mice were further randomly assigned to receive either miR-181c-5p mimic (7mg/kg i.v/day x drug) or a vehicle control administered through lateral tail vein at 24h and 48h after stroke. The effect of mimic treatment was evaluated at 1, 3, and 7 days after stroke using rotarod, open field test (OFT) and corner test. Mice were sacrificed 7 days after stroke for miRNA analysis. Results: Whole miRNone analysis of total RNA revealed miR 181-5p as one of the most affected miRNA in the brain tissue of post stroke socially isolated mice. Temporal profile expression data suggests that, miR-181c-5p was significantly down regulated (p&lt;0.05 vs STPH) 7 days after stroke as compared to ST-PH controls. Treatment with miR-181c-5p mimic successfully increased miR-181c-5p miRNA expression in the treatment group as validated by qPCR analysis. Isolation-induced reduction in miR-181c-5p expression was rescued by mimic treatment. Post treatment with mimic reduced stroke mortality by 25% and partially restored motor deficit in post stroke socially isolated mice. Summary: The present data suggest that social isolation reduces miR-181c-5p after stroke. Restoration of this miRNA improved post stroke mortality and behavioral recovery. This microRNA is a valid target to reduce the detrimental effects of social isolation after ischemic stroke.

Additional Specimens and a Critical Consideration of Eurypterid Opisthosoma Flexibility: (A Reply to Lamsdell et al.)

AbstractHerein additional and better-preserved instances of eurypterid specimens with opisthosoma preserved in strong lateral bends are documented, bolstering the evidence for potential lateral tail striking among various eurypterid species. Contrary to dissenting suggestions, such specimens remain fully articulated, and the opisthosoma exoskeleton of many eurypterids appears adapted to permit a wide range of lateral motion. Observation of long telson spines and prominent dorsal telson keels across the Eurypterida reveals that the two traits are negatively correlated. This indicates that the function of a spiniform telson was incompatible with an opisthosoma that generated strong lateral hydraulic resistance and therefore supports the hypothesis that, when present, eurypterids may have used their long telson spines as laterally striking weapons.

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20°, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20°, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Assessment of toxicity and tolerability of a combination vehicle; 5% Pharmasolve, 45% Propylene glycol and 50% Polyethylene glycol 400 in rats following repeated intravenous administration

The selection of a suitable vehicle for administration of NCEs in non-clinical studies is always a challenge for poorly soluble compounds. Challenge is increased if the dose formulation is intended for intravenous (i.v.) administration where isotonic, biologically compatible pH and solution form is an absolute requirement. Vehicle toxicity and tolerability data are not readily available for a number of combination vehicles therefore, an i.v. tolerability studies was planned in rats with 5% v/v Pharmasolve (NMP), 45% v/v Propylene glycol (PG) and 50% v/v Polyethylene glycol (PEG) 400 combination, at dose volume of 0.5, 1, 2 and 5 mL/kg body weight for 28 days. The vehicle combination was administered via lateral tail vein and effects on clinical signs, body weights, feed consumption, clinical pathology and histopathology were evaluated. Clinical signs of toxicity like tremors, convulsions and death were noticed at 5 mL/kg during the course of the study. At 2 mL/kg, injection site injury without systemic toxicity was noticed. In conclusion, 1 mL/kg of a combination vehicle of 5% NMP, 45% PG and 55% PEG 400 can be administered intravenously once-a-day up to 28 days without any discomfort or injury to rats.

Sleep restriction increased murine melanoma lung metastasis formation by reducing the number of cytotoxic cells and inducing a suppressive tumor microenvironment

Sleep restriction (SR) is an increasing condition in the modern society, and it leads to higher production of stress hormones in humans and rodents. These hormones, at high concentrations, can change many aspects of the immune system, including the production of antibodies, cytokine profiles, cell trafficking, among others, providing the development of diseases, like cancer. The aim of this study was to analyze the effects of sleep restriction (SR) in the development of melanoma lung metastases in male C57BL/6. To this, mice were inoculated with B16F10 in the lateral tail vein (day 0). In the next day (day 1), mice were submitted to sleep restriction (SR) by the modified multiple platforms method. At days 2, 7, 14 and 21, control and SR mice (5 per day, per group) were euthanized, the lung metastases were counted and tumor microenvironment cells were analyzed by flow cytometry. Results shown that, in SR mice, tumor appeared earlier and more lung metastatic colonies were formed. Cytotoxic NK and CD8 T cells were reduced and the ratio between effector and regulatory T cells were smaller in sleep restricted mice in day 14. The same results were observed when the ratio IFN-gamma/Il-4 and IFN-gamma/IL-10 were analyzed. Together, these data suggest that sleep restriction induced a suppressive tumor microenvironment and impaired anti- tumor effector cells infiltration, thus favoring rapid tumor installation and growth.

Temporal brow lift vs internal browpexy in females undergoing upper blepharoplasty: Effects on lateral brow lifting.

Lateral brow-lifting surgical procedures in conjunction with upper blepharoplasty may prevent secondary descent following upper blepharoplasty. To compare the results of internal browpexy (IBP) and temporal brow lift (TBL) in patients with dermatochalasis undergoing simultaneous upper blepharoplasty. This study was a single-center, parallel-group randomized controlled trial conducted on 32 female patients suitable for upper blepharoplasty. Patients were divided into two groups: the IBP group and the TBL group. The brow lift was measured using change in the distance between the ala nasi and lateral tail of the eyebrow as nasal ala to lateral brow (NALB) in millimeter (mm), and the vertical line between the lateral tail of eyebrow and horizontal line extending the lateral cantus as lateral brow plump line (LBPL) in mm before and after the surgery. The follow-up time was 6 months. The mean ± SD age of patients was 55.93 ± 7.1 years and 53.94 ± 7.7 years in the TBL and IBP groups, respectively (P > .05). No significant change in mean LBPL at 6 months compared to baseline was observed in the TBL group (baseline: 15.7 ± 1.6 mm vs 6 month: 15.8 ± 1.3 mm; P = .602). In the IBP group, a significant increase in mean LBPL at 6 months compared to baseline was observed (baseline: 15.09 ± 2.13 mm vs 6 months: 17. 43 ± 2.68 mm; P < .001). Internal browpexy combined with blepharoplasty could be considered the better procedure in patients with upper eyelid dermatochalasis in terms of long-lasting stability and lateral brow elevation.

Effects of the dimeric PSD-95 inhibitor UCCB01-144 on functional recovery after fimbria-fornix transection in rats

Pharmacological inhibition of PSD-95 is a promising therapeutic strategy in the treatment of stroke, and positive effects of monomeric and dimeric PSD-95 inhibitors have been reported in numerous studies. However, whether therapeutic effects will generalize to other types of acute brain injury such as traumatic brain injury (TBI), which has pathophysiological mechanisms in common with stroke, is currently uncertain. We have previously found a lack of neuroprotective effects of dimeric PSD-95 inhibitors in the controlled cortical impact model of TBI in rats. However, as no single animal model is currently able to mimic the complex and heterogeneous pathophysiology of TBI, it is necessary to assess treatment effects across a range of models. In this preliminary study we investigated the neuroprotective abilities of the dimeric PSD-95 inhibitor UCCB01-144 after fimbria-fornix (FF) transection in rats. UCCB01-144 or saline was injected into the lateral tail vein of rats immediately after sham surgery or FF-transection, and effects on spatial delayed alternation in a T-maze were assessed over a 28-day period. Task acquisition was significantly impaired in FF-transected animals, but there were no significant effects of UCCB01-144 on spatial delayed alternation after FF-transection or sham surgery, although decelerated learning curves were seen after treatment with UCCB01-144 in FF-transected animals. The results of the present study are consistent with previous research showing a lack of neuroprotective effects of PSD-95 inhibition in experimental models of TBI.