Establishment of withdrawal time and analysis of tiamulin residue in tissues of orally dosed broiler chickens

This study was conducted to investigate tiamulin (TML) residues in the edible tissues of orally dosed broiler chickens and to re-establish the withdrawal time (WT). Thirty-six healthy Ross broiler chickens were administered 0.5 (TML-1) and 2.5 kg (TML-2) per ton feed, respectively, of the drug containing TML 78 g/kg for 10 days. Twenty-four tissue samples were collected from 6 chickens in each of the TML-1 and TML-2 groups on 0, 1, 3, and 5 days after drug administration, respectively. The residual concentrations of TML were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The correlation coefficient of the calibration curves was 0.9978 to 0.9998, and the limits of detection and the limits of quantification (LOQ) were in the range of 0.03 to 0.06, and 0.1 to 0.2 µg/kg, respectively. Recoveries ranged between 89.0% to 116.7%, and the coefficients of variation were less than 13.9%. After the drug administration, TML in the TML-1 and TML-2 groups was detected above the LOQ in 1 and 6 samples of liver, respectively, at day 0, and in 1 liver sample from both groups on day one. At 3 days after administration, TML was detected below the LOQ in all samples of TML-1 and TML-2. The calculated WT of TML in both TML-1 and TML-2 using the WT calculation program WT 1.4 was 0 days. In conclusion, the developed analytical method is suitable for detection, and the calculated WT of TML in poultry edible tissues is shorter than the current recommended WT of 7 days for TML in broiler chickens.

Differential Gene Expression in the Hippocampi of Nonhuman Primates Chronically Exposed to Methamphetamine, Cocaine, or Heroin

Objective@#Methamphetamine (MA), cocaine, and heroin cause severe public health problems as well as impairments in neural plasticity and cognitive function in the hippocampus. This study aimed to identify the genes differentially expressed in the hippocampi of cynomolgus monkeys in response to these drugs. @*Methods@#After the monkeys were chronically exposed to MA, cocaine, and heroin, we performed large-scale gene expression profiling of the hippocampus using RNA-Seq technology and functional annotation of genes differentially expressed. Some genes selected from RNA-Seq analysis data were validated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). And the expression changes of ADAM10 protein were assessed using immunohistochemistry. @*Results@#The changes in genes related to axonal guidance (PTPRP and KAL1), the cell cycle (TLK2), and the regulation of potassium ions (DPP10) in the drug-treated groups compared to the control group were confirmed using RT-qPCR. Comparative analysis of all groups showed that among genes related to synaptic long-term potentiation, CREBBP and GRIN3A were downregulated in both the MA- and heroin-treated groups compared to the control group. In particular, the mRNA and protein expression levels of ADAM10 were decreased in the MA-treated group but increased in the cocaine-treated group compared to the control group. @*Conclusion@#These results provide insights into the genes that are upregulated and downregulated in the hippocampus by the chronic administration of MA, cocaine, or heroin and basic information for developing novel drugs for the treatment of hippocampal impairments caused by drug abuse.

Establishment of withdrawal time and analysis of dexamethasone residue in milk of intramuscularly dosed cows

This study investigated dexamethasone (DXM) residues in the milk from intramuscularly dosed dairy cows and established the withdrawal time (WT) of DXM in milk. Eighteen healthy Holstein cows were injected with 20 (DXM-1) or 40 mL (DXM-2) of a drug containing 1 mg/mL of DXM. After administering DXM, milk samples were collected from all cows at 12-hour intervals for five days. The DXM residue concentrations in milk were determined by liquid chromatography-mass spectrometry/mass spectrometry. The correlation coefficient of the calibration curve was 0.9966, and the limits of detection and quantification (LOQ) were 0.03 and 0.1 μg/kg, respectively. The recoveries were 97.0% to 104.0%, and the coefficient of variations was less than 7.22%. After treatment, DXM in DXM-1 was detected above the LOQ in two milk samples at 36 hours and below the LOQ in all milk samples of DXM-2 at 48 hours. Using the WT calculation program WT 1.4, the withdrawal periods of DXM-1 and DXM-2 in milk were established to be two days. In conclusion, the developed analytical method is sensitive and reliable for detecting DXM in milk. The estimated WT of DXM in bovine milk is shorter than the current milk WT recommendation of three days for DXM in lactating dairy cows.

Assessment of Hand Motor Function in a Non-human Primate Model of Ischemic Stroke

Ischemic stroke results from arterial occlusion and can cause irreversible brain injury. A non-human primate (NHP) model of ischemic stroke was previously developed to investigate its pathophysiology and for efficacy testing of therapeutic candidates; however, fine motor impairment remains to be well-characterized. We evaluated hand motor function in a cynomolgus monkey model of ischemic stroke. Endovascular transient middle cerebral artery occlusion (MCAO) with an angiographic microcatheter induced cerebral infarction. In vivo magnetic resonance imaging mapped and measured the ischemia-induced infarct lesion. In vivo diffusion tensor imaging (DTI) of the stroke lesion to assess the neuroplastic changes and fiber tractography demonstrated three-dimensional patterns in the corticospinal tract 12 weeks after MCAO. The hand dexterity task (HDT) was used to evaluate fine motor movement of upper extremity digits. The HDT was modified for a home cage-based training system, instead of conventional chair restraint training. The lesion was localized in the middle cerebral artery territory, including the sensorimotor cortex. Maximum infarct volume was exhibited over the first week after MCAO, which progressively inhibited ischemic core expansion, manifested by enhanced functional recovery of the affected hand over 12 weeks after MCAO. The total performance time decreased with increasing success rate for both hands on the HDT. Compensatory strategies and retrieval failure improved in the chronic phase after stroke. Our findings demonstrate the recovery of fine motor skill after stroke, and outline the behavioral characteristics and features of functional disorder of NHP stroke model, providing a basis for assessing hand motor function after stroke.

Age-related Effects of Heroin on Gene Expression in the Hippocampus and Striatum of Cynomolgus Monkeys

Objective@#The aim of this study was to investigate differentially expressed genes and their functions in the hippocampus and striatum after heroin administration in cynomolgus macaques of different ages. @*Methods@#Cynomolgus monkeys were divided by age as follows: 1 year (A1, n = 2); 3 to 4 years (A2, n = 2); 6 to 8 years (A3, n = 2); and older than 11 years (A4, n = 2). After heroin was injected intramuscularly into the monkeys (0.6 mg/kg), we performed large-scale transcriptome profiling in the hippocampus (H) and striatum (S) using RNA sequencing technology. Some genes were validated with real-time quantitative PCR. @*Results@#In the hippocampus, the gene expression of A1H was similar to that of A4H, while the gene expression of A2H was similar to that of A3H. Genes associated with the mitogen-activated protein kinase signaling pathway (STMN1, FGF14, and MAPT) and -aminobutyric acid-ergic synapses (GABBR2 and GAD1) were differentially expressed among control and heroin-treated animals. Differential gene expression between A1S and A4S was the least significant, while differential gene expression between A3S and A2S was the most significant. Genes associated with the neurotrophin signaling pathway (NTRK1 and NGFR), autophagy (ATG5), and dopaminergic synapses (AKT1) in the striatum were differentially expressed among control and heroin-treated animals. @*Conclusion@#These results suggest that even a single heroin exposure can cause differential gene expression in the hippocampus and striatum of nonhuman primates at different ages.

Comparative Evaluation of Hormones and Hormone-Like Molecule in Lineage Specification of Human Induced Pluripotent Stem Cells

BACKGROUND AND OBJECTIVES: Proficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidences had implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17-β (E2), progesterone (P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification. METHODS AND RESULTS: We used 10 nM E2, 3 μM P4, 10 nM DM, and 10 nM RA based on their functional in vivo developmental potential. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34⁺CD45⁺ cells with progenitor functions, even in the CD43⁻ population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision. CONCLUSIONS: Our findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.

Increased CD68/TGFβ Co-expressing Microglia/Macrophages after Transient Middle Cerebral Artery Occlusion in Rhesus Monkeys

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFβ) was found colocalized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206⁺ cells, may exert anti-inflammatory effects by secreting TGFβ after the subacute stage of ischemic stroke. CD68⁺ microglia/macrophages can therefore be used as a potential therapeutic target.

Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis)

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

Reference values of hematological and biochemical parameters in young-adult cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta) anesthetized with ketamine hydrochloride / 한국실험동물학회지

Nonhuman primate models are valuable in biomedical research. However, reference data for clinical pathology parameters in cynomolgus and rhesus monkeys are limited. In the present study, we established hematologic and biochemical reference intervals for healthy cynomolgus and rhesus monkeys anesthetized with ketamine hydrochloride. A total of 142 cynomolgus monkeys (28 males and 114 females) and 42 rhesus monkeys (22 males and 20 females) were selected and analyzed in order to examine reference intervals of 20 hematological and 16 biochemical parameters. The effects of sex were also investigated. Reference intervals for hematological and biochemical parameters were separately established by species (cynomolgus and rhesus) and sex (male and female). No sex-related differences were determined in erythrocyte-related parameters for cynomolgus and rhesus monkey housed in indoor laboratory conditions. Alkaline phosphatase and gamma glutamyltransferase were significantly lower in females than males in both cynomolgus and rhesus monkeys aged 48–96 months. The reference values for hematological and biochemical parameters established herein might provide valuable information for researchers using cynomolgus and rhesus monkeys in experimental conditions for biomedical studies.