Mouth cell collection device for newborn mice.

For efficient and accurate genotyping of transgenic and knockout mice, the ability to reduce pain and suffering and to obtain DNA early in life are critical. We have developed a novel method to sample buccal cells from neonatal mice to obtain DNA. Our mouse mouth cell collection process includes an oral speculum and collection device which enables rapid extraction of enough DNA for up to 50 PCRs from each buccal sampling. This cell collection device fills a clear need for buccal sampling from neonatal mice, greatly facilitating research in mouse models of human disease. Eliminating the pain, distress, and death caused by invasive and mutilating procedures lessens the potential for confounding variables between control and experimental animals. In conclusion, our mouse mouth cell collection process can be applied to very small animals for which there exists no current device.

Gene therapy for murine glycerol kinase deficiency: importance of murine ortholog.

A glycerol kinase (Gyk) knock-out (KO) mouse model permits improved understanding of glycerol kinase (GK) deficiency (GKD) pathogenesis, however, early death of affected mice limits its utility. The purpose of this work was to delay death of affected males to investigate thoroughly their phenotypes. An adenoviral vector carrying the human (Adeno-XGK) or mouse (Adeno-XGyk) GK gene was injected into KO mice within 24 h of birth. Adeno-XGK did not change KO mouse survival time despite liver GK activity greater than 100% of wild type. However, Adeno-XGyk improved KO mouse survival time greater than two-fold. These investigations demonstrate that gene replacement therapy for Gyk KO mice is more efficacious using murine Gyk than human GK. These studies expand our understanding of GKD pathogenesis in the murine model, and show that while murine GKD is more severe than in humans, GKD mice have similar metabolic disturbances to affected humans with hypoglycemia and acidemia.

Anaphylaxis with associated fibrinolysis, reversed with tranexamic acid and demonstrated by thrombelastography.

In the surgical setting, fibrinolysis can be a serious complication of anaphylaxis. We present four cases of anaphylaxis that were associated with fibrinolysis during anaesthesia, and the use of the thrombelastograph to demonstrate this haemostatic defect and its correction using tranexamic acid.

Uteroplacental insufficiency alters liver and skeletal muscle branched-chain amino acid metabolism in intrauterine growth-restricted fetal rats.

Uteroplacental insufficiency causes intrauterine growth restriction (IUGR) and decreases plasma levels of the branched-chain amino acids in both humans and rats. Increased fetal oxidation of these amino acids may contribute to their decline in the IUGR fetus. The rate-limiting step of branched-chain amino acid oxidation is performed by the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKAD), which is regulated by a deactivating kinase. We therefore hypothesized that uteroplacental insufficiency increases BCKAD activity through altered mRNA and protein levels of BCKAD and/or the BCKAD kinase. In IUGR fetal liver, BCKAD activity was increased 3-fold, though no difference in hepatic BCKAD protein or mRNA levels were noted. Hepatic BCKAD kinase mRNA and protein levels were significantly decreased in association with the increase in BCKAD activity. In IUGR fetal skeletal muscle, BCKAD mRNA levels were significantly increased. IUGR skeletal muscle BCKAD protein levels as well as BCKAD kinase mRNA and protein levels were unchanged. We also quantified mRNA levels of two amino acid transporters: LAT1 (system L) and rBAT (cysteine and dibasic amino acids). Both hepatic and muscle LAT1 mRNA levels were significantly increased in the IUGR fetus. We conclude that uteroplacental insufficiency significantly increases hepatic BCKAD activity in association with significantly decreased mRNA and protein levels of the deactivating kinase. We speculate that these changes contribute to the decreased serum levels of branched-chain amino acids seen in the IUGR fetus and may be an adaptation to the deprived milieu associated with uteroplacental insufficiency.