Production and rearing of germ-free X-SCID pigs.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/- pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

A swine model of acute thrombocytopenia with prolonged bleeding time produced by busulfan.

Animal models of thrombocytopenia are indispensable for evaluating the in vivo efficacy of hemostatic agents, cryopreserved platelets, and artificial platelets, but no large animal models are available. In this study, we generated a swine model of acute thrombocytopenia with prolonged bleeding times by administering the chemotherapeutic drug busulfan. First, we tested multiple doses of busulfan (4, 6, and 8 mg/kg) in pigs, and found that 6 mg/kg of busulfan is an optimal dose for producing a safe and moderate thrombocytopenia, with a platelet count of less than 30,000/µl. The pigs administered 6 mg/kg of busulfan (n=8) reached half their initial counts at day 7, counts below 30,000/µl at day 12, and their nadirs at day 15 (on average). The minimal platelet count was 14,000/µl. With this dose of busulfan (6 mg/kg), bleeding times were significantly prolonged in addition to the decrease in platelet counts (r=-0.63, P<0.01), while there were no cases of apparent hemorrhage. White blood cell counts were maintained at over 5,000/µl, and there were no infections or other adverse events including anemia or appetite or body weight loss. All pigs were sacrificed on day 16, with subsequent examination showing a significant reduction in cellularity and colony-forming units in the bone marrow, indicating that thrombocytopenia was the result of myelosuppression. In summary, administration with 6 mg/kg of busulfan induces safe and moderate thrombocytopenia with a prolonged bleeding time in swine.

A pilot study of quantitative capillary refill time to identify high blood lactate levels in critically ill patients.

INTRODUCTION: We developed a new device to quantify capillary refill time (CRT) by applying the pulse oximeter principle, and evaluated the correlation between quantitative CRT (Q-CRT) and hypoperfusion status, as represented by blood lactate levels, in critically ill patients. METHODS: A pilot study was undertaken in the intensive care unit (ICU) in a tertiary emergency medical centre. While the pulse oxygen saturation sensor was placed on the finger of the patients, transmitted light intensity (TLI) was measured with a pulse oximeter (OLV-3100; Nihon Kohden, Tokyo, Japan) before and during compression of the finger. Q-CRT was defined as the interval from the release of compression to the time when TLI reached 90% of baseline. RESULTS: Q-CRT was analysed in a total of 57 waveforms among 23 patients and statistically correlated with lactate levels (Spearman's rank correlation coefficient, 0.681; p<0.001). The cut-off value of Q-CRT for predicting a lactate level of ≥2.0 mmol/L was 6.81 s (area under the curve (AUC) (95% CI 1.000 (1.000 to 1.000), p<0.001), and the value for predicting a lactate level of ≥4.0 mmol/L was 7.27 s (AUC=0.989 (95% CI 0.954 to 1.000), p<0.001). CONCLUSIONS: Q-CRT correlated with blood lactate levels in this pilot study. The most useful threshold for Q-CRT was ∼6-8 s. Further study is needed to investigate the potential role of this modality as a non-invasive predictor of hypoperfusion in the emergency department, ICU and operating room settings.

[Micro-tourniquet method: a substitute or supporting technique for aneurysm clipping].

The micro-tourniquet method is designed as a substitute and/or supporting technique for obliterating aneurysms that are difficult to operate on using the conventional clipping technique. This method is useful for squeezing the aneurysm neck and making a detour around the neck to spare the branching vessels. These micro-tourniquet instruments are a ligature with both ends attached to a ligature guide, a guide holder, a silastic sheath, and a hemostatic clip or a small aneurysm clip set. The ligature is a 20cm long GORE-TEX suture CV-3, and is attached on both ends to a ligature guide. These ligature guides are made of 7mm wire, and are malleable enough to be bent intentionally during surgery. However, they are also rigid enough to be used as a micro dissector. The silastic sheath is made of a 20G infusion needle, and is cut to a length of 2cm. After branching vessels and perforators are dissected and spared with the aid of the ligature guide, the ligature is passed around the aneurysm neck, both ends of the ligature are passed through the sheath, the aneurysm neck is squeezed, and a clip is applied as a stopper on the ligatures adjacent to the distal end of the sheath. By gently displacing the distal end of the sheath, a conventional aneurysm clip is applied on the aneurysm neck just distal or proximal to the ligature on the neck. Then, the ligature is removed. Two demonstrable cases are presented and the usefulness of the micro-tourniquet method is discussed.