Kunihiro Seki

Preserving tardigrades under pressure

When an animal is exposed to high hydrostatic pressure, its cellular membranes,, proteins and DNA are damaged. At pressures of around 30 megapascals (MPa), proliferation and metabolism in micro-organisms stops; at 300 MPa, most bacteria and multicellular organisms die. But here we show that, in perfluorocarbon at pressures as high as 600 MPa, small terrestrial animals known as tardigrades can survive in a dehydrated state.

Cardiovascular and respiratory responses to passive leg cycle exercise in people with spinal cord injuries

The purpose of this study was to determine the effect of passive leg cycle exercise (PLE) on cardiovascular and respiratory responses in people with spinal cord injuries (PSCI). Eight PSCI with lesions from T8 to L1 and five control subjects (CS) performed PLE at pedalling frequencies of 20 or 40 rpm for 7 min at room temperature of about 25°C. We measured, at rest and during PLE, the pulmonary ventilation (VE), oxygen uptake (VO2), cardiac output (Q), stroke volume (SV), heart rate (HR) and arterial blood pressure, as well as the skin blood flow (SBF) in the lower limb after PLE. An increase in pedalling frequency promoted an increase in VE and VO2 in both groups. Compared with the CS, the PSCI showed significantly smaller increases in VO2 (P < 0.05). The Qc was significantly elevated during PLE at 20 and 40 rpm in CS, and at 40 rpm in PSCI (P < 0.05). In CS, it resulted from increases in both SV and HR, whereas in PSCI, it was contributed to by a greater increase in SV without a rise in HR. In CS, the increase in pedalling frequency promoted the increases in SV and HR and consequently in Qc In PSCI, however, the values remained constant irrespective of pedalling frequency. The arterial blood pressure and SBF in the lower limbs were unchanged by PLE in both groups. These results would suggest that passive leg exercise promotes venous return from the paralyzed lower limbs in PSCI.

Circadian, sleep and brain temperature rhythms in cats under sustained daily light-dark cycles and constant darkness

In adult cats, sleep-wake states and brain temperatures were determined under experimental LD cycles and prolonged DD. Convincing rhythms had not previously been well-established for this animal. The present results demonstrated daily fluctuation in total sleep time (TST) and in brain temperature (Tb). These comprise predominantly nocturnal wakefulness and high temperatures but include a bi-modal component marking dawn and dusk peaks. In addition there are ultradian variations with a period of 2-4 hour superimposed on the circadian rhythm. The latter continued at the beginning of prolonged DD, but gradually diminished and disappeared after about ten days. The TST circadian rhythm diminished more quickly than the Tb rhythm. This result suggests that the former is coupled more strongly to the circadian oscillator than the latter, and that the brain temperature rhythm could not depend on the circadian rhythm of sleep-wakefulness. These results encourage use of cats in further chronobiological studies of sleep and other physiological rhythms.

Effect of arm cranking exercise on skin blood flow of lower limb in people with injuries to the spinal cord

The purpose of this study was to examine whether arm cranking exercise induces changes in skin blood flow in the paralyzed lower limbs of people with injuries to the spinal cord (PISC). Ten PISC with lesions located between Th5 and L5 and six control subjects performed arm cranking exercise for 6 min at three intensities, 10, 30 and 50 W, at a room temperature of 25°C. Oxygen uptake (Vo2) and heart rate (HR) were measured for the last 2 min of each exercise period. The skin blood flow at the anterior thigh (BFsk,t) was continuously monitored using laser Doppler flowmetry for the whole 6-min period and for the first 10 min of recovery following exercise. During exercise, the PISC showed lower Vo2 and greater HR than the control subjects. No increase in BFsk,t was found in six of the PISC with lesions at or above Th12, irrespective of the exercise intensity. On the other hand, in PISC with lesions at L1 or below, BFsk,t increased significantly (P < 0.05) with an increase in Vo2 and HR, although the BFsk,t at a given Vo2 and HR was lower than that in the control subjects. These results would suggest that arm exercise can promote the blood circulation in the skin of the lower limbs if the injury level is below L1.

Relationship between core temperature and skin blood flux in lower limbs during prolonged arm exercise in persons with spinal cord injury.

The purposes of the present study were to examine the response of the skin blood flux (SBF) in the paralyzed lower limbs of persons with spinal cord injury (PSCI) and to clarify the relationship between the SBF and core temperature during prolonged arm exercise. Eight male PSCI with lesions from T6 to L5 and six male control subjects (CS) participated in this study. The subjects rested for 60 min and then performed arm-cranking exercise at 20 W for 30 min at 25 °C. The tympanic membrane temperature (Tty) and SBF in the anterior thigh (SBFT) and in the posterior calf (SBFC) were continuously measured throughout the experiment. The SBFC did not change in either PSCI or CS during the experiment. The SBFT in four PSCI with high lesions (T6 to T12), remained unchanged during exercise. The SBFT in the other four PSCI with low lesions (T12 to L5, ΔSBFT+) began to elevate markedly when the T t, exceeded a threshold temperature of 36.69 °C. The pattern of increase of SBFT in ΔSBFT+ was similar to that in CS, although onset of the increase in SBFT was delayed and the peak of SBFT during exercise was significantly lower in comparison with the CS. We consider that these differences between the ΔSBFT+ and CS were largely attributable to the lowerTty in the former group, which took a prolonged time to reach the threshold of 36.69 °C.

Effect of maximal arm exercise on skin blood flux in the paralyzed lower limbs in persons with spinal cord injury

The purpose of the present study was to examine the effect of maximal arm exercise on the skin blood circulation of the paralyzed lower limbs in persons with spinal cord injury (PSCI). Eight male PSCI with complete lesions located between T3 and L1 performed graded maximal arm-cranking exercise (MACE) to exhaustion. The skin blood flux at the thigh (SBFT) and that at the calf (SBFC) were monitored using laser-Doppler flowmeter at rest and for 15 s immediately after the MACE. The subjects mean peak oxygen uptake and peak heart rate was 1.41 ± 0.22 1·min−1 and 171.6 ± 19.2 beats·min−1, respectively. No PSCI showed any increase in either SBFT or SBFC after the MACE, when compared with the values at rest. These results suggest that the blood circulation of the skin in the paralyzed lower limbs in PSCI is unaffected by the MACE.

Study on the preservation with CO (PCO = 200-2,000 hPa), resuscitation, and heterotopic transplantation of an isolated rat heart.

In this experiment, CO was used as a gas mixture in a reversible relationship with O2. CO was added in a gas form mixed with O2. An isolated donor rat heart was obtained, exposed to a gas mixture such as PO2 = 1,800 hPa and PCO = 200 hPa, and PO2 = 1,000 hPa and PCO = 1,000 hPa in a 2 ATA high-pressure chamber and preserved in a refrigerator at 4°C. This report demonstrates that significant reproducibility has been verified. The heart was removed from the refrigerator 24 h later and heterotopic heart transplantation was performed in the right neck of a recipient rat and the pulsating of the transplanted heart was detected by an electrocardiogram.

Heterotopic transplant of an isolated rat heart preserved for 72 h in perfluorocarbon with CO2.

The inert fluid perfluorocarbon (PFC) has been used since about 1960 in liquid respiration and artificial blood for mammals. PFC has been used to successfully resuscitate tardigrades that had been dried and exposed to a high barometric pressure of 6,000 atmospheres. Next, scientists attempted to experimentally preserve organs that had been removed from animals, dried, and immersed in PFC. Since 1998 preservation and resuscitation experiments have been conducted with mammalian hearts using 2,015 rats and 70 pigs. Among those experiments, the maximum time after desiccation until successful resuscitation was 26 days for a rat heart and 37 days for a pig heart. However, these results could not be reproduced. Finally, in 2005, this laboratory demonstrated that a rat heart removed under 2 atmospheres pressure and a CO2 partial pressure of 400 hPa, followed by desiccation for 24 h, could be revived and heterotypically transplanted. Moreover, these results were reproducible. The preservation time can be extended to 72 h if, after immersing isolated rat hearts in PFC, they are dried by air exposure under a CO2 partial pressure of 100 hPa. The present report documents the resuscitation of this heart after 72 h of preservation followed by heterotrophic transplantation.

Preservation by desiccation of isolated rat hearts for 48 hours using carbon monoxide (PCO = 4,000 hPa) and oxygen (PO(2) = 3,000 hPa).

It is currently said that CO has anti-inflammatory and antiapoptosis effects and it has attracted attention as a medical gas. We used CO for rat hearts and conducted a preservation experiment. We isolated rat hearts, placed them into a specially made chamber, filled the chamber with a gas mixture of PCO (4,000 hPa) and PO2 (3,000 hPa), and preserved the hearts in a refrigerator at 4°C for 48 h. We then performed a heterotrophic transplantation on the neck of each recipient rat and resuscitated the preserved hearts. We herein report our findings.

A study on the perfusion preservation, resuscitation, and transplantation of a rat heart isolated for 96 hours.

Krebs-Henseleit (KH) solution was used to fill the heart chamber of an isolated rat heart before it was immersed in perfluorocarbon (PFC), which is an inert fluid. A gas mixture (PCO2 = 150 hPa and PO2 = 850 hPa) was then aerated at a constant rate into the PFC solution, and the isolated heart was thereafter preserved for 96 h with KH solution perfused continuously at a rate of 0.1 ml/h from the aorta of the isolated heart through a cannula. After preservation, the preserved heart was heterotopically transplanted into the neck of a recipient rat and then it was resuscitated. Using this method for preserving mammalian organs, we attained reproducibility after perfusion preservation for 96 h.