Histobiochemical Signs of apoptosis in Ground Squirrel Tissues During Awakening After Hibernation

Histological preparations of ground squirrel liver, heart and brain tissues were studied in the middle of the hibernation bout (body temperature of 1–4 °C), at self-heating (body temperature of 6–29 °C) and upon complete awakening (body temperature of 37–38 °C). In the state of deep hibernation, no signs of apoptosis were identified in ground squirrel tissues. We observed microcirculatory dysfunction manifested in abnormal blood rheology: acute hyperaemia of brain and myocardial capillaries, central veins and sinusoids of the liver, as well as mild tissue oedema and aggregations of red blood cells (rouleaux) in microvasculature. At self-heating, signs of the initial stage of apoptosis were detected: mild granularity in cardiomyocyte cytoplasm as a sign of protein dystrophy, and mild cytoplasmic vacuolization in hepatocytes. Morphological signs of these processes were revealed upon the animal’s complete awakening. Shrinkage of individual hepatocytes was observed; cells with pyknotic nuclei appeared, which can be explained by the condensation of chromatin and intracellular organelles; cell fragments with karyorrhectic phenomena resembling apoptotic bodies were identified. Thus, no signs of apoptosis were found in ground squirrel tissues during deep hibernation. At self-heating, signs of the initial stage of apoptosis were detected, and only in the state of complete awakening were single hepatocytes and neurons similar to apoptotic bodies identified. In addition, the absence of an inflammatory reaction in the tissues under study allows us to suggest that we are observing the stages of apoptosis. Noteworthy, ground squirrel awakening was accompanied by a significant increase in the activity of lysosomal membrane markers cathepsin D and acid phosphatase.

Serum Biochemical Reference Values for Adult and Non-adult Chinese Alligators during the Deep and Late Hibernation Periods

Background: The Chinese alligator (Alligator sinensis) is a critically endangered species. Due to the rapid growth of the captive population, the susceptibility to disease during the recovery period after winter hibernation, especially in young alligators, have detrimentally affected Chinese alligator populations. Serum biochemistry, which relates to metabolism, nutritional status and disease, is enormously helpful in evaluating physical conditions in reptile. Many studies have reported the serum biochemical reference values of various reptilian species, including several crocodilians. However, reference values for Chinese alligators have not yet been reported. For captive Chinese alligators, hibernation is a crucial period because winter management has a direct influence on the survival rate of juveniles and the reproduction rate of adults. The main object of the present study refore was to measure the serum biochemical values of captive Chinese alligators during hibernation.Materials, Methods & Results: As such, this study investigates the serum biochemistry as a factor of age and hibernation stage. During the deep and late hibernation periods blood samples were drawn from 30 healthy captive Chinese alligators (adults, sub-adults, and juveniles) at the Anhui Research Center of Chinese Alligator Reproduction (ARCCAR). Serum biochemical measurements were performed using an automated biochemical analyzer and compared based on the age group and hibernation stage via two-way ANOVA. During late hibernation, serum lactate dehydrogenase, alkaline phosphatase, and aspartate aminotransferase activity increased in all age groups in comparison to that in deep hibernation, while the concentration of calcium decreased. Meanwhile, the concentration of serum phosphorus, uric acid, total protein, and globulin in sub-adults and juveniles considerably increased in comparison to that in deep hibernation, while cholesterol and albumin declined. However, in adults only slight changes were noted. Based on comprehensive statistical analysis, our results indicate that sub-adults and juveniles are at risk of developing renal disease during artificial hibernation.Discussion: Chinese alligators, especially sub-adults and juveniles, are particularly vulnerable to disease when they wake from hibernation. They often display symptoms such as depression, anorexia, lethargy, sluggish movement, slow, incremental weight gain, progressive muscle wasting, and even death. The high rate of morbidity in non-adult Chinese alligators may be associated with the high density of UA and other changes in multiple biochemical markers that occur during late hibernation. These altered serum biochemical profiles may indicate kidney damage. One of the most common diseases among reptiles is nephropathy, the symptoms of which are non-specific and tend to agree with those observed post-hibernation. In summary, this study has reported the serum biochemical values of Chinese alligators of varying ages in the deep and late hibernation phases. Based on statistical analyses, interesting differences between the serum biochemical values of adults and non-adults during the deep and late hibernation have been found. The observed changes suggest that, under an artificial hibernation environment, the kidneys of sub-adults and juveniles may become impaired. We believe that the data reported in this study will provide clinical guidance to facilitate more appropriate artificial wintering conditions for Chinese alligators, and assist the breeding and management of these reptiles, as well as disease prevention, during hibernation and recovery.

Torpor and hibernation

A diurnal (circadian) rhythm in body temperature is a widespread, and possibly universal, feature of endotherms. Some mammals and birds down-regulate their metabolic rate significantly by night, allowing their body temperature to drop sufficiently that they become inactive and enter torpor. Both the minimum temperature achieved and the duration of torpor are highly variable. Daily torpor is principally a response to reduced energy intake, and a drop in ambient temperature. Hibernation is essentially an extreme form of torpor. Small mammals hibernating at high latitudes have regular arousals during which they urinate and may feed. Bears hibernate with relatively high body temperature, and do not undergo arousal. Only one bird, the poorwill, is known to hibernate. Rewarming during arousal may be fuelled exclusively by metabolism (for example in small mammals in the Arctic) or with significant energy input from basking (for example in subtropical arid areas). The capacity for torpor appears to be an ancestral character in both mammals and birds, possibly related to the origin of endothermy in small species subject to marked diurnal and/or seasonal variation in body temperature. Both deep hibernation and strict endothermy are probably derived characteristics.

Liver energy metabolism during hibernation in the golden-mantled ground squirrel, Spermophilus lateralis

Large changes in ATP production capacities and rates have been reported in mammalian hibernators throughout the different stages of the hibernation cycle. In this study we showed that total extractable liver [ATP], [ADP], and [ATP]/[ADP] do not differ among summer normothermic, hibernating, and aroused golden-mantled ground squirrels, Spermophilus lateralis, indicating that metabolism remains well balanced throughout the hibernation cycle. This implies that rates of ATP consumption must be down-regulated during deep hibernation in order to maintain this balance. Despite this, basal oxygen-consumption rates [Formula: see text] of hepatocytes isolated from hibernating, aroused, and summer cold-acclimated ground squirrels were 22.4–35.1% higher than those from ground squirrels in the summer normothermic condition when measured at 37 °C. The relatively high hepatocyte [Formula: see text] may help to minimize interbout arousal times, reducing energy demands during the hibernation season. At 7 °C, hepatocyte [Formula: see text] values do not differ among the four groups; however, the Q10 for hepatocyte [Formula: see text] is significantly lower for the summer group, suggesting lower temperature sensitivity. Despite the seasonal changes in thyroid hormone status known to occur in scuirid hibernators, the proportion of hepatocyte [Formula: see text] attributed to Na+,K+-ATPase, estimated by inhibition with 1 mM ouabain, is only around 15% and does not differ among hibernation/seasonal conditions.

Rates of glucose utilization in brain of active and hibernating ground squirrels

Rates of glucose utilization (CMRGlc) were determined in some cerebral structures of active warm- and cold-adapted ground squirrels and hibernating ground squirrels with [14C]deoxyglucose (DG) by direct chemical measurement of precursor and products in samples dissected from funnel-frozen brain. The rate of supply relative to demand of glucose and [14C]DG in brain of hibernating animals was similar to or greater than that of controls. [14C]DG cleared from the plasma in hibernators much more slowly than in active animals, and the level of unmetabolized [14C]DG in brain and the integrated specific activity of the precursor pool in plasma exceeded those of the active animals by 4- to 10-fold. At 45 min after an intravenous pulse of [14C]DG, the unmetabolized [14C]DG remaining in the brains of the hibernators accounted for approximately 96% of the total 14C compared with approximately 10-15% in the active animals. The value of lambda, a factor contained in the lumped constant of the operational equation of the [14C]DG method, was estimated for each animal and found to be relatively constant over the sixfold range of glucose levels in the brains of all animals. Calculated CMRGlc in squirrels in deep hibernation was only 1-2% of the values in active animals.

Thermal effects on neuronal activity in suprachiasmatic nuclei of hibernators and nonhibernators

The temperature sensitivity of neuronal firing rates in the suprachiasmatic nuclei (SCN) of the hypothalami of rats and ground squirrels was studied in vitro. SCN from euthermic squirrels were studied during the hibernation season (winter) and during the summer. SCN from hibernating squirrels were also studied. Most properties of SCN cells from hibernators and nonhibernators were similar. Warm- and cold-sensitive neurons were observed in all groups, but cold-sensitive neurons were more common in SCN from hibernating squirrels. No evidence for temperature compensation of firing rate was accumulated; no cell was observed to fire below 16.6 degrees C. If the persistence of circadian rhythmicity is a function of action potential-dependent neurotransmission from the SCN, these results suggest that deep hibernation (5-17 degrees C) should be characterized by an absence of circadian fluctuation in temperature. Two possible adaptations for the shallow torpor seen at somewhat higher temperatures were observed in the SCN: 1) a relatively large population of cold-sensitive neurons and 2) a population of neurons with very high activation energies. Activation energy analysis suggested that most of the temperature-sensitive properties of these cells could be explained in terms of the thermal sensitivity of the sodium channel.

Persistence of circadian rhythmicity in hibernating ground squirrels

The body temperatures (Tb) of golden-mantled ground squirrels maintained under constant dim light (< 20 1x red light) at an ambient temperature of 10 degrees C were monitored via telemetry throughout the hibernation season. During euthermia, when Tb ranged from 34 to 39 degrees C, these animals exhibited robust circadian Tb rhythms. During bouts of hibernation, when Tb rhythms persisted, although the amplitudes of the rhythms were considerably dampened compared with euthermia. The periods of the intrabout Tb rhythms were within the ranges observed during euthermia and were stable within an individual bout but varied between hibernation bouts. Arousals from hibernation occurred at a fixed phase angle of the Tb cycle. Once the period of an intrabout Tb rhythm was determined, it was possible to predict the timing of arousal from the hibernation bout to within 1 h of any 24-h period. This study confirms previous speculation about the persistence of circadian rhythms in golden-mantled ground squirrels during deep hibernation and demonstrates that the circadian system is involved in the timing of periodic arousals from hibernation.

Are ground squirrels sleep deprived during hibernation?

Hibernation is an adaptation for energy conservation, which probably evolved as an extension of non-rapid-eye-movement sleep mechanisms. Yet, during periodic arousals from bouts of deep hibernation, ground squirrels (Spermophilus lateralis) spend most of their time asleep. Spectral analysis of the electroencephalogram revealed that cortical slow-wave intensity during sleep is high at the beginning of a euthermic period and declines thereafter. Sleep slow-wave intensity is greater after longer bouts of hibernation than after shorter bouts. We hypothesize that low body temperatures during hibernation are incompatible with the restorative function of sleep as reflected in cortical slow-wave activity. Animals must incur the energetic costs of periodic arousals from hibernation to receive the restorative benefits of euthermic slow-wave sleep. The timing of arousals from hibernation may be a function of accumulated sleep debt.