Absence of cellular stress in brain after hypoxia induced by arousal from hibernation in Arctic ground squirrels

Although hypoxia tolerance in heterothermic mammals is well established, it is unclear whether the adaptive significance stems from hypoxia or other cellular challenge associated with euthermy, hibernation, or arousal. In the present study, blood gases, hemoglobin O2 saturation (S(O2), and indexes of cellular and physiological stress were measured during hibernation and euthermy and after arousal thermogenesis. Results show that arterial O2 tension (Pa(O2)) and S(O2) are severely diminished during arousal and that hypoxia-inducible factor (HIF)-1alpha accumulates in brain. Despite evidence of hypoxia, neither cellular nor oxidative stress, as indicated by inducible nitric oxide synthase (iNOS) levels and oxidative modification of biomolecules, was observed during late arousal from hibernation. Compared with rats, hibernating Arctic ground squirrels (Spermophilus parryii) are well oxygenated with no evidence of cellular stress, inflammatory response, neuronal pathology, or oxidative modification following the period of high metabolic demand necessary for arousal. In contrast, euthermic Arctic ground squirrels experience mild, chronic hypoxia with low S(O2) and accumulation of HIF-1alpha and iNOS and demonstrate the greatest degree of cellular stress in brain. These results suggest that Arctic ground squirrels experience and tolerate endogenous hypoxia during euthermy and arousal.     

Cholesterol and lipoprotein dynamics in a hibernating mammal

Hibernating mammals cease feeding during the winter and rely primarily on stored lipids to fuel alternating periods of torpor and arousal. How hibernators manage large fluxes of lipids and sterols over the annual hibernation cycle is poorly understood. The aim of this study was to investigate lipid and cholesterol transport and storage in ground squirrels studied in spring, summer, and several hibernation states. Cholesterol levels in total plasma, HDL and LDL particles were elevated in hibernators compared with spring or summer squirrels. Hibernation increased plasma apolipoprotein A-I expression and HDL particle size. Expression of cholesterol 7 alpha-hydroxylase was 13-fold lower in hibernators than in active season squirrels. Plasma triglycerides were reduced by fasting in spring but not summer squirrels. In hibernators plasma β-hydroxybutyrate was elevated during torpor whereas triglycerides were low relative to normothermic states. We conclude that the switch to a lipid-based metabolism during winter, coupled with reduced capacity to excrete cholesterol creates a closed system in which efficient use of lipoproteins is essential for survival.     

Lipids of the liver microsomal fraction in the ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis> during hibernation

Winter sleep of the ground squirrel Spermophilus undulatus was accompanied by a 20% decrease in phospholipid content (µg phospholipid per 1 mg protein) in microsomal fractions of the liver as compared with summer-active squirrels. The phosphatidylcholine level (mol %) in hibernating squirrels was lower than in summer-active squirrels, and the content of sphingomyelin (mol %) during the torpor bout was higher than in winter- and summer-active squirrels. The cholesterol, fatty acid, monoglyceride, and diglyceride levels in the microsomal fraction of the liver were elevated during hibernation. Pronounced seasonal changes in the lipid/protein ratio implicate the lipids of the liver microsomal fraction in adaptation of the ground squirrel to hibernation.     

Hibernation induces glutathione redox imbalance in ground squirrel intestine

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.     

Hibernation confers resistance to intestinal ischemia-reperfusion injury

The damaging effects of intestinal ischemia-reperfusion (I/R) on the gut and remote organs can be attenuated by subjecting the intestine to a prior, less severe I/R insult, a process known as preconditioning. Because intestines of hibernating ground squirrels experience repeated cycles of hypoperfusion and reperfusion, we examined whether hibernation serves as a model for natural preconditioning against I/R-induced injury. We induced intestinal I/R in either the entire gut or in isolated intestinal loops using rats, summer ground squirrels, and hibernating squirrels during natural interbout arousals (IBA; body temperature 37-39 degrees C). In both models, I/R induced less mucosal damage in IBA squirrels than in summer squirrels or rats. Superior mesenteric artery I/R increased MPO activity in the gut mucosa and lung of rats and summer squirrels and the liver of rats but had no effect in IBA squirrels. I/R in isolated loops increased luminal albumin levels, suggesting increased gut permeability in rats and summer squirrels but not IBA squirrels. The results suggest that the hibernation phenotype is associated with natural protection against intestinal I/R injury.     

Fat ablation and food restriction influence reproductive development and hibernation in ground squirrels

The role of white adipose tissue in development of the reproductive apparatus of male golden-mantled ground squirrels was assessed by surgical removal of fat (lipectomy) immediately prior to onset of hibernation or by manipulation of fat levels through food restriction for 5 wk preceding hibernation. Animals then were maintained without food at 6 degrees C, and they hibernated from November 1983 until April 1984. At that time, blood plasma was assayed for hormone levels, and body mass, body composition, and masses of the testes and seminal vesicle-prostate complex were determined. At autopsy, testes and sexual accessory organs were heavier in Control squirrels than in Food-Restricted or Lipectomized (LIPX) animals. Paired-testes mass was positively correlated with body mass. Testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were uniformly low in animals sampled during torpor, but were elevated in 2 animals that had regained the euthermic state. The Food-Restricted animals spent more time in torpor than did the other two groups, and the percentage of decrease in body mass over the hibernation season was less for Food-Restricted than for LIPX or Control animals. Adequate lipid stores may be essential for the normal development of the reproductive apparatus of male ground squirrels. In the field, animals with insufficient fat stores may forego reproduction and thereby conserve energy for survival.     

光照黑暗循环条件下达乌尔黄鼠的冬眠模式和能量消耗

为研究冬眠季节的光照条件对贮脂类冬眠动物入眠的影响,在达乌尔黄鼠腹腔埋植体温记录元件iButton,在体重高峰后的下降阶段置于5℃和12L:12D的光照条件下,观察测定其冬眠模式和能量消耗。达乌尔黄鼠冬眠模式出现深冬眠型、少冬眠型和不冬眠型,蛰眠阵包括深冬眠阵、短冬眠阵和日眠阵。不同冬眠阵中最低体温、冬眠阵的持续时间、阵间产热的持续时间、冷却速率和复温速率差异显著;阵间产热的最高体温基本相同。平均每日能量消耗在日眠阵中最高、短冬眠阵中居中、深冬眠阵中最低。入眠时间多集中于黑暗时相,觉醒时间多集中于光照时相。本实验结果提示,在冬眠季节施加光照黑暗循环条件可减少达乌尔黄鼠冬眠的时间,升高阵间最低体温,缩短冬眠阵与阵间产热的持续时间,降低复温速率;增加冬眠期间能量消耗。入眠与觉醒受光照条件影响,具有明显的光暗节律。     

Plasma androgen and gonadotropin levels during hibernation and testicular maturation in golden-mantled ground squirrels

The 4-5-mo hibernation season of golden-mantled ground squirrels consists of extended torpor bouts interspersed with brief, periodic intervals of normothermic arousal. Plasma levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and degree of scrotal pigmentation were measured in torpid and aroused male ground squirrels throughout a season of hibernation and in active animals after the termination of torpor. T was basal in torpid animals; beginning 3 weeks before torpor ended, T was elevated in normothermic males during the first half of periodic arousals but returned to basal levels before animals reentered torpor. After the last (terminal) arousal from torpor, T levels were moderately elevated for 4 wk and maximal for the next 6 wk before they returned to basal values. LH patterns were similar to those of T; however, levels of T and LH were positively correlated only in aroused or posthibernation males. FSH levels remained constant and low during most of the heterothermic season but increased in several torpid males within 3 days of terminal arousal. FSH levels peaked 2 wk after the end of heterothermy. Scrotal pigmentation developed over the first 4 wk after terminal arousal. Maturation of reproductive function occurs during the 4 wk after termination of heterothermy, but elevated levels of T during arousals and variable levels of FSH in the last days of torpor suggest that activation or increased sensitivity of the hypothalamic-pituitary-gonadal axis is important in the termination of heterothermy in ground squirrels.     

Modulation of apoptotic pathways in intestinal mucosa during hibernation

Mammalian hibernation is associated with several events that can affect programmed cell death (apoptosis) in nonhibernators, including marked changes in blood flow, extended fasting, and oxidative stress. However, the effect of hibernation on apoptosis is poorly understood. Here, we investigated apoptosis and expression of proteins involved in apoptotic pathways in intestinal mucosa of summer and hibernating ground squirrels. We used terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to identify possible apoptotic enterocytes in small intestine of summer squirrels and hibernating squirrels throughout the winter. Nuclear TUNEL staining increased as hibernation progressed, but the staining pattern was diffuse and not accompanied by chromatin condensation or apoptotic bodies. Electrophoresis of mucosal DNA revealed no ladders typical of apoptosis. Nuclear levels of proapoptotic p53 protein were fourfold less in hibernators compared with summer squirrels. A 12-fold increase in anti-apoptotic Bcl-x(L) compared with a 2-fold increase in proapoptotic Bax suggested a balance in favor of antiapoptotic signaling in hibernators. There was no change in Bcl-2 protein expression but phospho-Bcl-2 increased in mucosa of hibernators. Hibernation had minimal effects on expression of active caspase-8 or -9, whereas caspase-3-specific activity was lower in hibernators during an interbout arousal compared with summer squirrels. Expression of the prosurvival protein Akt increased 20-fold during hibernation, but phospho-Akt was not altered. These data provide evidence for enhanced expression of antiapoptotic proteins during hibernation that may promote enterocyte survival in a pro-oxidative, proapoptotic environment.     

The effects of caloric restriction on the body composition and hibernation of the golden-mantled ground squirrel (Spermophilus lateralis)

In preparation for hibernation, golden-mantled ground squirrels (Spermophilus lateralis) must deposit sufficient amounts of lipid during the summer to survive winter hibernation. We conducted an experiment from May 1998 to February 1999 to examine the effects of caloric restriction on the body composition (lipid and fat-free mass) and hibernation of golden-mantled ground squirrels. Ground squirrels were either provided with food ad lib. (controls) or with only enough food to maintain a constant body mass throughout the experiment (calorically restricted). Changes in body composition were followed using total body electrical conductivity (TOBEC). Implanted data loggers that recorded body temperature were used to determine when ground squirrels entered their first torpor bout and the lengths of torpor bouts. Body composition did not change in the calorically restricted ground squirrels between May and September, while both lipid and fat-free mass increased in the controls. However, from September to February, calorically restricted ground squirrels lost only fat-free mass, not lipid mass, but controls lost both lipid and fat-free mass. Calorically restricted ground squirrels entered their first torpor bout about 4 wk after controls, but the torpor bout duration (or length) during hibernation did not differ between the two groups. These results show that ground squirrels maintain body composition during caloric restriction, and the limited quantities of stored lipid have an effect on when hibernation begins but not on torpor bout length.     

Ca2+ cycling in heart cells from ground squirrels: adaptive strategies for intracellular Ca2+ homeostasis

Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca(2+) homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca(2+) current (I(Ca)) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I(Ca) did not compromise the Ca(2+)-induced Ca(2+) release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I(Ca). Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca(2+) removal were more rapid in ground squirrels. Ca(2+) sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I(Ca) threshold, low SR Ca(2+) leak and rapid cytosolic Ca(2+) clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca(2+) homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca(2+) overload-related heart diseases.     

Testis development and testosterone secretion in captive European ground squirrels before, during, and after hibernation

In some hibernating species, an extended euthermic period before spring emergence has been reported during which testicular maturation occurred. In this study, we investigated whether male European ground squirrels Spermophilus citellus (Linnaeus, 1766) develop scrotal testes before or after the termination of hibernation. The course of testis development and testosterone concentrations were examined in young-of-year male ground squirrels (n = 4) before, during, and after their first hibernation. In the environmental chamber, all animals showed regular hibernation patterns with decreasing torpor bout lengths prior to the end of hibernation. Four weeks before hibernation ended, testosterone levels were elevated during spontaneous arousals. Testosterone concentrations peaked during the first 2 months post heterothermy and decreased thereafter. In 2 experimental males, testes descended from the inguinal region into the scrotum shortly before the end of heterothermy. The state of testis development and testosterone secretion corresponded to that found in semi-free-living males at spring emergence. Testis size increased in active animals to maximum widths during weeks 3 and 4 post heterothermy. Scrotal skin pigmentation was absent until heterothermy ended and developed during the first month thereafter. The data suggest that male S. citellus do not need a pre-emergence euthermic period for reproductive development.     

Vaginal oestrus during the reproductive and non-reproductive period in European ground squirrels

Most hibernating animals are thought to be monoestrous because reproductive activity is subject to strong time constraints. In previous studies, female European ground squirrels (Spermophilus citellus) turned out to have elevated oestradiol levels during late lactation and after weaning, indicating ovarian activity during summer. Therefore, we monitored vaginal cytology, endocrine changes, and vulval development in semi-free-living female European ground squirrels throughout one active season. Vaginal oestrus, defined by the predominance of cornified cells in smear samples, was found during the mating period shortly after vernal emergence. This phase was followed by metoestrus, characterised by the appearance of leukocytes, and a subsequent anoestrous phase. During weaning or postlactation, a second vaginal oestrus was documented in all experimental females, again followed by a metoestrous and an anoestrous phase lasting until hibernation. In line with the second vaginal oestrus, plasma oestradiol concentrations peaked during postlactation. Progesterone levels were elevated from gestation to postlactation, and titres were marginally higher during vaginal oestrus in summer than in spring. Vulval swelling was more pronounced during the first than the second vaginal oestrus. The second oestrous cycle was non-reproductive, as males were sexually inactive with regressed testes during summer. We assume that the second oestrous cycle and the accompanying endocrine changes have beneficial effects on prehibernatory fattening and reproductive performance in the subsequent season. This might allow females to become oestrous immediately after emergence from hibernation in spring.     

The influence of hibernation on testis growth and spermatogenesis in the golden-mantled ground squirrel, Spermophilus lateralis

Testis size and spermatogenesis were monitored serially in individual golden-mantled ground squirrels before, during, and after the hibernation season. During hibernation, animals spent 81% of days in torpor at body temperatures of 3-4 degrees C. Torpor bouts of 6 days duration were interspersed with brief arousals from torpor during which animals were normothermic. In the 5 mo between December (when animals entered hibernation) and April (when torpor was spontaneously terminated), the estimated mass of testes increased gradually from 500 to 1100 mg, but spermatogenesis did not advance beyond pachytene spermatocytes, which were present before hibernation began. In contrast, during the month after torpor was terminated, testes increased rapidly to 3500 mg and after 31 days, spermatozoa were found in the epididymides. We suggest that the limited testis growth that occurred during the hibernation season was restricted to intervals during which squirrels were aroused from torpor. The major portion of gonadal growth and spermatogenesis in the laboratory, and presumably in the field, occurs after ground squirrels have regained the normothermic state. Since males are reproductively mature when first trapped in spring, these findings suggest that males are normothermic for several weeks before they emerge from their hibernacula in the spring.     

达乌尔黄鼠入眠准备期的体温、代谢率及能量特征

贮脂类动物在冬眠前大量积累脂肪来准备冬眠,并在入眠时迅速降低体温和代谢率。为探究入眠准备期达乌尔黄鼠体温、代谢率、呼吸商及能量代谢的变化,将其入眠准备期分为育肥期、体重高峰期、育肥后期和冬眠前的试降期,使用植入式半导体温度记录元件iButton、开放式代谢仪和改进的代谢笼,监测其体温、代谢率及呼吸商和能量摄入的变化。结果显示:(1)达乌尔黄鼠体温在冬眠前13 - 34 d 开始下降,远早于冬眠但晚于体重高峰期;体重高峰期体温有降低的趋势,持续时间为1 - 3 d;育肥后期体温显著下降,体温日波动幅度增加。(2)体重高峰期的静止代谢率高于育肥期,育肥后期有降低的趋势,试降期最低。(3)呼吸商在体重高峰期先升高,之后迅速衰减;入眠准备期的能量摄入在体重达高峰期前达到最大值。结果表明,达乌尔黄鼠在入眠准备期,其体温和代谢率已开始降低,能源物质已开始转变;体重高峰期可能是达乌尔黄鼠入眠的一个转折点或启动入眠的开关。