Ultrastructure of juxtaglomerular cells correlated with biochemical parameters in a hibernator

Ultrastructural features of juxtaglomerular cells have been correlated with plasma and kidney analyses from non-hibernating, hibernating and awakening ground squirrels. Juxtaglomerular cells in kidneys from hibernating animals show signs of increased activity. Plasma samples from hibernating animals show a significant increase in magnesium. Kidney analyses from hibernating animals, show glycogen increases and lactate and inorganic phosphate decrease significantly. Adenosine triphosphate remains the same. Maintenance of high-energy phosphates in the hibernating kidney is essential to maintaining sodium transport and osmotic pressure. This coupled with a functional renin-angiotensin system regulates water and electrolyte balance.     

Effects of hibernation on somatostatin-like immunoreactivity in the brain of the ground squirrel (Spermophilus richardsonii) and European hedgehog (Erinaceus europaeus)

Summary The localization of the somatostatin system in the brains of Richardson's ground squirrels (Spermophilus richardsonii) and European hedgehogs (Erinaceus europaeus) was described by use of immunocytochemical methods. In addition, (i) chemically differing types of somatostatin and (ii) different activity phases of the somatostatin system during the hibernation cycle were investigated in the ground squirrel by means of high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA).In both species, the hypothalamic component of the somatostatin system (periventricular nuclei, fiber projections to the median eminence) is more prominent than the widespread extrahypothalamic representation of the system displaying mainly scattered perikarya and nerve fibers. The reactivity pattern of the somatostatin system varied among hibernating, aroused, and non-hibernating animals; moreover, the interspecific differences were pronounced. The activity of the hypothalamic somatostatin system in the hibernating ground squirrel appeared to be suppressed when compared to non-hibernating controls, whereas in the hibernating hedgehog this system showed signs of increased activity in comparison to non-hibernating controls. In contrast, in the present material the extrahypothalamic components of the somatostatin system did not exhibit significant changes in their activity.     

Ultrastructure of the renal corpuscle of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals

The renal corpuscle of hibernating and non-hibernating Testudo graeca was studied by means of light and electron microscopy. Renal corpuscles are small and have a glomerular architecture similar to that found in other vertebrates with a limited glomerular filtration rate. In hibernating animals, unlike non-hibernating, some morphological changes took place. The cells of the renal corpuscle were densely packed, podocytes and parietal cells showed a marked cytoplasmic vacuolization, there was a highly developed capillary basement membrane and the endothelial and mesangial cells showed abundant dense granules. These morphological features apparently correspond to a vacuolar degeneration. They may also be the morphological basis of the decrease in the glomerular filtration rate observed during this period.     

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel,Spermophilus richardsonii

Summary In the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Overwintering and prevalence of Neozygites floridana (Zygomycetes: Neozygitaceae) in hibernating females of Tetranychus urticae (Acari: Tetranychidae) under cold climatic conditions in strawberries

To evaluate overwintering strategies of the fungus Neozygites floridana, an important natural enemy of Tetranychus urticae, hibernating T. urticae females were investigated for the presence of fungal structures throughout one winter (October 12, 2006 to February 19, 2007) in field-grown strawberries in a cold climate in Norway (min. ambient temp -15.3 degrees C). Neozygites floridana was present as hyphal bodies inside live, hibernating females in T. urticae populations throughout the sampling period. The lowest percentages of hibernating females with hyphal bodies were found at the two first dates of sampling at 5.5 and 0% on October 12 and 19, respectively. The prevalence then increased and peaked at 54.4% on January 14. Resting spores (immature) were also found in live hibernating females at some dates, but at lower prevalence than for hyphal bodies and predominantly only until November 8. Prevalence of resting spores in live hibernating females ranged from 2.5 to 13.8%. Total number of T. urticae was also recorded, and most mites of all four categories (nymphs, males, non-hibernating and hibernating females) were found at the first sampling date. At this date non-hibernating females were the most abundant. A sharp decrease in non-hibernating females, nymphs and males was, however, seen from mid-October to mid-November; also numbers of hibernating females decreased, but not as fast. The relative abundance of hibernating females compared to non-hibernating females increased from 32.2% at the first collection (October 12) to 97.7% at the last collection (February 2). This study confirms that N. floridana survives the winter as a semi-latent hyphal body infection, protected inside live hibernating females. It is therefore ready to develop and sporulate as soon as climatic conditions permit, resulting in early season infection of T. urticae.     

Hibernation activates glyoxylate cycle and gluconeogenesis in black bear brown adipose tissue

Biochemical studies on brown adipose tissue removed from a hibernating black bear and a non-hibernating control animal demonstrate that this tissue: (1) can carry out cyanide-insensitive fatty acid oxidation, and (2) possesses catalase activity and the enzyme activities unique to the glyoxylate cycle, isocitrate lyase and malate synthase. These activities are all markedly increased in brown fat obtained from the hibernating animal. Additionally, hibernation enhances the ability of the tissue to synthesize glycogen in the presence of a fatty acid substrate. The glyoxylate cycle enzymes and the ability to convert fatty acid carbons to glucose have been generally regarded as being absent from vertebrate cells and tissues.     

Ultrastructure of the tubular nephron of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals

The tubular nephron of hibernating and non-hibernating specimens of Testudo graeca (Chelonia) was studied by means of conventional light and electron microscopy and histochemistry. The tubular nephron was composed of proximal, intermediate, distal and collecting tubules in both hibernating and non-hibernating animals. The cells of the proximal tubule showed long microvilli, cytoplasmic vacuoles, a developed endoplasmic reticulum and abundant mitochondria. Fat droplets were also observed. The intermediate segment was lined by ciliated and non-ciliated cells. The lining cells of the distal tubule presented few microvilli, abundant dense mitochondria and clear vesicles of mucous appearance in the terminal portion. Collecting ducts are composed of mucous and non-mucous cells. Mucous cells presented strong reaction to the histochemical techniques detecting sialo- and sulpho-mucins. During hibernation, a progressive vacuolar degeneration of the endoplasmic reticulum was observed in all the segments of tubular nephron, which may be caused by a massive intake of extracellular water into the cell.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.     

Changes in renal morphology and renin secretion in the golden-mantled ground squirrel (Spermophilus lateralis) during activity and hibernation

Summary Chronological changes in renal glomerular morphology and plasma renin activity were investigated during active and hibernating periods in the golden-mantled ground squirrel Spermophilus lateralis. The objective of this study was to determine whether the glomerular endothelium, visceral epithelium (podocytes), basement membrane, mesangial cells, proximal convoluted tubule cells and plasma renin activity exhibit measurable sequential differences between as well as within active and hibernating states at various time points. Limitations in the size of the experimental population prevented an evaluation of changes in these parameters during other important periods such as periodic arousal between hibernation bouts. In this study, glomerular endothelial pore number and epithelial filtration slit number significantly decreased by early hibernation when compared to those during summer activity, and then they increased back toward summer levels by late hibernation. In contrast, podocytic pedicel width along the glomerular basement membrane increased from summer activity to early hibernation, before significantly decreasing again by late hibernation. Mesangial cell and proximal convoluted tubule cell activity appeared increased during hibernation as compared to summer activity, whereas the width of the glomerular basement membrane showed no significant alterations throughout. Plasma renin activity significantly increased during early hibernation and mid-hibernation when compared to summer levels but had decreased by late hibernation toward summer values. The glomerular and plasma renin activity changes observed in this study clearly illustrate the drastic structural and functional adjustments which hibernating species make during torpor and also correlate well with the reported decrease in renal perfusion pressure and urine formation during hibernation. The observed morphological changes during hibernation do not appear to be temperature-dependent, because significant alterations in most of the parameters studied occurred during this period despite the fact that cold-room temperatures were kept constant throughout. The chronological approach to this study and its morphometric evaluation represent a pilot attempt at accurately documenting these changes during two critical states in the hibernator's cycle and may eventually lead to the characterization of these changes during the entire circannual cycle.     

Normalization of aortic function during arousal episodes in the hibernating ground squirrel

Hypothermia is commonly used to restrict organ damage during preservation of tissue, but does not offer complete protection. Organ damage after reperfusion/rewarming is amongst others caused by an impairment of vascular properties, particularly endothelium-dependent vasodilatation. We hypothesized that hibernating small animals, which frequently cycle through periods of deep cooling (torpor) and full rewarming (arousal), employ specific mechanisms to preserve vascular function after cooling and reperfusion. Therefore we measured contraction of aortic tissue of hibernating European ground squirrels after 24 h and 7 days of torpor, arousal (1.5 h) and in non-hibernating animals. To assess the role of nitric oxide (NO), experiments were performed in the absence and presence of the NO-synthesis inhibitor, L-NMMA (10(-4) M). Maximum contraction to phenylephrine and angiotensin II was doubled in 7-days torpid animals without a shift in EC50, compared to the other 3 groups. Maximum contraction to KCl was doubled in 7-days torpid animals compared to the arousal group and non-hibernating animals. Relaxation to acetylcholine (ACh) and sodium nitrite in phenylephrine precontracted rings did not differ between groups. In the presence of L-NMMA, the maximum of concentration-response curves for all three vasoconstrictors was increased by about 30% in the arousal group, but unaffected in other groups. L-NMMA completely inhibited ACh-induced relaxation in 24-h torpid animals and non-hibernating animals, but only partially in 7-days torpid animals and in the arousal group. From this we conclude that vascular adaptation proceeds during torpor. Further, increased contractility of aortic tissue during long torpor returns to normal within 1.5 hours of arousal, which is associated with an increased basal NO synthesis. In addition, involvement of NO in agonist-mediated relaxation differs between the various stages of hibernation.Thus, hibernating animals have effectively developed mechanisms to preserve vascular function after cooling and rewarming.     

An analysis of the peripheral blood picture in the suslik and the white rat at different seasons of the year

New data are presented concerning seasonal changes in the peripheral blood of hibernating and non-hibernating rodents. In non-hibernating rats, seasonal changes in the blood are mainly phenotypical. Hibernating mammals are capable periodically to renew cellular composition of the blood during winter and summer hibernation.     

The influence of hypothermia on the restoration of neural activity of brain slices from hibernating ground squirrels

1. 1. Neural activity was recorded in hippocampal slices from deep hibernating Yakut ground squirrels and in hippocampal and septal slices from non-hibernating animals.

2. 2. Slices were placed immediately after preparation in hypothermic conditions (3–4°C). Their activity was tested under standard conditions at 31°C in the incubation chamber. Some of the prepared slices were tested after maintenance in hypothermia for 2 or 24 h.

3. 3. In the hippocampal slices of hibernating ground squirrels, neural activity was present, irrespective of the period in hypothermia.

4. 4. Slices from guinea-pigs and hamsters did not possess neural activity after either 2 or 24 h of hypothermic treatment.

     

Mitochondrial alpha-glycerophosphate dehydrogenase activity in active and torpid ground squirrels, Spermophilus richardsoni.

1. Mitochondrial alpha-glycerophosphate dehydrogenase (GPD) activity was assayed in liver homogenates from active and torpid ground squirrels. 2. Arrhenius plots of GPD activity were linear in non-hibernating animals and discontinuous in hibernators. Compared with non-hibernators, the energy of activation in hibernators was reduced between 37 and 25 degrees C, but increased between 25 and 6 degrees C. 3. A dose-response relation between GPD activity and injected thyroxine was determined in active animals. No correlation was found between enzyme activity at 37 or 6 degrees C and circulating titres of thyroid hormones, in ground squirrels sampled during the preparative and hibernating phases.     

Cloning, sequence analysis and phylogeny of connexin43 isolated from American black bear heart.

Conduction in the heart requires gap junctions. In mammalian ventricular myocytes these consist of connexin43 (Cx43). Hearts of non-hibernating species display conduction disturbances at reduced temperatures. These may exacerbate into lethal arrhythmias. Hibernating species are protected against these arrhythmias by a non-resolved mechanism. To analyze whether the amino acid composition of Cx43 from the hibernating American black bear displays specific features, we cloned the full coding sequence of Ursus americanus Cx43 and compared with that of other (non)hibernating species. UaCx43 displays 99.7% identity to rabbit Cx43 at the amino acid level. No specific features were observed in UaCx43 when compared to previously cloned Cx43 from hibernating and non-hibernating mammals. Phylogenetic tree reconstruction of this and other published full-length Cx43 sequences reveals a very high level of conservation from fish to men. Finally, one of the previously identified six mammalian characteristic amino acids, is not conserved in the black bear.     

Studies on the juxtaglomerular apparatus

The juxtaglomerular apparatus of the rat was studied after freeze-fracturing with special respect to intercellular junctions. It was found that juxtaglomerular granulated cells of the vas afferens are interconnected by gap junctions to adjacent cells (granulated cells, possibly also smooth muscle cells). Gap junctions have also been found on the surface of lacis cells and mesangial cells. It is therefore concluded that these cells of the juxtaglomerular apparatus and the glomerulus--granulated cells (possibly also smooth muscle cells) of the vas afferens, lacis cells and mesangium cells--form a functional system reacting in a coordinated manner to physiological stimuli.     

黑龙江林蛙冬眠和非冬眠消化道内分泌细胞的比较研究

目的应用5-羟色胺(5-HT)、生长抑素(SS)、胃泌素(GAS)、胰高血糖素(Glu)、胰多肽(PP)和P-物质(SP)6种特异性抗血清,对黑龙江林蛙冬眠期和非冬眠期消化道内分泌细胞进行检测。方法应用卵白素-生物素-过氧化物酶复合物(avidin-biotin-peroxidase complex,ABC)免疫组织化学法。结果在冬眠期和非冬眠期黑龙江林蛙消化道内检测到5-HT细胞、SS细胞和Glu细胞,而GAS细胞、PP细胞和SP细胞均未检测到。5-HT细胞分布广泛,存在于黑龙江林蛙冬眠期和非冬眠期从食管到直肠的整个消化道中,在冬眠期分布密度高峰为胃幽门部和十二指肠,而非冬眠期则为胃幽门部;SS细胞分布也较为广泛,在黑龙江林蛙冬眠期和非冬眠期消化道中,除了直肠以外,均有分布,且这两个时期SS细胞的分布密度高峰均在胃幽门部。Glu细胞在冬眠期仅在胃贲门部、胃体、空肠和回肠检测到,而在非冬眠期除以上四个部位外直肠部也检测到了Glu细胞,并且Glu细胞分布密度高峰都在胃体部。结论黑龙江林蛙在冬眠期和非冬眠期消化道中内分泌细胞分布型的不同,可能与机体适应不同时期的生理功能有关。     

泰和鸡肾小球旁器的观察

本文用光镜和透射电镜对泰和鸡(乌骨鸡)的肾小球旁器进行了观察。结果表明,泰和鸡的肾小球旁器由球旁细胞、过渡型致密斑、球外间膜细胞和极周细胞所组成。极周细胞在鸟类还属首次报道,它位于肾小囊脏层与壁层上皮移行处,环绕着肾小体的血管极,其结构与哺乳动物的相似。本文还就泰和鸡肾小球旁器的结构与功能的关系作了讨论。     

Dynamics of renin production by the juxtaglomerular and mesangial cells of individual rat kidney glomeruli following adrenalectomy]

Adrenal incompetence developed in rats 6 weeks after adrenalectomy without any salt and hormonal compensation. In individual fragments of the isolated glomeruli containing juxtaglomerular cells (JGC) renin activity increased 1.2 times on the average, and there was revealed renin-like activity (RLA) in the fragments containing mesangial cells (MC). Signs of intensified renin secretion (expressed in reduction of granule count, marked development of granular endoplasmic reticulum, Golgi complex and microtubules) were noted in the JGC. In MC such organoids were well developed, but no granules were revealed. The following occurred in 8 to 12 weeks with the restoration of the 11-OCS and sodium level in the plasma: renin JGC activity became normal, RLA activity in MC disappeared, and the initial ultrastructure of both of these cells was restored. The reserve role of MC as the source of renin-like substances was confirmed.     

Ultrastructural changes in Paneth cells during hibernation in the ground squirrel Spermophilm lateralis

Summary The ultrastructure of Paneth cells from jejuno-ileal segments of the small intestine of the ground squirrel, S. lateralis, was examined under normal euthermic conditions and during the profoundly depressed metabolic conditions of natural hibernation. Paneth cells obtained from hibernating animals gave evidence of markedly reduced activity when compared to Paneth cells from euthermic animals. In hibernating animals, the nuclei were smaller, with less prominent nucleoli and with an increased proportion of heterochromatin. In hibernating animals, the rough endoplasmic reticulum was fragmentary and poorly organized, in contrast to the typical arrangement of concentric lamellae seen in euthermic animals. Although the total number of ribosomes was decreased in hibernating animals, there were proportionally more free ribosomes than in euthermic animals. Paneth cells from hibernating animals also contained a greater number of apical secretory granules which were smaller and more variable in electron density than granules from control animals. These ultrastructural features indicate that during hibernation the Paneth cell is relatively quiescent.Supported by U.S.P.H.S. grants RR 05411 and RR 05583 from the N.I.H.