A Novel Model of Chronic Kidney Disease in Rats: Dietary Adenine in Combination with Unilateral Nephrectomy.

BACKGROUND: Adenine at 0.75% in the diet (AD) triggers renal impairment in rats. This model of kidney disease is largely reversible when AD feeding is stopped. Testing of novel drugs parallel to AD administration may result in unwanted interference. OBJECTIVES: We hypothesized that combining AD with unilateral nephrectomy (UNx) would result in progressive chronic kidney disease (CKD) even after cessation of AD. METHODS: In an explorative study, 16 rats with UNx (AD-1K rats) and 10 sham-operated rats (AD-2K rats) received AD-supplemented feed for 3 weeks, followed by 4 weeks of standard chow. Ten sham-operated rats receiving only standard chow served as controls. Laboratory parameters in blood and urine were frequently assessed during and after cessation of AD feeding. Comprehensive pathological examinations were performed in all rats at the end of the experiment. RESULTS: Rats with UNx were more affected by impaired glomerular filtration rate, anemia, hyperphosphatemia, and hypocalcemia. After cessation of AD feeding, recovery was poorest in AD-1K rats, paralleled by increased proteinuria indicative of progressive CKD. Scores in histopathological damage of the kidneys indicative of CKD were seen in both AD-fed groups, with key parameters being more affected in AD-1K rats. Histopathological changes in the heart were most prominent in AD-1K rats. CONCLUSIONS: Combining AD feeding with UNx provides a time window after cessation of AD feeding for the testing of drugs without interference. Our findings in rats may have implications for research in other target animal species as well.

Neuronal Activation After Prolonged Immobilization: Do the Same or Different Neurons Respond to a Novel Stressor?

Despite extensive research on the impact of emotional stressors on brain function using immediate-early genes (e.g., c-fos), there are still important questions that remain unanswered such as the reason for the progressive decline of c-fos expression in response to prolonged stress and the neuronal populations activated by different stressors. This study tackles these 2 questions by evaluating c-fos expression in response to 2 different emotional stressors applied sequentially, and performing a fluorescent double labeling of c-Fos protein and c-fos mRNA on stress-related brain areas. Results were complemented with the assessment of the hypothalamic-pituitary-adrenal axis activation. We showed that the progressive decline of c-fos expression could be related to 2 differing mechanisms involving either transcriptional repression or changes in stimulatory inputs. Moreover, the neuronal populations that respond to the different stressors appear to be predominantly separated in high-level processing areas (e.g., medial prefrontal cortex). However, in low-hierarchy areas (e.g., paraventricular nucleus of the hypothalamus) neuronal populations appear to respond unspecifically. The data suggest that the distinct physiological and behavioral consequences of emotional stressors, and their implication in the development of psychopathologies, are likely to be closely associated with neuronal populations specifically activated by each stressor.

What can we know from pituitary-adrenal hormones about the nature and consequences of exposure to emotional stressors?

Exposure to stress induces profound physiological and behavioral changes in the organisms and some of these changes may be important regarding stress-induced pathologies and animal models of psychiatric diseases. Consequences of stress are dependent on the duration of exposure to stressors (acute, chronic), but also of certain characteristics such as intensity, controllability, and predictability. If some biological variables were able to reflect these characteristics, they could be used to predict negative consequences of stress. Among the myriad of physiological changes caused by stress, only a restricted number of variables appears to reflect the intensity of the situation, mainly plasma levels of ACTH and adrenaline. Peripheral hypothalamic-pituitary-adrenal (HPA) hormones (ACTH and corticosterone) are also able to reflect fear conditioning. In contrast, the activation of the HPA axis is not consistently related to anxiety as evaluated by classical tests such as the elevated plus-maze. Similarly, there is no consistent evidence about the sensitivity of the HPA axis to psychological variables such as controllability and predictability, despite the fact that: (a) lack of control over aversive stimuli can induce behavioral alterations not seen in animals which exert control, and (b) animals showed clear preference for predictable versus unpredictable stressful situations. New studies are needed to re-evaluate the relationship between the HPA axis and psychological stress characteristics using ACTH instead of corticosterone and taking advantages of our current knowledge about the regulation of this important stress system.

Behavioral and endocrine consequences of simultaneous exposure to two different stressors in rats: interaction or independence?

Although behavioral and endocrine consequences of acute exposure to stressors have been extensively studied, little is known about how simultaneous exposure to two different stressors interacts to induce short- and long-term effects. In the present experiment we studied this interaction in adult male rats exposed to cat fur odor (impregnated cloth) or immobilization on boards either separately or simultaneously. We reasoned that exposure to the odor of a potential predator while immobilized, may potentiate its negative consequences as compared to exposure to only one of the stressors. Exposure to cat odor elicited the expected reduction of activity and avoidance of the area where the impregnated cloth was located. The endocrine response (plasma levels of ACTH and corticosterone, as a measure of the hypothalamic-pituitary-adrenal axis, HPA) was markedly greater after immobilization than after cat fur odor and no additive effects were found by simultaneous exposure to both stressors. Cat odor, but not immobilization, increased anxiety-like behavior as evaluated in the elevated plus-maze 7 days after the stressors, with no evidence of enhanced HPA activation. In addition, cat odor exposure resulted in long-lasting (8 days later) fear conditioning to the box containing a clean cloth, which was reflected by hypoactivity, avoidance of the cloth area and enhanced HPA activation. All these effects were similarly observed in rats exposed simultaneously to cat odor and immobilization. In rats only exposed to immobilization, only some weak behavioral signs of fear conditioning were found, but HPA activation in response to the context paired to immobilization was enhanced to the same extent as in cat odor-exposed animals, supporting a certain degree of endocrine conditioning. The present results did not reveal important behavioral interactions between the two stressors when animals experienced both simultaneously, whereas some interactions were found regarding HPA activation. Theoretical implications are discussed.

Repeated exposure to immobilization or two different footshock intensities reveals differential adaptation of the hypothalamic-pituitary-adrenal axis.

Factors involved in adaptation to repeated stress are not well-characterized. For instance, acute footshock (FS) of high intensity appears to be less severe than immobilization (IMO) in light of the speed of post-stress recovery of the hypothalamic-pituitary-adrenal (HPA) axis and other physiological variables. However, repeated exposure to IMO consistently resulted in reduction of the HPA response to the same stressor (adaptation), whereas failure to adapt has been usually reported after FS. Thus, in the present work we directly compared the activation of HPA axis and other physiological changes in response to both acute and repeated exposure to IMO and two intensities of FS (medium and high) in adult male rats. Control rats were exposed to the FS boxes but they did not receive shocks. Daily repeated exposure to IMO resulted in significant adaptation of the overall ACTH and corticosterone responses to the stressor. Such a reduction was also observed with repeated exposure to FS boxes and FS-medium, whereas repeated exposure to FS-high only resulted in a small reduction of the corticosterone response during the post-stress period. This suggests that some properties of FS-high make adaptation to it difficult. Interestingly, overall changes in food intake and body weight gain throughout the week of exposure to the stressors reveal a greater impact of IMO than FS-high, indicating that factors other than the intensity of a stressor, at least when evaluated in function of the above physiological variables, can influence HPA adaptation. Since FS exposure is likely to cause more pain than IMO, activation of nociceptive signals above a certain level may negatively affect HPA adaptation to repeated stressors.

Adaptation of the hypothalamic-pituitary-adrenal axis and glucose to repeated immobilization or restraint stress is not influenced by associative signals.

Repeated exposure to the same stressor very often results in a reduction of some prototypical stress responses, namely those related to the hypothalamic-pituitary-adrenal (HPA) and sympatho-medullo-adrenal (SMA) axes. This reduced response to repeated exposure to the same (homotypic) stressor (adaptation) is usually considered as a habituation-like process, and therefore, a non-associative type of learning. However, there is some evidence that contextual cues and therefore associative processes could contribute to adaptation. In the present study we demonstrated in two experiments using adult male rats that repeated daily exposure to restraint (REST) or immobilization on boards (IMO) reduced the HPA (plasma levels of ACTH and corticosterone) and glucose responses to the homotypic stressor and such reduced responses remained intact when all putative cues associated to the procedure (experimenter, way of transporting to the stress room, stress boxes, stress room and colour of the restrainer in the case of REST) were modified on the next day. Therefore, the present results do not favour the view that adaptation after repeated exposure to a stressor may involve associative processes related to signals predicting the imminence of the stressors, but more studies are needed on this issue.

Do odors from different cats induce equivalent unconditioned and conditioned responses in rats?

Since cats are predators of rats and mice, interest in the consequences of exposure to cat-associated odors has increased in the last decade, particularly regarding the development of putative animal models of post-traumatic stress disorder (PTSD). Although in the literature there are some comments on the variability of the effects depending on the individual cat used, there are no reports on this subject. In the present study, we demonstrated, using male Sprague-Dawley rats and cloths impregnated with fur/skin odors from three different cats (one ovariectomized female and two intact males), that the unconditioned endocrine (release of corticosterone) and behavioral (inhibition of activity and avoidance of the cloth area) responses to the presence of the cat odors were statistically significant and similar among the three cats. The conditioned behavioral response to a clean cloth, studied 7 days after the initial exposure to odors, was also evident with the three cats, with minor differences among them. In contrast, only the fur odor from one of the male cats clearly increased anxiety-like behavior in the elevated plus-maze 6 days later. These data indicate that: (i) evaluation of unconditioned and conditioned responses to cat odors does not appear to strongly predict long-lasting increases in anxiety-like behavior; and, therefore, both types of responses are partially dissociated; and (ii) differences among cats mainly affected the induction of long-lasting changes in anxiety-like behavior. The ultimate reasons for these differences are not known, but their characterization is critical for a proper understanding of putative PTSD models.

Cat odor causes long-lasting contextual fear conditioning and increased pituitary-adrenal activation, without modifying anxiety.

A single exposure to a cat or cat odors has been reported by some groups to induce contextual and auditory fear conditioning and long-lasting changes in anxiety-like behaviour, but there is no evidence for parallel changes in biological stress markers. In the present study we demonstrated in male rats that exposure to a novel environment containing a cloth impregnated with cat fur odor resulted in avoidance of the odor, lower levels of activity and higher pituitary-adrenal (PA) response as compared to those exposed to the novel environment containing a clean cloth, suggesting increased levels of stress in the former animals. When re-exposed 9 days later to the same environment with a clean cloth, previously cat fur exposed rats again showed avoidance of the cloth area and lower levels of activity, suggesting development of contextual fear conditioning, which again was associated with a higher PA activation. In contrast, unaltered both anxiety-like behaviour and PA responsiveness to an elevated plus-maze were found 7 days after cat odor exposure. It is concluded that: (i) PA activation is able to reflect both the stressful properties of cat fur odor and odor-induced contextual fear conditioning; (ii) development of cat odor-induced contextual fear conditioning is independent of the induction of long-lasting changes in anxiety-like behaviour; and (iii) greater PA activation during exposure to the odor context is not explained by non-specific sensitization of the PA axis caused by previous exposure to cat fur odor.