Cortisol up-regulates corticotropin releasing factor gene expression in the fetal ovine brainstem at 0.70 gestation.

Glucocorticoids are important for the development of the central nervous system. In the ovine fetus, increased levels of plasma cortisol at term provide a stimulus to initiate parturition. CRF is central to this event in that it is one of the main modulators of the hypothalamic-pituitary-adrenal (HPA) axis. The purpose of the present study was to determine the effect of physiological increases in fetal plasma cortisol levels on corticotropin-releasing factor (CRF) gene expression in the developing ovine brain. Fetal plasma cortisol levels were chronically elevated at 0.70 gestation (100 days) to physiological levels found at 0.90 gestation (130 days; term 145 +/- 2 days) when glucocorticoid-induced maturational changes are known to occur in the HPA axis. The 3' end of the ovine CRF gene encodes 4 putative polyadenylation (poly(A)) signals that may post-transcriptionally regulate gene expression through stability, translation and localization of the mRNA in a temporal and spatial manner. To determine whether CRF mRNA levels or poly(A) site usage are differentially regulated by cortisol in a region-specific manner, we used an RNase protection assay with an antisense CRF RNA probe from the 3' coding and untranslated regions of the gene to quantify changes in mRNA levels in the hypothalamus (Hypo), hippocampal-amygdala complex (H and A), frontal cerebral cortex (FCC) and brainstem. Our novel finding was a 3.5-fold increase in CRF mRNA levels in the medulla oblongata of fetuses from the cortisol group compared to those from the saline group (P = 0.001). CRF mRNA levels in the Hypo, H and A and FCC did not change significantly in fetuses from the cortisol group.(ABSTRACT TRUNCATED AT 250 WORDS)

Water maze performance of aged Sprague-Dawley rats in relation to retinal morphologic measures.

The spatial learning ability of aged male and female Sprague-Dawley rats was assessed using the Morris water maze. To determine the influence of age-related visual deficits on performance levels, retinal morphologic measures were correlated with water maze performance for each rat. Rats were first trained on the water maze task at 21 months of age and were retrained 3 or 4 times at 6-week intervals. After the last training session the rats were killed and their eyes were removed for histopathologic and morphometric evaluation of the retinas. There was a large degree of retinal degeneration in all of the aged Sprague-Dawley rats with an average decrease in the thickness of the retinal outer nuclear layer (photoreceptor nuclei containing layer) of 85% in old males and 95% in old females. Some rats, however, had less degeneration of the retinas than others, and the degree of retinal degeneration was strongly related to performance levels on the water maze task. Among the aged rats in this study with the least retinal degeneration, there was little evidence for a subset of rats that were unable, with extensive training, to learn a platform position. Of the 41 rats with the least retinal degeneration (out of a total of 81), only one was a clear non-learner on the water maze task, whereas, of the 27 rats with the most retinal degeneration, 20 were non-learners. These results illustrate the potentially serious confounding effects of deteriorating visual ability on attempts to assess cognitive functioning of aged albino rats on tasks requiring utilization of visual cues.

Analysis of severe photoreceptor loss and Morris water-maze performance in aged rats.

In a study of aging and memory in 25-27-month-old albino rats, performance on a Morris water maze was found to be dependent on the structural integrity of the retina. Generally, as expected, 'learners' had intact retinas, while 'non-learners' had retinas with severe photoreceptor loss and a non-continuous outer nuclear layer, consisting of scattered cell nuclei. However, contrary to this general correlation between learning ability and photoreceptor presence, some learners had severely degenerated retinas and occasionally, non-learners had photoreceptor populations that apparently were comparable to those of learners. Rat retinas from these unpredictable, borderline response categories were examined histopathologically and morphometrically with the purpose of determining the minimal number of photoreceptors (PRs) necessary for animals to be rated as learners on the Morris water maze. However, among these severely damaged retinas of borderline groups, total number of surviving photoreceptors did not vary significantly among the learner, ambiguous or marginal and non-learner groups. The population of surviving PRs in learners was as low as 0.04% and in non-learners as high as 0.4%, as compared to that of young, adult rats. Therefore, borderline learners and non-learners had overlapping surviving PR numbers and the results did not clarify the response difference between these groups in the Morris water maze. It is suggested that the pattern of surviving PRs over the retinal surface, as well as the ratio of surviving rods to cones and their connectivity with other retinal neurons, may be related to the residual function of degenerated retinas of learner rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin releasing factor mRNA and peptide levels are differentially regulated in the developing ovine brain.

The regulation of CRF mRNA and protein in the developing ovine brain has been studied to assess the hypothesis that CRF is differentially regulated in the hypothalamus (Hypo), hippocampal-amygdala complex (H & A), frontal cerebral cortex (FCC) and brainstem (BS). We used a quantitative RNase protection assay and radioimmunoassay to determine mRNA and peptide concentrations, respectively, from the last third of gestation until term (i.e., from 95 to 142 days gestation (dg); term approximately 145 days). The major findings from this study are: (1) Hypothalamic CRF mRNA was increased by 2-fold in 140-142 dg fetuses compared to 128-138 and 95-123 dg fetuses; P = 0.016. (2) In the hypothalamus of 140-142 dg fetuses, there was a 2.5-fold increase in CRF mRNA derived from polyadenylation at poly(A) sites 2, 3 or 4; P = 0.005. (3) In 128-138 dg fetuses, CRF mRNA in the frontal cortex was 2-fold higher than in the other brain regions during this time period; P = 0.008. (4) CRF peptide concentrations in the Hypo were 2.5-fold higher in 140-142 dg fetuses compared to 95-106 and 128-138 dg fetuses; P = 0.007. (5) CRF peptide concentrations in the frontal cortex were 5.5-fold higher in 140-142 dg fetuses compared to fetuses at 95-106 dg; P = 0.004. (6) CRF peptide concentrations in the H & A were 5-fold higher in 140-142 dg fetuses compared to 95-106 dg fetuses; P = 0.029. The results from the present study demonstrate for the first time that CRF mRNA and peptide are differentially regulated in a region-specific manner during development.

Significance of gaseous microemboli in the cerebral circulation during cardiopulmonary bypass in dogs.

BACKGROUND: Gaseous microemboli during cardiac surgery may damage the brain by reducing cerebral blood flow (CBF). We examined whether the incidence of gaseous microemboli during 150-minute hypothermic (28 degrees C) cardiopulmonary bypass (CPB) adversely affects CBF (radioactive microspheres). METHODS AND RESULTS: Thirty anesthetized dogs were placed on CPB using bubble oxygenators with 50% O2 (n = 10) or 100% O2 (n = 10) to produce a wide range in the number of gaseous microemboli or membrane oxygenators with 50% O2 (n = 10) to avoid microemboli. The number of carotid artery microemboli occurring in a 1-minute interval was counted using a 5-MHz Doppler probe every 15 minutes for the duration of CPB, which lasted 258 +/- 5 minutes. With bubbled 100% O2, the number of microemboli averaged 4.1 +/- 1.7 emboli per minute on normothermic bypass and increased with cooling to 18.3 +/- 4.9 emboli per minute (P < .001). With bubbled 50% O2, the microemboli number was greater on normothermic bypass (19.8 +/- 9.8 emboli per minute, P = .0653 compared with bubbled 100% O2) and increased with cooling (100.3 +/- 18.7 emboli per minute, P < .001) to a greater extent than with bubbled 100% O2 (P < .001). In contrast, with membrane 50% O2, the emboli number was small (0.6 +/- 0.1 emboli per minute) and did not change with CPB temperature. CBF values were not reduced after termination of CPB, even when compared with prebypass values, being 48.3 +/- 7.5 mL/min per 100 g (bubbled 50% O2), 49.6 +/- 4.1 mL/min per 100 g (bubble 100% O2), and 44.5 +/- 2.8 mL/min per 100 g (membrane 50% O2, P = .7581). Similarly, regional perfusion to the cerebellum, hippocampus, and caudal brainstem was not adversely affected by microemboli. After CPB, cortical biopsies demonstrated no difference among groups with respect to lactate (P = .1753), energy charge (P = .5179), and brain water content (P = .939). Retinal histopathology indicated no differences among groups. CONCLUSIONS: These results indicate that: (1) the incidence of gaseous microemboli during hypothermia increases when a bubble oxygenator is used, and (2) global CBF and regional brain perfusion are not adversely affected by numerous gaseous microemboli.

Melatonin increases photoreceptor susceptibility to light-induced damage.

Melatonin is an indolamine hormone synthesized in the retina and pineal gland. It is thought to act as a paracrine neurohormone in the mammalian retina. Pinealectomy has been shown to protect photoreceptors from light-induced damage, and melatonin treatment has been reported to increase the degree of photoreceptor damage in albino rats. To determine how melatonin influences photoreceptor survival, the effect of melatonin administration on light-induced retinal damage was studied. Melatonin was administered to albino rats by intraperitoneal injections at various times before or after light exposure. The rats were exposed to high-intensity illumination (1600 lux) for 24 hr to induce photodamage, then returned to cyclic lighting for 12 days. After this, they were killed, and their eyes were removed and examined histologically. Measurements of the outer nuclear layer (ONL) thickness were taken at 12 different loci around the circumference of the retinal sections. The animals that received daily melatonin injections (100 micrograms) in the late afternoon (3 hr before lights off) for 1-3 days before photodamage showed an approximate 30% greater reduction compared with sham control animals in ONL thickness in the superior quadrant, the area most susceptible to light damage. Melatonin injections given after the photodamage did not affect ONL thickness. Although retinal susceptibility to light damage varied with time of day, the degree to which melatonin increased the degree of damage appeared unaffected by the time of day. These results suggest that melatonin may be involved in some aspects of photoreceptor sensitivity to light damage.

Cerebral perfusion during canine hypothermic cardiopulmonary bypass: effect of arterial carbon dioxide tension.

Cerebral blood flow (radioactive microspheres), intracranial pressure (subdural bolt), and retinal histopathology were examined in 20 dogs undergoing 150 minutes of hypothermic (28 degrees C) cardiopulmonary bypass to compare alpha-stat (arterial carbon dioxide tension, 40 +/- 1 mm Hg; n = 10) and pH-stat (arterial carbon dioxide tension, 61 +/- 1 mm Hg; n = 10) techniques of arterial carbon dioxide tension management. Pump flow (80 mL.kg-1.min-1), mean aortic pressure (78 +/- 2 mm Hg), and hemoglobin level (87 +/- 3 g/L [8.7 +/- 0.3 g/dL]) were maintained constant. During bypass, intracranial pressure progressively increased in the alpha-stat group from 6.0 +/- 1.0 to 13.9 +/- 1.8 mm Hg (p less than 0.05) and in the pH-stat group from 7.7 +/- 1.1 to 14.7 +/- 1.4 mm Hg (p less than 0.05), although there was no evidence of loss of intracranial compliance or intracranial edema formation as assessed by brain water content. With cooling, cerebral blood flow decreased by 56% to 62% in the alpha-stat group (p less than 0.05) and by 48% to 56% in the pH-stat group (p less than 0.05). However, 30 minutes after rewarming to 37 degrees C, cerebral blood flow in both groups failed to increase and remained significantly depressed compared with baseline values. Both groups showed similar amounts of ischemic retinal damage, with degeneration of bipolar cells found in the inner nuclear layer in 67% of animals. We conclude that, independent of the arterial carbon dioxide tension management technique, (1) cerebral perfusion decreased comparably during prolonged hypothermic bypass, (2) intracranial pressure increases progressively, (3) ischemic damage to retinal cells occurs despite maintenance of aortic pressure and flow, and (4) a significant reduction in cerebral perfusion persists after rewarming.

Dietary restriction does not alter retinal aging in the Fischer 344 rat.

In the present study, the effects of long-term dietary restriction (60% of the calories in the ad lib diet, beginning at 16 weeks of age) on quantitative morphometric measures and histopathologic indications of aging have been investigated in the retina of Fischer 344 male rats. The animals were maintained by the NIA Biomarkers Program, National Center for Toxicological Research. Group size ranged from 8 to 15 rats. A gradual thinning of the outer nuclear layer (ONL) of photoreceptor nuclei occurred with aging in control ad lib groups. The restricted diet did not affect retinal aging in 18-, 21-, 26-, or 27-month-old rats, as judged by photoreceptor cell death, ONL thickness, and pattern of cell loss. Retinal thickness (RT) was unaffected by restricted diet, except in the 21-month-old group; in that group, the RT was reduced significantly in thickness as compared to ad lib animals. These results are in contrast to studies of dietary restriction on most nonneuronal markers of aging and suggest that a different mechanism may modulate at least some aspects of brain aging.

Water deprivation protects photoreceptors against light damage.

Photoreceptor cell death after light-damage and during aging in rats is associated with the hormonal status of the animal, as well as other environmental and intrinsic factors. Restricted caloric intake extends the life of rodents and is usually accompanied by a reduction in water consumption. In this study, male and female rats were placed on restricted water intake for either 3 or 7 days to induce dehydration. Following exposure to damaging visible light, the retinas were evaluated for severity of damage and photoreceptor survival, heat shock (stress) protein (HSP) and total protein synthesis, and plasma arginine vasopressin (AVP) levels. Photoreceptor cells of 7-day, dehydrated male and female rats survived light-damage significantly better than those allowed water ad libitum; however, after 3 days of water restriction, only the male rats demonstrated protection from photodamage. Severity of photoreceptor damage could not be correlated with retinal HSP synthesis and content, although the latter was significantly reduced in dehydrated animals. Total retinal protein content and synthesis were unchanged by restricted water intake. AVP increased by 350% during the 7-day period of dehydration. Protection of photoreceptors from light-damage in this study may be correlated with osmotically stimulated changes in the retinas of dehydrated animals.

Scleral calcification and photoreceptor cell death during aging and exposure to chronic stress.

Male and female Fischer 344 rats of three different ages (12, 18, and 25 months) have been examined for the presence of photoreceptor (PR) cell loss and for occurrence of scleral cartilage and bone formation. In addition, male and female rats, aged 11 months at the beginning of the experiments, were exposed to chronic stress for either 0.5, 2, 4, or 6 months. Photoreceptor cell death gradually increases during the aging process and is exacerbated by exposure to chronic stress. It is more severe in the peripheral than the central retina and exposure to stress increases this pattern of cell loss. The superior retina is more severely affected than the inferior hemisphere in aging and during stress. The incidence of scleral cartilage or bone formation increases with age in male and female rats, but with stress exposure an increase is seen in males only. Bone formations occur more frequently in male than in female animals and are almost always (97%) located in the superior hemisphere of the eye. Although there appears to be a direct relationship between photoreceptor cell death and the occurrence of scleral ossifications in group data, in individual eyes the bone formations are not always associated with severity of PR cell loss. The relationship of PR cell death and incidence of scleral ossification to gender and to exposure to stress supports a hypothesis for an endocrine basis of ocular aging.

Hydroxyindole-O-methyltransferase in rat retinal bipolar cells: persistence following photoreceptor destruction.

The presence of hydroxyindole-O-methyltransferase (HIOMT) activity and localization of HIOMT immunoreactivity was examined in albino rat retinas following photoreceptor destruction. Male Sprague-Dawley rats were exposed to high intensity fluorescent light for 4 consecutive days, then placed on a 14:10 h light:dark cycle for two weeks to allow for phagocytic removal of damaged cells from the retina. Histologic examination revealed almost complete destruction and removal of all photoreceptors. The damaged retinas exhibited an increase in HIOMT activity relative to controls, when expressed as activity per mg of protein. HIOMT activity in the pineal glands was not affected. When control and light damaged retinas were examined for HIOMT localization by immunocytochemistry, the control retinas displayed intense HIOMT immunoreactivity in all photoreceptors, and a somewhat lighter labeling in a population of bipolar cells, whereas the light damaged retinas (lacking photoreceptors) showed intense HIOMT immunoreactivity in bipolar cells. These results suggest that the increase in HIOMT activity following photoreceptor destruction is due to increased synthesis of this enzyme in a population of bipolar cells. These HIOMT-immunoreactive bipolar cells may perhaps respond in a compensatory manner to changing levels of melatonin in the retina.

Neuroendocrine aspects of cerebral aging.

In their study of the neuroendocrine aspects of cerebral aging the authors review the effect of glucocorticoids, their participation in the mechanisms of neuronal loss and their beneficial and destructive effects. They discuss the treatment strategies for showing the destructive aspects of aging and the effect of acetyl-L-carnitine in rats. They conclude that age-related degeneration of neural tissue is the complex result of multiple factors which synergize to cause neural destruction, including endogenous excitatory amino acids, calcium ions, endogenous proteolytic enzymes, free radicals and circulating glucocorticoids. It is considered that acetyl-L-carnitine may have protective effects in rats.

Effects of acute and chronic stress on the neural retina of young, mid-age, and aged Fischer-344 rats.

Male Fischer-344 rats at 5 (young), 11 (mid-age) and 18 (aged) months of age were exposed either to a single, 1-h period of acute stress, or to daily 4-h periods (chronic) of escapable footshock stress for 6 months, and subsequently allowed a one month interval without stress. The influence of age and exposure to stress on the neural retina was examined by histopathologic and morphometric methods. Age changes in the retina of unstressed control animals included reduction in the thickness of the outer nuclear layer (ONL; photoreceptor nuclei) and of the retina, especially in the peripheral areas. The superior hemisphere was more severely affected than the inferior retina. Exposure to acute stress did not influence retinal histopathology. However, in mid-age and aged rats exposed to chronic stress, the ONL and retinal thicknesses were reduced significantly. Our results indicate for the first time that exposure of rats to chronic stress produces changes in retinal morphology that are associated commonly with aging, such as extensive loss of peripheral photoreceptor cells. In addition, the results show that the effects of chronic stress exposure are greatest in aged rats. The effect of light exposure on the aging retina was not investigated since all rats were exposed to the same total photoperiod.

Gender and chronic stress effects on the neural retina of young and mid-aged Fischer-344 rats.

Young (5 months) and mid-aged (11 months) male and female Fischer rats were exposed to daily (5 days/week) chronic escapable foot-shock stress for 6 months. Following a subsequent 1-month rest period, by which time the animals were 12 and 18 months old, neural retinas were evaluated histopathologically and morphometrically. A significant reduction in the thickness of the retina occurred in the mid-aged, as compared to the young animals. A severe age-related loss in photoreceptor cells, particularly in the peripheral zones of the retina, occurred in a pattern resembling that described for aging humans. The effect of stress was to increase photoreceptor loss in a pattern resembling that of age-related cell loss. Stress-associated photoreceptor cell death was observed in males and females of both ages, but was more pronounced and statistically significant for mid-aged males (a five-fold increase in cell loss over females). The results demonstrated that exposure of rats to chronic escapable foot-shock stress exacerbates retinal changes commonly associated with aging and that the deleterious effects of chronic stress exposure were greater in the older age, male group.

Photically-induced retinal damage in diabetic rats.

The present study examines the interaction of light damage to the retina and streptozotocin (SZ)-induced diabetes in male and female rats during the early development of the disease, when changes occur in the blood-retinal barrier and in pigment cell membranes. Exposure of rats to low illuminance was used to determine the relationship between photically-induced cell death and diabetes. Other groups of animals were exposed to a greater illuminance for shorter time periods (24 hours) in attempts to identify a specific post-treatment day for the effect of diabetes. Blood glucose levels were monitored to indicate the severity of the diabetes. Morphometric analyses and histopathologic observations demonstrated that the outer nuclear layer (ONL, photoreceptor nuclei) was reduced significantly in thickness in female rats exposed to light during a 9 day period after SZ injection, but was unchanged from the control groups when exposed beginning at 12 days after SZ treatment. Removal of the pituitary gland prior to SZ treatment and light exposure resulted in the survival of more photoreceptor cells and prevented the differential in ONL thickness observed between control and diabetic intact animals. Attempts to establish a period of greatest susceptibility of the diabetic retina to photic damage were unsuccessful, but results indicate that prior light history and/or shipment stress might be related to retinal damage from light exposure.

The diurnal susceptibility of rat retinal photoreceptors to light-induced damage.

Exposure of albino rats to high intensity light results in rapid, graded loss of photoreceptors. The hormonal status and age of an animal at the time of exposure affect the severity of light-induced retinal damage. The adrenal axis and pituitary hormones (prolactin) have been demonstrated previously to affect the degree of cell death in the retina. Because circadian rhythms for adrenal and pituitary secretion have been demonstrated in the rat, a series of experiments was undertaken to determine if a diurnal pattern of retinal susceptibility to light damage exists which might be related to endogenous endocrine rhythms. Male Sprague-Dawley rats were exposed to 4 hr of high intensity fluorescent light for 8 consecutive days during different phases of the 14:10 hr light: dark animal room light cycle. Morphometric analysis performed at the light microscopic level 2 weeks after exposure demonstrated a differential susceptibility to light-induced cell death depending upon the period during the light-dark cycle when animals received their daily light exposure. Neuronal cell death was confined to the outer nuclear layer as previously described. The retinas of animals exposed during the middle of the dark period or during the first 5 hr of the light period were significantly more damaged than the retinas of animals exposed during the last 9 hr of the light period. Control groups for the relative amounts of dark-adaptation between groups suggested that the diurnal susceptibility to light damage was not solely dependent upon the degree of dark adaptation. These results demonstrate a diurnal susceptibility of photoreceptors to light-induced cell death.

Neuronal damage in the rat retina after chronic stress.

Long-term exposure to escapable foot shock has been used to determine if chronic stress influences neuronal cell death in the retina of albino and pigmented rats. Histopathologic and morphometric approaches analyzed changes in photoreceptors and neurons of the bipolar and ganglion cell layers of the retina. Albino Fischer rats when exposed to chronic stress for 4-8 h daily for 1 week to 6 months, developed severe retinal damage, as compared to unstressed control retinas, with reduction in photoreceptor and bipolar neurons, particularly in the superior central retina. The damage was observed in male and female rats, but males appeared to be more susceptible to the influence of stress than female animals. Ganglion cells were unaffected. Photoreceptor destruction did not occur in Long-Evans pigmented rats under identical experimental conditions. The results suggest that: input of the sensory stimulus, light, to the retina of stressed rats augmented neuronal damage and might be required for its initiation; and hormones and/or neurotransmitters associated with long-term chronic stress might be related to increased neuronal cell death in the mammalian retina.

Effect of morphine on 3H-thymidine incorporation in the subependyma of the rat: an autoradiographic study.

Following morphine treatment, an autoradiographic study investigated the uptake of 3H-thymidine by the subependymal cells in the rat brain. 3H-thymidine was administered subcutaneously to adult, male Sprague-Dawley rats 30 minutes after saline or morphine (19 mg/kg) injection. The animals were sacrified 1 hour after 3H-thymidine administration. In some experiments the opioid antagonist, naloxone, was given alone 45 minutes before 3H-thymidine or 125 minutes before morphine treatment. Three areas of the subependyma were evaluated in terms of the percentage labeled cells and number of grains per nucleus, and a dorsal-to-ventral gradiant was described. Morphine treatment significantly increased the number of 3H-thymidine labeled subependymal cells and number of grains/nucleus within labeled cells. Examination of the distribution of grains/nucleus showed that morphine-treated animals had significantly more cells labeled with 30 or more grains than did saline-injected controls. Prior administration of naloxone blocked the increased 3H-thymidine uptake in morphine-treated animals but had no significant influence on cell proliferation when administered alone. The data are discussed in terms of morphine's possible dual influence on mechanisms which enhance cell transition from G to S phase and/or which accelerate DNA synthesis once these cells have entered the S phase of cell replication.

Chronologic analysis of variations in retinal damage in two strains of rats after short-term illumination.

In order to determine whether an optimal period existed for evaluating retinal photodamage in rats, a chronologic analysis was made of quantifiable changes in the outer nuclear layer (ONL) and retinal thicknesses in Sprague-Dawley (SD) and Wag/Rij rats after a single, 24 hr exposure to fluorescent light. A gradual reduction in ONL and retinal thicknesses occurred between postexposure days 0 and 10 and appeared to stabilize by day 14 in both strains. The percent difference in both ONL and retinal thickness in unexposed and exposed rats was greater in Wag/Rij than in SD rats. That the superior hemisphere of the retina was more susceptible to photodamage than the inferior was substantiated, and the difference was greater in the Wag/Rij than in SD rats. This finding emphasizes a significant variability between rat strains and is possibly a manifestation of the slowly progressing genetic retinopathy in the Wag/Rij rats. Results indicated that the optimal period for assessing photodamage under these experimental conditions was not sooner than 10 to 14 days after exposure. Quantifiable analyses prior to that time would not provide an accurate evaluation of the total influence of a short-tern exposure period.