Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

A refined method for sequential blood sampling by tail incision in rats

Levels of endogenous or administered substances can be estimated by blood sampling. This allows an evaluation of the relationship between clinical signs, physiological parameters, pharmacological treatments and behaviour of the animal. We show that blood samples can be taken occasionally as well as sequentially by means of a small incision at the end of the rats' tails. Up to 300 microl of blood can be collected within 90 s. The advantages of this method are: (i) anaesthesia and surgery or restraint of the animal are not necessary; (ii) the procedure can be considered stress-free as indicated by the low, basal levels of the stress hormone corticosterone, even with frequent sequential blood sampling over 3 h; and (iii) it can be used for longitudinal studies allowing intra-individual comparisons over months and even years. Blood samples collected via an intravenous catheter and, at the same time, by our tail incision method resulted in comparable amounts of corticosterone. Moreover, we consider the tail incision method for rats to be 'animal-friendly' and a real alternative to other conventionally used blood sampling techniques.     

Water‐borne and plasma corticosterone are not correlated in spotted salamanders

Water‐borne hormone measurement is a noninvasive method suitable for amphibians of all sizes that are otherwise difficult to sample. For this method, containment‐water is assayed for hormones released by the animal. Originally developed in fish, the method has expanded to amphibians, but requires additional species‐specific validations. We wanted to determine physiological relevance of water‐borne corticosterone in spotted salamanders (Ambystoma maculatum) by comparing concentrations to those taken using established corticosterone sampling methods, such as plasma. Using a mixture of field and laboratory studies, we compared water‐borne corticosterone levels to other traditional methods of sampling corticosterone for spotted salamander larvae, metamorphs, and adults. Despite multiple attempts, and detecting differences between age groups, we found no correlations between water‐borne and plasma corticosterone levels in any age group. Water‐borne sampling measures a rate of release; whereas plasma is the concentration circulating in the blood. The unique units of measurement may inherently prevent correlations between the two. These two methods may also require different interpretations of the data and the physiological meaning. We also note caveats with the method, including how to account for differences in body size and life history stages. Collectively, our results illustrate the importance of careful validation of water‐borne hormone levels in each species in order to understand its physiological significance.     

Comparative analysis of ACTH and corticosterone sampling methods in rats

A frequently debated question for studies involving the measurement of stress hormones in rodents is the optimal method for collecting blood with minimal stress to the animal. Some investigators prefer the implantation of indwelling catheters to allow for frequent sampling. Others argue that the implantation of a catheter creates a chronic stress to the animal that confounds stress hormone measures and therefore rely on tail vein sampling. Moreover, some investigators measure hormones in trunk blood samples obtained after anesthesia, a practice that may itself raise hormone levels. To address these controversies, we 1) compared plasma ACTH and corticosterone (Cort) concentrations in pre- and poststress rat blood samples obtained via previously implanted vena cava catheters, tail vein nicks, or clipping the tip off the tail and 2) compared plasma ACTH and Cort in rat blood samples obtained by decapitation with and without anesthesia. Rats sampled via indwelling catheters displayed lower prestress ACTH levels than those sampled by tail vein nick if the time to acquire samples was not limited; however, elevated basal ACTH was not observed in samples obtained by tail clip or tail nick when the samples were obtained within 3 min. Baseline Cort levels were similar in all groups. After restraint stress, the profile of the plasma ACTH and Cort responses was not affected by sampling method. Decapitation with prior administration of CO2 or pentobarbital sodium increased plasma ACTH levels approximately 13- and 2-fold, respectively, when compared with decapitation without anesthesia. These data indicate that tail vein nicking, tail clipping, or indwelling venous catheters can be used for obtaining plasma for ACTH and Cort during acute stress studies without confounding the measurements. However, the elevation in basal ACTH seen in the tail vein nick group at baseline suggests that sampling needs to be completed rapidly (<3 min) to avoid the initiation of the pituitary stress response. Death by CO2 and pentobarbital sodium injection before trunk blood collection cause significant stress to animals, as reflected in the elevated plasma ACTH levels. These results support the use of either chronic vascular cannulas or sampling from a tail vein. However, collection of blood under pentobarbital sodium or CO2 anesthesia is likely to confound the results of stress studies when ACTH is an important endpoint.     

Blood sampling by puncture in the cavernous sinus from hunted wild boar

A new method of sampling based on the extraction of blood from the cavernous sinus of the dura mater has been assessed in hunted wild boar. Blood from 139 animals was obtained by two different extraction methods: the harvesting from thoracic cavity (TC) and intracavernous venipuncture (IV). Sera obtained by the IV method had higher volume (mean 2.85 vs 1.85 ml), were less hemolytic (mean absorbance at 450 nm: 1.01 vs 2.41 nm). A higher number of samples and a higher proportion of sera collected by IV (90.6 %) compared to those obtained using the TC method (78.4 %), could be analyzed against Aujeszky’s disease using blocking ELISA. No statistically significant differences in seroprevalences between samples obtained using both extraction methods were observed. The results obtained indicate that the IV is an easy, fast, reliable, clean, and safe method to collect blood samples from hunted wild boar, proving a real alternative to the traditional collection method.     

Gamma amino butyric acid (GABA) levels in hypophyseal stalk plasma of rats

To determine the possible physiologic contribution of GABA to the tonic hypothalamic inhibition of adenohypophyseal prolactin secretion, we compared GABA levels in hypophyseal stalk plasma with those found in the peripheral circulation. Hypophyseal stalk blood was collected $\text{via}$ a parapharyngeal approach from 8 urethane anesthetized diestrus rats. Peripheral blood was collected simultaneously from the external jugular vein of the same rat at a rate similar to hypophyseal stalk blood flow. Blood samples resulting from a single 4 hr collection per animal were centrifuged, and the plasma stored frozen before ethanol extraction and assay using a radioreceptor method. GABA levels in hypophyseal stalk plasma ($\text{909±171}\text{pmol/ml}\text{;}\text{X}\text{±}\text{S.E.M.}$) were not significantly higher than levels in peripheral plasma (845±182; p>0.05), indicating little or no secretion of GABA by the median eminence.     

Adrenal splanchnic innervation contributes to the diurnal rhythm of plasma corticosterone in rats by modulating adrenal sensitivity to ACTH

Activity of the hypothalamic-pituitary-adrenal axis is characterized by a diurnal rhythm with an AM nadir and PM peak. Splanchnic nerve transection disrupts the diurnal rhythm in plasma corticosterone; however, there is a controversy as to whether the nerve-mediated effect is 1) via inhibition in the AM vs. excitation in the PM, or 2) involves changes in adrenal sensitivity to ACTH. The present studies were designed to address these issues. Adult male rats were anesthetized and underwent bilateral transection of the thoracic splanchnic nerve or sham transection. One week after surgery, rats were killed in the AM or PM with collection of nonstress plasma for measurement of corticosterone and ACTH. Plasma corticosterone was increased in the PM relative to the AM; however, plasma corticosterone in the PM was attenuated by splanchnic nerve transection, without affecting plasma ACTH. This decrease in PM plasma corticosterone after nerve-transection was 1) associated with decreased adrenal responsivity to ACTH, 2) associated with decreased adrenal cAMP content, 3) prevented by adrenal demedullation, and 4) not affected by removal of adrenal capsaicin-sensitive afferent fibers. Repeated serial blood sampling from individual rats confirmed the excitatory effect of splanchnic innervation in the PM. These results support the hypothesis that the adrenal splanchnic innervation modulates the diurnal rhythm in plasma corticosterone by increasing adrenal responsivity to ACTH and augmenting steroidogenesis in the PM and suggest that alterations in adrenal corticosterone secretion obscured by pulsatile secretion are more clearly revealed with repeated serial blood sampling.     

A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm

Plasma corticosterone (CORT) measures are a common procedure to detect stress responses in rodents. However, the procedure is invasive and can influence CORT levels, making it less than ideal for monitoring CORT circadian rhythms. In the current paper, we examined the applicability of a non-invasive fecal CORT metabolite measure to assess the circadian rhythm. We compared fecal CORT metabolite levels to circulating CORT levels, and analyzed change in the fecal circadian rhythm following an acute stressor (i.e. blood sampling by tail veil catheter). Fecal and blood samples were collected from male adolescent rats and analyzed for CORT metabolites and circulating CORT respectively. Fecal samples were collected hourly for 24 h before and after blood draw. On average, peak fecal CORT metabolite values occurred 7-9 h after the plasma CORT peak and time-matched fecal CORT values were well correlated with plasma CORT. As a result of the rapid blood draw, fecal production and CORT levels were altered the next day. These results indicate fecal CORT metabolite measures can be used to assess conditions that disrupt the circadian CORT rhythm, and provide a method to measure long-term changes in CORT production. This can benefit research that requires long-term glucocorticoid assessment (e.g. stress mechanisms underlying health).     

Capillary blood sampling as an alternative to venipuncture in the assessment of serum 25 hydroxyvitamin D levels

This study compared 25-hydroxyvitamin D [25(OH)D] measurements in capillary and venous blood samples collected, respectively by fingerprick and venipuncture. Capillary blood for measuring 25(OH)D has potential advantages by reducing blood volume required (2mL versus 0.3mL for venipuncture and capillary sampling, respectively), facilitating blood collection for those populations in whom venipuncture is difficult (e.g. infants and children), improving patient convenience and reducing costs associated with phlebotomy. The results demonstrated a highly significant relationship between 25(OH)D levels in serum derived from venous and capillary blood samples (r(2)=0.901). Despite statistically higher 25(OH)D levels in fingerprick samples (108+/-9nmol/L) compared with venipuncture samples (90+/-7nmol/L), the correlation between venous and capillary samples provides support for this approach as a practical alternative to venipuncture for vitamin D determination. However, clinical application may require the incorporation of a correction factor for the assessment of insufficiency, and research studies should avoid using the two methods interchangeably. Studying vitamin D's role in health and disease requires collection techniques and measurement methods that are reliable, reproducible, easily accessible, inexpensive and minimally burdensome to the patient. The option to collect patient samples by fingerprick may facilitate the collection process.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Blood Substrate Collection and Handling Procedures under Pseudo-Field Conditions: Evaluation of Suitability for Inflammatory Biomarker Measurement

Routine incorporation of blood-based biomarker measurements in population studies has been hampered by challenges in obtaining samples suitable for biomarker assessment outside of laboratory settings. Here, we assessed the suitability of venous blood left unprocessed for 4, 24, or 48 hours post-collection at either room temperature or 4°C for quantification of two biomarkers, Interleukin-6 (IL-6) and C-reactive protein (CRP). Blood samples were collected in both K₂EDTA tubes and a dedicated plasma-preservation tube, P100. Dried blood spot (DBS) samples from the same subjects were also collected in order to compare delayed-processing plasma performance against a popular alternative collection method. We found that K₂EDTA mean plasma concentrations of both IL-6 and CRP were not significantly different from concentrations in plasma processed immediately; this was observed for tubes stored up to 48 hours pre-processing at either temperature. Concentrations of IL-6 measured in P100 tubes showed significant time-dependent increases when stored at room temperature; otherwise, levels of IL-6 and CRP were similar to those found in samples processed immediately. Levels of CRP in DBS were correlated with plasma CRP levels, even when pre-processed blood was stored for up to 48 hours. These data indicate that plasma is suitable for IL-6 and CRP estimation under data collection conditions that involve processing delays.     

Effects of chronic stress on nicotine-induced locomotor activity and corticosterone release in adult and adolescent rats.

We examined nicotine-induced locomotion and increase in corticosterone plasma levels in adolescent and adult animals exposed to chronic restraint stress. Adolescent [postnatal day (P) 28-37] and adult (P60-67) rats were restrained for 2 hours once daily for 7 days. Three days after the last exposure to stress, the animals were challenged with saline or nicotine (0.4 mg/kg subcutaneously). Nicotine-induced locomotion was recorded in an activity cage. Trunk blood samples were collected in a subset of adolescent and adult rats and plasma corticosterone levels were determined by radioimmunoassay. Exposure to stress did not affect the nicotine-induced locomotor- or corticosterone-activating effects in both ages.     

Episodic corticosterone secretion in the female rat

Studies conducted in several laboratories have shown primate glucocorticoid secretion to occur episodically. In light of the methodological, as well as physiological importance of this finding, the present study was undertaken to determine whether the rat corticosteroid secretion also occurs episodically. Female rats were outfitted with chronic intravenous cannulas, and 1 week later 200 units of heparin were injected through the implanted cannula and blood samples (0.3-0.4 ml) were collected from each rat every 10 min for 3 h during the morning (06.00-10.00 h) or during the afternoon (16.00-19.00 h) (lights on from 05.00 to 19.00 h). Plasma corticosterone levels in cannulated rats showed fluctuations indicative of episodic secretion. The pattern of plasma corticosterone levels was characterized by periodic rapid increases in hormone concentration during both the morning and afternoon sampling periods; the occurrence of these hormonal fluctuations did not have a characteristic frequency. When the data were grouped to obtain single morning and afternoon values, the AM-PM difference was significant (p < 0.005). Collectively, these data suggest that in the rat, adrenal corticosteroids are secreted episodically.     

Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [²H₄]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.     

Effect of bupropion on hippocampal neurotransmitters and on peripheral hormonal concentrations in the rat.

The purpose of the present study was to administer an acute dose of the dual dopamine norepinephrine reuptake blocker bupropion in freely moving rats and to monitor the extracellular neurotransmitter concentrations in the hippocampus via in vivo microdialysis and the peripheral hormonal concentrations via catheterization. A microdialysis probe was inserted in the hippocampus, and samples for serotonin, dopamine, and norepinephrine were collected every 20 min before and after the injection of 17 mg/kg of bupropion, for a total sampling time of 180 min. A catheter was placed in the vena femoralis of the second group of rats, and blood samples were collected before and after bupropion injection for quantification of growth hormone, prolactin, corticosterone, adrenocorticotropin hormone, and beta-endorphins. All neurotransmitter levels (dopamine, norepinephrine, and serotonin) significantly increased after bupropion injection. This was accompanied by a significant decrease in prolactin concentrations, whereas the other hormones showed no statistically significant variation. It can, therefore, be concluded that, although bupropion has dual reuptake proprieties, the observed effects both at the central and at the peripheral level seem to be ruled by the dopaminergic system.     

Reevaluation of immunoreactive gonadotropin-releasing hormone (GnRH) levels in general circulation in women: changes in levels and episodic patterns before, during and after gonadotropin surges

In order to reevaluate the earlier varying data regarding circulatory gonadotropin-releasing hormone (GnRH), we assayed extracted GnRH from the plasma frequently collected at mid-cycle in 11 women. For the analysis of episodic GnRH patterns and basal levels, blood samples were obtained at 6 h intervals for 72 h and at 15 min intervals for 2 h every 12 h throughout the experimental period. All blood samples were assayed for GnRH and selected samples for LH, FSH, estradiol and progesterone. For GnRH assay, 5 or 6 ml of blood was mixed with 60 mg of ethylenediaminetetraacetic acid, disodium salt, and 3 mg of phenylmethylsulfonyl floride immediately after blood collection. These enzyme inhibitors prevented the destruction of GnRH in the blood at room temperature for at least 4 h. Plasma GnRH was extracted through several steps including florisil absorption, acidic extraction and washing with organic solvent. Nonspecific immunoreactivity in the plasma was markedly decreased through this extraction process. Our assay values (approximate range, 0.1-2.0 pg/ml) of plasma GnRH in normal women corresponded to the low range of those obtained by others who used the alcohol extraction method. The basal levels of GnRH did not change significantly throughout 3 different periods, i.e., before, during and after the LH surges, and fluctuated between a small range of 0.11 and 1.44 pg/ml. Although the peak levels of GnRH observed in its episodic patterns did not change between the periods before and during the LH surges, they decreased significantly after the LH surge compared with those seen during the LH surges (0.93 +/- 0.07 vs 1.17 +/- 0.09 pg/ml, p less than 0.05). The present data demonstrate that immunoreactive GnRH in the extracted peripheral plasma does not change significantly in its mean, basal and peak levels during the periovulatory period except for a minor but significant decrease in the peak levels shortly after an LH surge.     

Steroid concentrations in plasma, whole blood and brain: effects of saline perfusion to remove blood contamination from brain

The brain and other organs locally synthesize steroids. Local synthesis is suggested when steroid levels are higher in tissue than in the circulation. However, measurement of both circulating and tissue steroid levels are subject to methodological considerations. For example, plasma samples are commonly used to estimate circulating steroid levels in whole blood, but steroid levels in plasma and whole blood could differ. In addition, tissue steroid measurements might be affected by blood contamination, which can be addressed experimentally by using saline perfusion to remove blood. In Study 1, we measured corticosterone and testosterone (T) levels in zebra finch (Taeniopygia guttata) plasma, whole blood, and red blood cells (RBC). We also compared corticosterone in plasma, whole blood, and RBC at baseline and after 60 min restraint stress. In Study 2, we quantified corticosterone, dehydroepiandrosterone (DHEA), T, and 17β-estradiol (E₂) levels in the brains of sham-perfused or saline-perfused subjects. In Study 1, corticosterone and T concentrations were highest in plasma, significantly lower in whole blood, and lowest in RBC. In Study 2, saline perfusion unexpectedly increased corticosterone levels in the rostral telencephalon but not other regions. In contrast, saline perfusion decreased DHEA levels in caudal telencephalon and diencephalon. Saline perfusion also increased E₂ levels in caudal telencephalon. In summary, when comparing local and systemic steroid levels, the inclusion of whole blood samples should prove useful. Moreover, blood contamination has little or no effect on measurement of brain steroid levels, suggesting that saline perfusion is not necessary prior to brain collection. Indeed, saline perfusion itself may elevate and lower steroid concentrations in a rapid, region-specific manner.     

Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals'' physiological condition, which should be considered whenever blood samples are obtained.     

Effect of aging on corticosterone secretion in diestrous rats

The roles of age and prolactin (PRL) in regulating glucocorticoid secretion in diestrous rats were investigated. Adrenal zona fasciculata-reticularis (ZFR) cells from young, adult, middle (mid)-aged, and old female rats were isolated. Estrous cycle stage was determined by light microscopy after vaginal smears. Blood samples were collected from right jugular vein at 0, 30, 60, and 120 min after challenge with adrenocorticotropin (ACTH). During the diestrous phase, plasma levels of estradiol and progesterone were lower in mid-aged and old rats than in either young or adult rats. Age-dependent increases of the basal levels of plasma PRL and corticosterone were observed. No difference of ACTH-increased plasma concentrations of corticosterone was observed among young, adult, mid-aged, and old rats. Aging increased the basal, ACTH-, PRL-, forskolin (an adenylate cyclase activator)-, and 3-isobutyl-l-methylxanthine (IBMX, a non-selective phosphodiesterase inhibitor)-stimulated release of corticosterone and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. However, the 8-Br-cAMP (a membrane-permeable cAMP)-stimulated release of corticosterone was not affected by age. Taken together, these data indicated that aging increased corticosterone secretion in female rats during diestrous phase, which is in part due to an increase in cAMP accumulation. In conclusion, aging and PRL play a stimulatory role in the co-regulation of corticosterone secretion.     

Stress-induced decrease of the afternoon prolactin surge the influence of the adrenal gland

Blood sampling initiated during the afternoon in ovariectomized, estrogen-treated rats resulted in a suppression of the prolactin (PRL) surge. The administration of 2.5 μg or 25 μg of corticosterone (B) immediately after the first blood sample did not alter the course of the PRL suppression. Plasma B levels of adrenal intact controls were not acutely by blood sampling and the normal afternoon increase was evident. Similar treatment of adrenalectomized rats resulted in a similar pattern of PRL suppression. The data indicated that adrenal secretions were not responsible for the decrease in plasma PRL induced by blood sampling initiated in the afternoon.