Evidence of lasting dysregulation of neuroendocrine and HPA axis function following global cerebral ischemia in male rats and the effect of Antalarmin on plasma corticosterone level.

Abnormal function of the neuroendocrine stress system has been implicated in the behavioral impairments observed following brain ischemia. The current study examined long-term changes in stress signal regulation 30days following global cerebral ischemia. Experiment 1 investigated changes in the expression of corticotropin releasing hormone (CRH) and its subtype 1 receptor (CRHR1), glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala (CeA), and the CA1 subfield of the hippocampus. Tyrosine hydroxylase (TH) was determined at the locus coeruleus (LC). Experiment 2 investigated the role of central CRHR1 activation on corticosterone (CORT) secretion at multiple time intervals following global ischemia after exposure to an acute stressor. Findings from Experiment 1 demonstrated a persistent increase in GR, CRH and CRHR1 immunoreactivity (ir) at the PVN, reduced GR and CRHR1 expression in pyramidal CA1 neurons, and increased LC TH expression in ischemic rats displaying working memory errors in the radial arm Maze. Findings from Experiment 2 revealed increased CORT secretion up to 7 days, but no longer present 14 and 21 days post ischemia. However upon an acute restraint stress induced 27 days following reperfusion, ischemic rats had increased plasma CORT secretions compared to sham-operated animals, suggesting HPA axis hypersensitivity. Antalarmin (2 µg/2 µl) pretreatment significantly attenuated post ischemic elevation of basal and stress-induced CORT secretion. These findings support persistent neuroendocrine dysfunctions following brain ischemia likely to contribute to emotional and cognitive impairments observed in survivors of cardiac arrest and stroke.

A refined blood collection method for quantifying corticosterone.

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.

Changes in HPA reactivity and noradrenergic functions regulate spatial memory impairments at delayed time intervals following cerebral ischemia.

This study investigates the association of ischemia-induced spatial memory impairment to alterations of the HPA axis and noradrenergic activation post insult. Experiment 1 characterized the effects of 10 min forebrain ischemia on corticosterone (CORT) secretion following ischemia and in response to spatial memory assessment in the Barnes maze, as well as the impact of pre-ischemia treatment with the glucocorticoid inhibitor metyrapone (175 mg/kg; s.c.). The results showed that cerebral ischemia represents a significant physiological stressor that upregulated CORT secretion 1, 24 and 72 h post-ischemia but not at 7 days. In response to testing in the Barnes maze ischemic animals showed elevated CORT secretion simultaneously with spatial memory deficits. The single dose of metyrapone attenuated the ischemia-induced adrenocortical hyper-responsiveness and subsequent memory deficits despite not providing neuroprotection in the hippocampal CA1 pyramidal cells. To complement these findings, we examined whether norepinephrine which provides positive feedback to the HPA axis and is upregulated following brain ischemia could influence memory performance at delayed intervals after ischemia. Experiment 2 demonstrated that pre-testing administration of the alpha2-adrenoceptor agonist clonidine (.04 mg/kg, s.c.) attenuated ischemia-induced working memory impairments in a radial maze while opposite effects were obtained with the antagonist yohimbine (.3 mg/kg, s.c.). Post-testing administration of clonidine produced spatial reference memory impairments in ischemic rats. The findings from the current study demonstrate increased sensitization and responsiveness of systems regulating stress hormones at long intervals post ischemia. Importantly, we demonstrate that these effects contribute to post ischemic cognitive impairments which can be attenuated pharmacologically even in the presence of hippocampal degeneration at time of testing.

How right is the righting reflex? The risk of false positives in neuroprotection studies using behavioral measures to certify forebrain ischemia.

The righting reflex (RR) as a behavioral index of incomplete ischemia during four-vessel occlusion (4VO) remains a common exclusion criterion. In the present study different drugs (aniracetam, ondansetron, and metyrapone) were administered to Wistar rats at a variety of doses prior to unanesthetized forebrain ischemia lasting 10 minutes forebrain ischemia in. Using the RR as an exclusion criterion, their effects on neuronal survival and functional recovery were investigated. Our observations revealed dose-related suppression of RR expression leading to a higher proportion of drug-treated rats categorized as having experienced successful ischemia compared to the vehicle-treated groups. If uncontrolled, this effect led to assessments of increased neuronal survival (in CA1 hippocampal subfield) or improved cognitive behavioral recovery in treated rats. These observations appeared to be related to failure in identifying rats subjected to incomplete forebrain ischemia producing minimal neuronal damage rather than protective effects associated to drug administration. These findings highlight the risk of falsely reporting treatment-related neuronal protection with the unanesthetized 4VO model in rodents.

Time-dependent effects of global cerebral ischemia on anxiety, locomotion, and habituation in rats.

Although changes in emotionality represent common features of post-ischemic recovery in humans, little is known about the effects of global cerebral ischemia on standard behavioral measures of emotionality in rodents. The present study investigated anxiety, locomotor activity, and habituation in test-naïve ischemic (subjected to 10 min global ischemia) and sham-operated rats tested 1, 5, 15, and 30 days post-reperfusion in the elevated plus-maze and the open-field. Although rats tested on day 1 post-reperfusion showed increased anxiety relative to sham-operated controls, they demonstrated decreased anxiety on day 5. Anxiety levels were normal on days 15 and 30 following ischemia. Similarly, time-dependent changes in locomotor activity were observed with ischemic rats showing increased activity level on days 1, 5, and 30 post-reperfusion. Surprisingly, locomotor activity was suppressed at day 15. Habituation deficits in the open-field were apparent only on day 1 despite the lack of CA1 neuronal degeneration at this time interval. These findings suggest that both the nature and extent of the effects of global ischemia on behavioral measures of emotionality, locomotion, and habituation in rats are time-dependent.