Lovisa Holm

Different methods for administering 17β-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage

BackgroundNumerous stroke studies have controversially shown estrogens to be either neuroprotective or neurodamaging. The discordant results observed in rat brain ischemia models may be a consequence of discrepancies in estrogen administration modes resulting in plasma concentration profiles far from those intended. To test this hypothesis we reproduced in detail and extended an earlier study from our lab using a different mode of 17β-estradiol administration; home-made silastic capsules instead of commercial slow-release 17β-estradiol pellets. Four groups of female rats (n = 12) were ovariectomized and administered 17β-estradiol or placebo via silastic capsules. All animals underwent MCAo fourteen days after ovariectomy and were sacrificed three days later.ResultsIn contrast to our earlier results using the commercial pellets, the group receiving 17β-estradiol during the entire experiment had significantly smaller lesions than the group receiving placebo (mean ± SEM: 3.85 ± 0.70% versus 7.15 ± 0.27% of total slice area, respectively; p = 0.015). No significant neuroprotection was found when the 17β-estradiol was administered only during the two weeks before or the three days immediately after MCAo.ConclusionsThe results indicate that different estrogen treatment regimens result in diametrically different effects on cerebral ischemia. Thus the effects of estrogens on ischemic damage seem to be concentration-related, with a biphasic, or even more complex, dose-response relation. These findings have implications for the design of animal experiments and also have a bearing on the estrogen doses used for peri-menopausal hormone replacement therapy.

Estrogen induces a rapid increase in galanin levels in female rat hippocampal formation − possibly a nongenomic/indirect effect

Administration of 17β‐estradiol to ovariectomized rats increased the concentrations of galanin‐like immunoreactivity (LI) in the hippocampal formation by 215% (P < 0.001) within 1 h. An increase of 125% (P < 0.05) was observed in the same brain region in the proestrous phase of a normal estrous cycle. Tamoxifen® did not block the 17β‐estradiol‐induced increase in the concentration of galanin‐LI but resulted in a 62% decrease in the hypothalamus within 1 h. In vivo microdialysis in the dorsal hippocampal formation showed a decrease of extracellular galanin‐LI (P < 0.001) 1−2 h after treatment with 17β‐estradiol, indicating a decreased release of galanin. For comparision, we studied the concentrations of neuropeptide Y, which were not influenced significantly in any of the regions studied. Taken together our results suggest that 17β‐estradiol inhibits galanin release, presumably from noradrenergic nerve terminals, and primarily via a nongenomic/indirect action, not necessarily involving the classical nuclear receptors ER‐α or ER‐β. These rapid estrogen‐induced changes in galanin release could influence transmitter signalling and plasticity in the hippocampal formation.

Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain

The effects of hypothermia on galanin concentrations and the relation between ischemic brain lesions, hypothermia and galanin concentrations in a transient and focal rat stroke model were investigated in order to elucidate whether hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective effect of hypothermia. Female rats were allocated to normothermia (37 degrees C) or hypothermia (33 degrees C) treatments during a 60 min microclip middle cerebral artery occlusion. The ischemic lesions were visualized after observation periods of 2 or 7 days and the concentration of galanin measured by radioimmunoassay in extracts of punch biopsies from both the lesioned and the contralateral control hemisphere. Hypothermia-induced an overall increase in the concentrations of immunoreactive galanin (p<0.001). The elevated galanin levels were predominantly found in the non-ischemic control hemisphere, in the hippocampus, thalamus and the posterior part of parietal cortex. The galanin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contra lateral intact hemisphere (p=0.049). The factor of time, 2 respectively 7 days, did not show any significant difference regarding the galanin concentrations (p=0.844). Multivariate analyses of variance revealed significant effect of ischemia on the size of the ischemic brain lesions (p=0.001) but no overall effect of temperature when data from both 2 and 7 days observation periods were analyzed together. The ischemic lesions were generally larger at 33 degrees after 2 days (p=0.230). Prolonged observation time of 7 days resulted in a significant reduction of the ischemic brain lesion (p=0.011) with smaller ischemic lesions in the hypothermic group. Our data support the notion that hypothermia-induced increase in the tissue concentrations of galanin in the brain are the result of changes from optimal homeostatic conditions - the hypothermia-induced stress - rather than the ischemia/re-perfusion lesion induced changes in galanin concentrations. Hypothermia-induced elevation in galanin concentration is therefore not likely to be amongst the major protective mechanisms of hypothermia.

Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats

Several studies have shown that injury to the central and peripheral nervous system can increase expression of galanin, a 29 amino acid neuropeptide. Moreover, there is evidence that galanin, especially through its galanin receptor 2 (GalR2) receptor, plays a neuroprotective role in different injury models. However, direct studies of a possible neuroprotective effect of galanin in experimental stroke models are lacking. Galanin, a GalR2/3 agonist or artificial CSF was continuously infused intracerebroventricularly (i.c.v.) in naïve female rats after a 60min transient and focal occlusion of the middle cerebral artery. The animals were sacrificed, and the ischemic lesion was visualized using 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The lesion was 98% larger after i.c.v. administration of the GalR2/3 agonist (2.4nmol/day) seven days after occlusion compared to artificial CSF (p=0.023). No statistically significant differences were found after seven days in the groups treated with galanin in three different concentrations (0.24, 2.4 and 24nmol/day; p=0.939, 0.715 and 0.977, respectively). There was no difference in the size of the ischemic lesions measured after three days in the galanin-treated group (2.4nmol/d) compared to artificial CSF (p=0.925). The present results show, surprisingly, that a GalR2/3 agonist doubled the size of the ischemic lesion. Whether this effect primarily reflects the properties of the current model, species, gender and/or the mode of galanin administration, e.g. causing desensitization, or whether galanin indeed lacks neuroprotective effect of its own, remains to be corroborated.

Rapid change of neuropeptide Y levels and gene-expression in the brain of ovariectomized mice after administration of 17β-estradiol

Estrogen alters excitability and changes synaptic morphology in the rat hippocampal formation. We have compared, by means of radioimmunoassay and in situ hybridization, the effects of short-term treatment with 17beta-estradiol on neuropeptide Y (NPY) in the brain of ovariectomized mice. A highly significant reduction in concentrations of NPY-like immunoreactivity (LI) was observed in the hippocampal formation, some cortical areas and the caudate nucleus 1h after administration of 17beta-estradiol as compared to the control group. In contrast, NPY transcript levels increased in the hippocampal formation (dentate gyrus) and the caudate nucleus, possibly representing a compensatory increase of NPY synthesis following increased estradiol-induced NPY release. These data suggest that 17beta-estradiol, via membrane-related mechanisms, increases NPY release and synthesis in forebrain areas involved in cognition, mood and motor functions.

Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats

Injury to neurons results in up-regulation of galanin in some central and peripheral systems, and it has been suggested that this neuropeptide may play a protective and trophic role, primarily mediated by galanin receptor 2 (GalR2). The objective of the present study was to investigate galanin, GalR1, GalR2 and GalR3 gene expression in the female rat brain 7 days after a 60-min unilateral occlusion of the middle cerebral artery followed by reperfusion. Quantitative real-time PCR was employed in punch-biopsies from the locus coeruleus, somatosensory cortex and dorsal hippocampal formation, including sham-operated rats as controls. Galanin gene expression showed a ∼2.5-fold increase and GalR1 a ∼1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side. Furthermore, the GalR1 mRNA levels decreased by 35% in somatosensory cortex of the ischemic hemisphere. Immunohistochemical analysis indicated a depletion of galanin from cell bodies and dendrites in the locus coeruleus after middle cerebral artery occlusion. The present results suggest that a stroke-induced forebrain lesion up-regulates synthesis of galanin and GalR1 in the locus coeruleus, a noradrenergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation. These results support the notion that galanin may play a role in the response of the central nervous system to injury.

Preclinical evaluation of the kappa-opioid receptor antagonist CERC-501 as a candidate therapeutic for alcohol use disorders

Prior work suggests a role of kappa-opioid signaling in the control of alcohol drinking, in particular when drinking is escalated due to alcohol-induced long-term neuroadaptations. Here, we examined the small molecule selective kappa antagonist CERC-501 in rat models of alcohol-related behaviors, with the objective to evaluate its potential as a candidate therapeutic for alcohol use disorders. We first tested the effect of CERC-501 on acute alcohol withdrawal-induced anxiety-like behavior. CERC-501 was then tested on basal as well as escalated alcohol self-administration induced by 20% alcohol intermittent access. Finally, we determined the effects of CERC-501 on relapse to alcohol seeking triggered by both stress and alcohol-associated cues. Control experiments were performed to confirm the specificity of CERC-501 effects on alcohol-related behaviors. CERC-501 reversed anxiety-like behavior induced by alcohol withdrawal. It did not affect basal alcohol self-administration but did dose-dependently suppress self-administration that had escalated following long-term intermittent access to alcohol. CERC-501 blocked relapse to alcohol seeking induced by stress, but not when relapse-like behavior was triggered by alcohol-associated cues. The effects of CERC-501 were observed in the absence of sedative side effects and were not due to effects on alcohol metabolism. Thus, in a broad battery of preclinical alcohol models, CERC-501 has an activity profile characteristic of anti-stress compounds. Combined with its demonstrated preclinical and clinical safety profile, these data support clinical development of CERC-501 for alcohol use disorders, in particular for patients with negatively reinforced, stress-driven alcohol seeking and use.

Acute effects on brain cholecystokinin-like concentration and anxiety-like behaviour in the female rat upon a single injection of 17β-estradiol

BACKGROUND The neuropeptide cholecystokinin (CCK) has been implicated in the neurobiology of anxiety and panic disorders, as well as in dopamine-related behaviours. Anxiety and panic-disorders are twice as common in females compared to males, but studies of females are rare, although increasing in number. Limited studies have found that CCK fluctuates in limbic regions during the estrous cycle, and that CCK and its receptors are sensitive to estrogen. AIM/PURPOSE The aim of the present work was to study the acute effects of 17β-estradiol on anxiety-like behaviour and on CCK-like immunoreactivity (LI) in the female rat brain (amygdala, hippocampus, nucleus accumbens, and cingulate cortex). METHODS Four groups of female Sprague-Dawley rats were used: ovariectomized, ovariectomized+17β-estradiol-replacement, sham, and sham+17β-estradiol-replacement. The effect of 17β-estradiol-replacement on anxiety-related behaviour was measured in all animals on the elevated plus maze 2-24 h after injection. CCK-LI concentration was measured in punch biopsies by means of radioimmunoassay. RESULTS 17β-estradiol decreased anxiety-like behaviour 2 h after administration in ovariectomized and sham-operated animals, as demonstrated by increased exploration of the open arms compared to respective sesame oil-treated controls. This effect was not present when testing occurred 24 h post-treatment. The rapid behavioural effect of 17β-estradiol was accompanied by changes in CCK-LI concentrations in regions of the limbic system including cingulate cortex, hippocampus, amygdala and nucleus accumbens. CONCLUSION Although the interpretation of these data requires caution since the data were collected from two different experiments, our results suggest that estrogen-induced anxiolytic effects may be associated with changes of the CCK-system in brain regions controlling anxiety-like behaviour.