Long-term cerebral cortex protection and behavioral stabilization by gonadal steroid hormones after transient focal hypoxia

doi:10.1016/j.jsbmb.2012.01.007

The Journal of Steroid Biochemistry and Molecular Biology

Volume 131, Issues 1–2, August 2012, Pages 10-16

https://doi.org/10.1016/j.jsbmb.2012.01.007 Get rights and content

Abstract

Sex steroids are neuroprotective following traumatic brain injury or during neurodegenerative processes. In a recent short-term study, we have shown that 17β-estradiol (E) and progesterone (P) applied directly after ischemia reduced the infarct volume by more than 70%. This protection might primarily result from the anti-inflammatory effects of steroids. Here, we focus on the long-term neuroprotection by both steroids with respect to the infarct volume, functional recovery, and vessel density in the penumbra. The application of E/P during the first 48 h after stroke (transient middle cerebral artery occlusion, tMCAO) revealed neuroprotection after two weeks. The infarct area was reduced by 70% and motor activity was preserved compared to placebo-treated animals. Blood vessel density in the penumbra using immunohistochemistry for von Willebrand factor showed increased vessel density after tMCAO which was not affected by hormones. Expression of vascular endothelial growth factor (VEGF) and its receptor (R1) was increased at 24 h after tMCAO and up-regulated by E/P but not changed 14 days after stroke. These findings suggest that the neuroprotective potency of both steroids is sustained and persists for at least two weeks. Besides anti-inflammatory and anti-apoptotic actions, angiogenesis in the damaged area appears to be initially affected early after ischemia and is manifested up to two weeks.

This article is part of a Special Issue entitled ‘Neurosteroids’.

Introduction

The gonadal steroid hormones 17β-estradiol (E) and progesterone (P) limit neural damage and improve functional outcome after different types of brain damages including traumatic brain injury and focal ischemia [1], [2]. The administration of E and P, single or in combination, immediately before or after transient focal cerebral ischemia decreases the infarct volume and ameliorates stroke-related behavioral deficits in both sexes [3], [4], [5]. Several studies have contributed to a better understanding of the protective mechanisms and the related cellular and molecular cascades controlled by both steroids. Defensive steroid hormone actions include stabilizing the blood-brain-barrier (BBB), alleviating brain edema, dampening pro-inflammatory processes, activating anti-apoptotic molecules, stimulating survival-promoting factors, counteracting oxidative stress, promoting respiratory chain function, and reducing glutamate excitotoxicity [1], [3], [6], [7], [8], [9]. Genomic- and non-genomic steroid signaling pathways appear to be implicated in mediating such effects side by side and additive [10], [11].

Irrespective of the large variety of possible mechanisms which might contribute to steroid-dependent tissue protection, another aspect needs to be highlighted. Most of the above mechanisms such as decreased central inflammation and oxidative stress represent fast cellular actions which might directly rescue damaged neurons during the early acute oxidative and energetic challenges created by reduced or suppressed oxygen and glucose supply. Since many stroke studies have been performed under a short-term protection paradigm, it is not fully clear to which extent these activated individual pathways contribute to an improved functional long-term outcome. Moreover, long-term protection studies were often performed using repeated steroid applications for up to several weeks, and it also turned out that the method of administration and dosage is causally linked to the study outcome [12]. We assume that the multitude of beneficial steroid effects act in concert to achieve sufficient neuroprotection but are aware that each hormone also transmits selective cellular actions. In addition to the above mentioned neuroprotective mechanisms, angiogenesis, the growth of new blood vessels, is an essential prerequisite for optimal long-term recovery and protection of the neurovascular unit after ischemic injury. This is supported by data showing that the above steroids increase cerebral blood flow and cerebral capillary density in the cortex under physiological conditions [1], [13].

These findings have prompted us to analyze the long-term neuroprotective potential (two weeks after stroke) of these two steroids after transient middle cerebra artery occlusion (tMCAO) in adult male rats using four-times repeated steroid supplementation during the first two days after transient ischemia. The combinatory application was chosen, since previous findings document a maximum protective efficacy only when both steroids are applied together. In addition to the measurement of the infarct volume and detailed behavioral analysis, we have studied the density of blood vessels of the ipsi-/contralateral cerebral cortex and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF receptor 1 (VEGFR 1) in the penumbra.

Section snippets

Materials and methods

Experiments were performed with male Wistar rats (11–14 weeks old, 330–410 g, Charles-River, Germany) which underwent tMCAO or sham surgery as described earlier [3]. A total of twenty rats were used, four rats (3 tMCAO, 1 sham) died during recovery, and 16 were included in the study.

For hormone treatment, 25 μg 17β-estradiol (E) and 10 mg progesterone (P) per kg body weight (Sigma–Aldrich, Munich, Germany, initially diluted in pure ethanol) were given as neck depots in 500 μl sesame oil at 0 h, 12 h,

Results and discussion

Transient MCAO caused a well-defined infarct area comprising major parts of the ipsilateral cerebral cortex and the adjacent basal ganglia (Fig. 1A–C). The resulting infarcted tissue can be comparably demonstrated by HE-staining and NeuN immunocytochemistry. The sharp border line between intact and injured tissue is shown in Fig. 1C at higher magnification. Treatment with E/P significantly ameliorated the tissue damage concerning mainly the cerebral cortex and to a much lesser extent the basal

Acknowledgments

This study was in part supported by grants from B. Braun Melsungen AG and the B. Braun foundation as well as the START funding of the UKA (RWTH Aachen).

References (31)

D.G. Stein
Progesterone exerts neuroprotective effects after brain injury
Brain Res. Rev.
(2008)
J. Dang et al.
Gonadal steroids prevent cell damage and stimulate behavioral recovery after transient middle cerebral artery occlusion in male and female rats
Brain Behav. Immun.
(2011)
S. Suzuki et al.
Neuroprotective effects of estrogens following ischemic stroke
Front. Neuroendocrinol.
(2009)
L.M. Garcia-Segura et al.
Neuroprotection by estradiol
Prog. Neurobiol.
(2001)
J. Pawlak et al.
Regulation of glutamate transporter GLAST and GLT-1 expression in astrocytes by estrogen
Mol. Brain Res.
(2005)
W. Cai et al.
Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage
Neuropharmacology
(2008)
S.C. Fagan et al.
Combination treatment for acute ischemic stroke: a ray of hope?
J. Stroke Cerebrovasc. Dis.
(1999)
I. Sayeed et al.
Progesterone as a neuroprotective factor in traumatic and ischemic brain injury
Prog. Brain Res.
(2009)
D. Amantea et al.
From clinical evidence to molecular mechanisms underlying neuroprotection afforded by estrogens
Pharmacol. Res.
(2005)
I. Sayeed et al.
Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism for better neuroprotective effects of allopregnanolone over progesterone
Brain Res.
(2009)

M. Liu et al.

Neuroprotection by sex steroids

Minerva Endocrinol.

(2010)

G.E. Hoffmann et al.

Neuroprotection by ovarial hormones in animal models of neurological disease

Endocrine

(2006)

S. Arnold et al.

Neuroprotection by estrogen in the brain: the mitochondria compartment as presumed therapeutic target

J. Neurochem.

(2009)

M.E. O’Donnell et al.

Estradiol reduces activity of the blood-brain-barrier Na–K–Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion

J. Cereb. Blood Flow Metab.

(2006)

Q.G. Zhang et al.

Estrogen attenuates ischemic oxidative damage via an estrogen receptor alpha-mediated inhibition of NADPH oxidase activation

J. Neurosci.

(2009)

Cited by (43)

The effect of female sex hormones on Hsp27 phosphorylation and histological changes in prefrontal cortex after tMCAO
2021, Pathology Research and Practice
Female sex hormones are protective factors against many neurological disorders such as brain ischemia. Heat shock protein like HSP27 is activated after tissue injury. The main purpose of the present study is to determine the effect of a combined estrogen / progesterone cocktail on the morphology of astrocytes, neurons and Hsp27 phosphorylation after cerebral ischemia.
One hour after the MCAO induction, a single dose of estrogen and progesterone was injected. The infarct volume was calculated by TTC staining 24 h after ischemia. Immunohistochemistry was used to show the effects of estrogen and progesterone on astrocyte and neuron morphology, as well as the Western blot technique used for the quantitation of phosphorylated Hsp27.
The combined dose of estrogen and progesterone significantly decreased astrocytosis after ischemia and increased neuron survival. There was a large increase in Hsp27 phosphorylation in the penumbra ischemic region after stroke, which was significantly reduced by hormone therapy.
Our results indicate that the neuroprotective effect of neurosteroids in the brain may be due to the modulation of heat shock proteins.
Modulatory effect of 17β-estradiol on myeloid cell infiltration into the male rat brain after ischemic stroke
2020, Journal of Steroid Biochemistry and Molecular Biology
Ischemic stroke is the leading cause of human disability and mortality in the world. Neuroinflammation is the main pathological event following ischemia which contributes to secondary brain tissue damage and is driven by infiltration of circulating immune cells such as macrophages. Because of neuroprotective properties against ischemic brain damage, estrogens have the potential to become of therapeutic interest. However, the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In the current study, 12-week-old male Wistar rats underwent an experimental ischemia by occluding the middle cerebral artery transiently (tMCAO) for 1 h. Male rats subjected to tMCAO were randomly assigned to receive 17β-estradiol or vehicle treatment. The animals were sacrificed 72 h post tMCAO, transcardially perfused and the brains were proceeded either for TTC staining and gene analysis or for flow cytometry (CD45, CD11b, CD11c, CD40). We found that 17β-estradiol substitution significantly reduced the cortical infarct which was paralleled by an improved Garcia test scoring. Flow cytometry revealed that CD45⁺ cells as well as CD45⁺CD11b⁺CD11c⁺ cells were massively increased in tMCAO animals and numbers were nearly restored to sham levels after 17β-estradiol treatment. Gene expression analysis showed a reperfusion time-dependent upregulation of the markers CD45, CD11b and the activation marker CD40. The reduction in gene expression after 72 h of reperfusion and simultaneous 17β-estradiol substitution did not reach statistical significance. These data indicate that 17β-estradiol alleviated the cerebral ischemia-reperfusion injury and selectively suppressed the activation of the neuroinflammatory cascade via reduction of the number of activated microglia or infiltrated monocyte-derived macrophages in brain.
EPO regulates neuroprotective Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury
2019, Experimental Neurology
Citation Excerpt :
Total infarct volumes were calculated by adding the mean area of each section and multiplied by the thickness of the sections. The infarct volume was measured using the 2,3,5-triphenyltetrazolium chloride staining method as described earlier (Ulbrich et al., 2012). In brief, 72 h after surgery mice were deeply anesthetized and EDTA blood of each animal was taken transcardially for a blood count using the Celltac α MEK-6450 K (Nihon Kohden Europe, Rosbach, Germany) in the Institute for Experimental Animal Science and Central Laboratory for Experimental Animals (RWTH Aachen) (Supplementary Fig. S4).
Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members exert inhibitory activities in apoptosis and necroptosis. FAIM2 (TMBIM-2) is neuroprotective against murine focal ischemia and is regulated by erythropoietin (EPO). Similar to FAIM2, GRINA (TMBIM-3) is predominantly expressed in the brain. The role of GRINA in transient brain ischemia, its potential synergistic effects with FAIM2 and its regulation by EPO treatment were assessed.
We performed transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 72 h of reperfusion in GRINA-deficient (GRINA^−/−), FAIM2-deficient (FAIM2^−/−), double-deficient (GRINA^−/-FAIM2^−/−) and wildtype littermates (WT) mice. We administered EPO or saline 0, 24 and 48 h after tMCAo. We subjected primary murine cortical neurons (pMCN) of all mouse strains to oxygen–glucose deprivation (OGD) after GRINA and/or FAIM2 gene transfection.
Compared to wildtype controls GRINA deficiency led to a similar increase in infarct volumes as FAIM2 deficiency (p < .01). We observed the highest neurological deficits and largest infarct sizes in double-deficient mice. EPO administration upregulated GRINA and FAIM2 mRNA levels in wildtype littermates. EPO decreased infarct sizes and abrogated neurological impairments in wildtype controls. GRINA and/or FAIM2 deficient mice showed increased expression levels of cleaved-caspase 3 and of pro-apoptotic BAX mRNA. Further, caspase 8 was upregulated in FAIM2^−/− and caspase 9 in GRINA^−/− mice. Overexpression of GRINA and FAIM2 in wildtype and in double deficient pMCN decreased cell death rate after OGD.
GRINA and FAIM2 are highly expressed in the brain and convey EPO-mediated neuroprotection after ischemic stroke involving different caspases.
Role of Steroid Therapy after Ischemic Stroke by N-Methyl-D-Aspartate Receptor Gene Regulation
2018, Journal of Stroke and Cerebrovascular Diseases
Stroke is the main cause of cerebrovascular disease mortality. Prolonged stimulation of n-methyl-d-aspartate (NMDA) receptor subtypes by the accumulation of glutamate neurotransmitter in the extracellular space after a stroke could activate cell death pathways. It is reported that progesterone provides different mechanisms of neuroprotection and could be considered as a candidate for stroke treatment. This study aimed to investigate progesterone impact on the expression of NMDA receptor subunits NR1, NR2 (A and B), NR3 (A and B) after an experimental model of ischemic stroke which is followed by an in silico analysis.
Progesterone was introduced subcutaneously after transient middle cerebral artery occlusion in male rats. After a period of reperfusion, a set of behavioral tests was performed to evaluate the postischemic neurological deficits. The 2,3,5-triphenyltetrazolium chloride staining method was done for quantification of infarct volume and gene expression analysis was performed in the penumbra region using reverse transcription polymerase chain reaction for NMDA receptor subunits. An AutoDock tool was employed to perform molecular docking analyses for evaluation of progesterone interaction with NMDA receptor.
Cerebral ischemia caused a significant downregulation in NR1, NR2A, NR2B and a profound upregulation of NR3B in cortical penumbra region. Treatment with progesterone resulted in upregulation of NR1, NR2A, and NR3B which could explain a possible the neuroprotection of steroids via binding to NMDA glutamate receptor. In addition, in silico analysis revealed that progesterone could strongly interact with NR1/NR2B and NR2A.
The findings elucidate a new aspect of the neuroprotective mechanism of progesterone via NMDA receptors gene regulation.
Treatment targets for M2 microglia polarization in ischemic stroke
2018, Biomedicine and Pharmacotherapy
As the first line of defense in the nervous system, resident microglia are the predominant immune cells in the brain. In diseases of the central nervous system such as stroke, Alzheimer’s disease, and Parkinson’s disease, they often cause inflammation or phagocytosis; however, some studies have found that despite the current controversy over M1, M2 polarization could be beneficial. Ischemic stroke is the third most common cause of death in humans. Patients who survive an ischemic stroke might experience a clear decline in their quality of life, owing to conditions such as hemiplegic paralysis and aphasia. After stroke, the activated microglia become a double-edged sword, with distinct phenotypic changes to the deleterious M1 and neuroprotective M2 types. Therefore, methods for promoting the differentiation of microglia into the M2 polarized form to alleviate harmful reactions after stroke have become a topic of interest in recent years. Subsequently, the discovery of new drugs related to M2 polarization has enabled the realization of targeted therapies. In the present review, we discussed the neuroprotective effects of microglia M2 polarization and the potential mechanisms and drugs by which microglia can be transformed into the M2 polarized type after stroke.
Aucubin promotes angiogenesis via estrogen receptor beta in a mouse model of hindlimb ischemia
2017, Journal of Steroid Biochemistry and Molecular Biology
Aucubin (AU) is an iridoid glycoside that has been shown to display estrogenic properties and has various pharmacological effects. Herein, we described the angiogenic properties of AU. In the study, hindlimb ischemia was induced by ligation of femoral artery on the right leg of ovariectomized mice. AU treatment significantly accelerated perfusion recovery and reduced tissue injury in mice muscle. Quantification of CD31-positive vessels in hindlimb muscles provided evidences that AU promoted angiogenesis in peripheral ischemia. In addition, results from quantitative PCR and western blot suggested AU induced angiogenesis via vascular endothelial cell growth factor (VEGF)/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway. More interestingly, AU’s angiogenic effects could be completely abolished in estrogen receptor beta (ERβ) knockout mice. In conclusion, the underlying mechanisms were elucidated that AU produced pro-angiogenic effects through ERβ-mediated VEGF signaling pathways. These results expand knowledge about the beneficial effects of AU in angiogenesis and blood flow recovery. It might provide insight into the ERβ regulating neovascularisation in hindlimb ischemia and identify AU as a potent new compound used for the treatment of peripheral vascular disease.

View all citing articles on Scopus

¹: These authors contributed equally.

View full text

ReviewLong-term cerebral cortex protection and behavioral stabilization by gonadal steroid hormones after transient focal hypoxia

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgments

Brain Res. Rev.

Brain Behav. Immun.

Front. Neuroendocrinol.

Prog. Neurobiol.

Mol. Brain Res.

Neuropharmacology

J. Stroke Cerebrovasc. Dis.

Prog. Brain Res.

Pharmacol. Res.

Brain Res.