Elizabeth B. Engler-Chiurazzi

Estrogens as neuroprotectants: Estrogenic actions in the context of cognitive aging and brain injury

HIGHLIGHTSThe biological impacts of estrogen extend beyond the gonads to other bodily systems, including the brain and behavior.Conflicting preclinical and clinical data regarding actions of estrogens on the nervous system suggests that estrogen is a conditional neuroprotectant.We provide a summary of the biological actions of estrogen and estrogen‐containing formulations in the context of cognition and brain injury. ABSTRACT There is ample empirical evidence to support the notion that the biological impacts of estrogen extend beyond the gonads to other bodily systems, including the brain and behavior. Converging preclinical findings have indicated a neuroprotective role for estrogen in a variety of experimental models of cognitive function and brain insult. However, the surprising null or even detrimental findings of several large clinical trials evaluating the ability of estrogen‐containing hormone treatments to protect against age‐related brain changes and insults, including cognitive aging and brain injury, led to hesitation by both clinicians and patients in the use of exogenous estrogenic treatments for nervous system outcomes. That estrogen‐containing therapies are used by tens of millions of women for a variety of health‐related applications across the lifespan has made identifying conditions under which benefits with estrogen treatment will be realized an important public health issue. Here we provide a summary of the biological actions of estrogen and estrogen‐containing formulations in the context of aging, cognition, stroke, and traumatic brain injury. We have devoted special attention to highlighting the notion that estrogen appears to be a conditional neuroprotectant whose efficacy is modulated by several interacting factors. By developing criteria standards for desired beneficial peripheral and neuroprotective outcomes among unique patient populations, we can optimize estrogen treatments for attenuating the consequences of, and perhaps even preventing, cognitive aging and brain injury.

From the 90's to now: A brief historical perspective on more than two decades of estrogen neuroprotection.

UNLABELLED Historical perspective abstract:From the 90s to now: a historical perspective on more than two decades of estrogen neuroprotection: In the early 90s, estrogens were known to exert organizational and activational effects on reproductive tissues and sexual behavior. As well, the role of sex and gonadal hormones in altering the risk for developing Alzheimers Disease (AD) was only beginning to be elucidated. Preliminary investigations suggested that estrogen-containing therapies typically given for the management of disruptive menopausal symptoms could reduce AD risk, attenuate disease-associated cognitive deficits, and modulate brain substrates known to be dysregulated by the condition, such as the cholingeric system. The findings from our seminal paper demonstrating cognitive benefits and cholinergic impacts with exogenous estrogen treatment in a rodent model of surgical hormone depletion provided initial support for use of estrogen-containing therapies as a treatment for age-related brain disorders. We then went on to demonstrate neuroprotective actions of estrogen in several other in vivo and in vitro models of neurological challenge, including stroke and AD. Further, our findings of the chemical structure requirements for estrogens neuroprotective effects identified a novel approach for optimizing future estrogen-containing hormone therapy options. These early efforts laid the groundwork for later, large-scale clinical investigations into the potential of estrogen-based menopausal hormone therapies for the prevention of a variety of age-related disorders. Although findings of these studies were equivocal, the neuroprotective actions of estrogen, and specifically 17β-estradiol, identified by early investigations, remain well-documented. Future development of interventions that optimize cognitive aging are crucial and, with proper understanding of the factors that influence the realization of beneficial impacts, estrogen-containing treatments may still be among these. ORIGINAL ARTICLE ABSTRACT Ovarian steroid deprivation results in a reversible learning impairment and compromised cholinergic function in female Sprague-Dawley rats: We hypothesized that estradiol (E2) serves as a neurotrophomodulatory substance for basal forebrain cholinergic neurons thought to be involved in learning and memory. Learning/memory was assessed using the two-way active avoidance paradigm and the Morris water task. Female Sprague-Dawley rats were either ovariectomized (OVX) or OVX for 3 weeks, followed by s.c. implantation of a Silastic pellet containing 17-βE2 (E2 pellet), resulting in a replacement of E2 to physiological levels. Ovary-intact (INTACT) animals served as our positive control. Active avoidance behavior and choline acetyltransferase (ChAT) activity in the frontal cortex and hippocampus were assessed at 5 and 28 weeks postovariectomy while performance on the Morris water task and high-affinity choline uptake (HACU) were measured only at the 5-week time point. At the 5-week time point, E2 replacement caused a significant elevation in the level of active avoidance performance relative to OVX animals. At the 28-week time point, OVX animals demonstrated a significantly lower number of avoidances relative to controls (61%) whereas E2-pellet animals not only demonstrated superior performance relative to OVX animals but also showed an accelerated rate of learning. Morris water task performance, on the other hand, was not significantly affected by estrogenic milieu despite a trend towards better performance in the E2-pellet group. Neurochemical analyses revealed that 5 weeks of ovariectomy was sufficient to reduce HACU in both the frontal cortex and hippocampus by 24 and 34%, respectively, while E2 replacement was successful in elevating HACU relative to OVX animals in both regions. ChAT activity was decreased in the hippocampus but not the frontal cortex of 5-week OVX animals. E2 replacement resulted in a reversal of this effect. At the 28-week time period, an unexpected decrease in ChAT activity was observed across all treatment groups. Interestingly, E2-pellet animals demonstrated the least severe decline in ChAT. This phenomenon was most evident in the frontal cortex where ChAT decreased by 61 and 56% in INTACT and OVX animals, respectively, whereas the decline in E2-pellet animals was only 16% over the same time period, suggesting a previously unreported cytoprotective effect of E2. Taken together, these findings demonstrate important effects of estrogens on cholinergic neurons and support the potential use of estrogen therapy in treatment of dementias in postmenopausal women.

Paper-Based Surface-Enhanced Raman Scattering Lateral Flow Strip for Detection of Neuron-Specific Enolase in Blood Plasma

An inexpensive and disposable paper-based lateral flow strip (PLFS) has been developed as an immunoassay, in which surface-enhanced Raman scattering (SERS) is utilized for sensing signal transduction. The Au nanostar@Raman Reporter@silica sandwich nanoparticles are developed as the SERS probes, which is the key to the high sensitivity of the device. Compared with a colorimetric PLFS, the SERS-PLFS exhibits superior performance in terms of sensitivity and limit of detection (LOD) in a blood plasma-containing sample matrix. In addition, the SERS-PLFS has been successfully used for detection of neuron-specific enolase (NSE), a traumatic brain injury (TBI) protein biomarker, in diluted blood plasma samples, achieving a LOD of 0.86 ng/mL. Moreover, the SERS-PLFS was successfully employed to measure the NSE level in clinical blood plasma samples taken from deidentified TBI patients. This work demonstrates that the SERS-PLFS has great potential in assisting screening of TBI patients in the point-of-care setting.

Impacts of prenatal nanomaterial exposure on male adult Sprague-Dawley rat behavior and cognition.

ABSTRACT It is generally accepted that gestational xenobiotic exposures result in systemic consequences in the adult F1 generation. However, data on detailed behavioral and cognitive consequences remain limited. Using our whole-body nanoparticle inhalation facility, pregnant Sprague-Dawley rats (gestational day [GD] 7) were exposed 4 d/wk to either filtered air (control) or nano-titanium dioxide aerosols (nano-TiO2; count median aerodynamic diameter of 170.9 ± 6.4 nm, 10.4 ± 0.4 mg/m3, 5 h/d) for 7.8 ± 0.5 d of the remaining gestational period. All rats received their final exposure on GD 20 prior to delivery. The calculated daily maternal deposition was 13.9 ± 0.5 µg. Subsequently, at 5 mo of age, behavior and cognitive functions of these pups were evaluated employing a standard battery of locomotion, learning, and anxiety tests. These assessments revealed significant working impairments, especially under maximal mnemonic challenge, and possible deficits in initial motivation in male F1 adults. Evidence indicates that maternal engineered nanomaterial exposure during gestation produces psychological deficits that persist into adulthood in male rats.

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment.

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimers disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimers disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

A novel mechanism of non-feminizing estrogens in neuroprotection ☆

&NA; Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in an in vitro assay using a panel of > 70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200‐fold through modifications that positioned a large bulky group at the C2 or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C or D rings either reduced or did not markedly change neuroprotection. Collectively, there was a negative correlation between binding to ERs and neuroprotection with the more potent compounds showing no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro‐oxidant insult. We demonstrated that these non‐feminizing estrogens engage in a redox cycle with glutathione, using the hexose monophosphate shunt to apply cytosolic reducing potential to cellular membranes. Together, these results demonstrate that non‐feminizing estrogens are neuroprotective and protect brain from the induction of ischemic‐ and Alzheimers disease (AD)‐like neuropathology in an animal model. These features of non‐feminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, as they are not expected to show the side effects of chronic estrogen therapy that are mediated by ER actions in the liver, uterus and breast. HighlightsEstrogen neuroprotection can be independent of estrogen receptors.The phenolic A ring of estrogen molecule is essential to its neuroprotective activity.Allophalic substitutions at the 2 and 4 carbon of the A ring enhance neuroprotective potency.Non‐feminizing estrogens represent a novel target for post‐menopausal brain aging.Yet, with these agents, peripheral estrogenic benefits will not be observed.

Structure–activity studies of non-steroid analogues structurally-related to neuroprotective estrogens

Estrone and 17β-estradiol are phenolic steroids that are known to be neuroprotective in multiple models of neuronal injury. Previous studies have identified the importance of their phenolic steroid A-ring for neuroprotection and have identified ortho substituents at the C-2 and C-4 positions on the phenol ring that enhance this activity. To investigate the importance of the steroid ring system for neuroprotective activity, phenolic compounds having the cyclopent[b]anthracene, cyclopenta[b]phenanthrene, benz[f]indene, benz[e]indene, indenes linked to a phenol, and a phenolic spiro ring system were prepared. New synthetic methods were developed to make some of the cyclopent[b]anthracene analogues as well as the spiro ring system. Compounds were evaluated for their ability to protect HT-22 hippocampal neurons from glutamate neurotoxicity and their activity relative to a potent neuroprotective analogue of 17β-estradiol was determined. An adamantyl substituent placed ortho to the phenolic hydroxyl group gave neuroprotective analogues in all ring systems studied.

Locomotor effects of a low-frequency fire alarm on C57BL/6 male mice: a preliminary study

Maintaining appropriate acoustic conditions for animal welfare and data collection are crucial in biomedical research facilities. Negative impacts of disruptive sound are known and can include auditory damage, immune function changes, and behavioral alterations. One type of disruptive sound occurring in research facilities is that of fire alarms. To ameliorate this problem, many facilities have incorporated the use of low-frequency fire alarms that emit tones outside the rodent audible range. The impact of these devices has been assumed to be negligible. However, this has yet to be evaluated with controlled behavioral experiments. Thus, our objective was to investigate the impact of low-frequency fire alarm exposure on locomotor behavior in the open field, a test sensitive to acoustic stimuli disruption. Male mice were randomized to three alarm exposure groups (No-Alarm; Alarm-During; and Alarm-After) and placed in individual photobeam-activated locomotor chambers. The Alarm-During group displayed significantly reduced horizontal locomotion, with a trend towards reduced vertical locomotion. These data suggest that a low-frequency brief alarm tone can temporarily disrupt movement, a valuable insight should an alarm be deployed. Further, findings support close collaboration between researchers and institutional facility staff to ensure appropriate acoustic conditions are maintained, whenever possible, for research animals.

Absolute lymphocyte and neutrophil counts in neonatal ischemic brain injury.

Objectives: This study aimed to identify differences in absolute neutrophils, lymphocytes, and neutrophil-to-lymphocyte ratio between neonates with two forms of ischemic brain injury, hypoxic-ischemic encephalopathy, and acute ischemic stroke, compared to controls. We also aimed to determine whether this neutrophil/lymphocyte response pattern is associated with disease severity or is a consequence of the effects of total-body cooling, an approved treatment for moderate-to-severe hypoxic-ischemic encephalopathy. Methods: A retrospective chart review of 101 neonates with hypoxic-ischemic encephalopathy + total-body cooling (n = 26), hypoxic-ischemic encephalopathy (n = 12), acute ischemic stroke (n = 15), and transient tachypnea of the newborn (n = 48) was conducted; transient tachypnea of the newborn neonates were used as the control group. Absolute neutrophil count and absolute lymphocyte count at three time-intervals (0–12, 12–36, and 36–60 h after birth) were collected, and neutrophil-to-lymphocyte ratio was calculated. Results: Hypoxic-ischemic encephalopathy + total-body cooling neonates demonstrated significant time-interval-dependent changes in absolute lymphocyte count and neutrophil-to-lymphocyte ratio levels compared to transient tachypnea of the newborn and acute ischemic stroke patients. Pooled analysis of absolute lymphocyte count for neonates with acute ischemic stroke and hypoxic-ischemic encephalopathy (not hypoxic-ischemic encephalopathy + total-body cooling) revealed that absolute lymphocyte count changes occurring at 0–12 h are likely due to disease progression, rather than total-body cooling treatment. Conclusion: These data suggest that the neutrophil/lymphocyte response is modulated following neonatal ischemic brain injury, representing a possible target for therapeutic intervention. However, initial severity of hypoxic-ischemic encephalopathy among these patients could also account for the observed changes in the immune response to injury. Thus, additional work to clarify the contributions of cooling therapy and disease severity to neutrophil/lymphocyte response following hypoxic-ischemic encephalopathy in neonates is warranted.

MiR-34a and stroke: Assessment of non-modifiable biological risk factors in cerebral ischemia

Aging of the nervous system, and the occurrence of age-related brain diseases such as stroke, are associated with changes to a variety of cellular processes controlled by many distinct genes. MicroRNAs (miRNAs), short non-coding functional RNAs that can induce translational repression or site-specific cleavage of numerous target mRNAs, have recently emerged as important regulators of cellular senescence, aging, and the response to neurological insult. Here, we focused on the assessment of the role of miR-34a in stroke. We noted increases in miR-34a expression in the blood of stroke patients as well as in blood and brain of mice subjected to experimental stroke. Our methodical genetic manipulation of miR-34a expression substantially impacted stroke-associated preclinical outcomes and we have in vitro evidence that these changes may be driven at least in part by disruptions to blood brain barrier integrity and mitochondrial oxidative phosphorylation in endothelial cells. Finally, aging, independent of brain injury, appears to be associated with shifts in circulating miRNA profiles. Taken together, these data support a role for miRNAs, and specifically miR-34a, in brain aging and the physiological response to age-related neurological insult, and lay the groundwork for future investigation of this novel therapeutic target.