The Prodrug DHED Delivers 17ß-Estradiol into the Retina for Protection of Retinal Ganglion Cells and Preservation of Visual Function in an Animal Model of Glaucoma.

We report a three-pronged phenotypic evaluation of the bioprecursor prodrug 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED) that selectively produces 17ß-estradiol (E2) in the retina after topical administration and halts glaucomatous neurodegeneration in a male rat model of the disease. Ocular hypertension (OHT) was induced by hyperosmotic saline injection into an episcleral vein of the eye. Animals received daily DHED eye drops for 12 weeks. Deterioration of visual acuity and contrast sensitivity by OHT in these animals were markedly prevented by the DHED-derived E2 with concomitant preservation of retinal ganglion cells and their axons. In addition, we utilized targeted retina proteomics and a previously established panel of proteins as preclinical biomarkers in the context of OHT-induced neurodegeneration as a characteristic process of the disease. The prodrug treatment provided retina-targeted remediation against the glaucomatous dysregulations of these surrogate endpoints without increasing circulating E2 levels. Collectively, the demonstrated significant neuroprotective effect by the DHED-derived E2 in the selected animal model of glaucoma supports the translational potential of our presented ocular neuroprotective approach owing to its inherent therapeutic safety and efficacy.

Isolated Congenital Mitral Regurgitation Repair in Children: Long-term Outcomes of Artificial Rings.

BACKGROUND: Because isolated congenital mitral regurgitation (ICMR) is rare, the role of artificial rings in annuloplasty for ICMR is still debated. In this study we compared long-term results of annuloplasty with and without rings for ICMR. METHODS: We performed a retrospective review of all patients ≤ 18 years old undergoing repair for ICMR from 1993 to 2019 at our hospital. Techniques of annuloplasty depended on individual lesions of the mitral valve (MV). One hundred twelve cases were divided into 3 groups: group A (43 cases) with ≥26-mm ring, group B (35 cases) with <26-mm ring, and group C (34 cases) with a band. Study endpoints were overall survival and MV reoperation. RESULTS: Patients in group A were older than the others (median age 11 compared with 5 and 4 years, respectively). The MV lesions were complicated, but annular dilatation existed in 91% of cases. Three patients died, and there were 25 reoperations during a median follow-up period of 11 years. Groups B and C had higher rates of reoperation than group A (hazard ratios, 5.35 [95% confidence interval, 1.71-17.75] and 3.61 [95% confidence interval, 1.03-12.60], respectively). Most reoperations in group B (13/14 cases) were due to stenosis, whereas 6 of 7 reoperations in group C were due to recurrent regurgitation. CONCLUSION: MV reconstruction for ICMR in children had good long-term survival. Annuloplasty with ≥26-mm ring had the lowest risk of reoperation. In cases of young children bands are more favored than <26-mm rings, but they carry a risk of recurrent regurgitation.

[ß-Glu2]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain.

Selective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu2]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [ß-Glu2]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [ß-Glu2]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [ß-Glu2]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.

Neuroprotective effects of Sonic hedgehog agonist SAG in a rat model of neonatal stroke.

BACKGROUND: Neonatal stroke affects 1 in 2800 live births and is a major cause of neurological injury. The Sonic hedgehog (Shh) signaling pathway is critical for central nervous system (CNS) development and has neuroprotective and reparative effects in different CNS injury models. Previous studies have demonstrated beneficial effects of small molecule Shh-Smoothened agonist (SAG) against neonatal cerebellar injury and it improves Down syndrome-related brain structural deficits in mice. Here we investigated SAG neuroprotection in rat models of neonatal ischemia-reperfusion (stroke) and adult focal white matter injury. METHODS: We used transient middle cerebral artery occlusion at P10 and ethidium bromide (EB) injection in adult rats to induce damage. Following surgery and SAG or vehicle treatment, we analyzed tissue loss, cell proliferation and fate, and behavioral outcome. RESULTS: We report that a single dose of SAG administered following neonatal stroke preserved brain volume, reduced gliosis, enhanced oligodendrocyte progenitor cell (OPC) and EC proliferation, and resulted in long-term cognitive improvement. Single-dose SAG also promoted proliferation of OPCs following focal demyelination in the adult rat. CONCLUSIONS: These findings indicate benefit of one-time SAG treatment post insult in reducing brain injury and improving behavioral outcome after experimental neonatal stroke. IMPACT: A one-time dose of small molecule Sonic hedgehog agonist protected against neonatal stroke and improved long-term behavioral outcomes in a rat model. This study extends the use of Sonic hedgehog in treating developing brain injury, previously shown in animal models of Down syndrome and cerebellar injury. Sonic hedgehog agonist is one of the most promising therapies in treating neonatal stroke thanks to its safety profile and low dosage.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications.

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17ß-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Bilateral ultrasound-guided thoracic erector spinae plane blocks using a programmed intermittent bolus improve opioid-sparing postoperative analgesia in pediatric patients after open cardiac surgery: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Postoperative pain after pediatric cardiac surgery is usually treated with intravenous opioids. Recently, the focus has been on postoperative regional analgesia with the introduction of ultrasound-guided erector spinae plane blocks (ESPBs). We hypothesized that bilateral ESPB with a programmed intermittent bolus (PIB) regimen decreases postoperative morphine consumption at 48 hours and improves analgesia in children who undergo cardiac surgery. METHODS: This randomized, double-blind, placebo-controlled study comprised 50 children who underwent cardiac surgery through midline sternotomy. The patients were allocated randomly into two groups: ultrasound-guided bilateral ESPB at the level of T3-T4 transverse process then PIB with saline infusion (group 1, n=23) or PIB with 0.2% ropivacaine (group 2, n=27). Intravenous morphine at 30 µg/kg/hour was used as rescue analgesia. Postoperative pain was assessed using the COMFORT-B score for extubation, drain removal, and mobilization, and the FLACC (Face, Legs, Activity, Cry, Consolability) scale at 0, 2, 4, 6, 8, 12, 16, 20, 24, 36, and 48 hours after surgery. Adverse events were noted. RESULTS: The total dose of morphine in 48 hours was significantly decreased in patients receiving a bilateral ESPB with ropivacaine (120±320 µg/kg) compared with patients with saline infusion (512±560 µg/kg; p=0.03). Fourteen per cent of patients required rescue analgesia with morphine in group 2 compared with 41% in group 1 (p=0.05). The patients in group 2 demonstrated significantly reduced COMFORT-B scores at extubation, drain removal, and mobilization compared with those in group 1 and had reduced FLACC scale levels at 20 and 24 hours postoperatively (p=0.05 and p=0.001, respectively). No differences were reported for extubation and drain removal times or for length of hospital stay. In addition, vomiting episodes were decreased in group 2 (p=0.01). CONCLUSIONS: In pediatric cardiac surgery, the results of this study confirm our hypothesis that bilateral ESPB analgesia with ropivacaine decreases the postoperative morphine consumption at 48 hours and demonstrates better postoperative analgesia compared with a control group. Trial registration number NCT03593642.

Brain-Selective Estrogen Therapy Prevents Androgen Deprivation-Associated Hot Flushes in a Rat Model.

Hot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17ß-estradiol, E2) are produced from testosterone. Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10ß, 17ß-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.

Oxygen Tension and the VHL-Hif1α Pathway Determine Onset of Neuronal Polarization and Cerebellar Germinal Zone Exit.

Postnatal brain circuit assembly is driven by temporally regulated intrinsic and cell-extrinsic cues that organize neurogenesis, migration, and axo-dendritic specification in post-mitotic neurons. While cell polarity is an intrinsic organizer of morphogenic events, environmental cues in the germinal zone (GZ) instructing neuron polarization and their coupling during postnatal development are unclear. We report that oxygen tension, which rises at birth, and the von Hippel-Lindau (VHL)-hypoxia-inducible factor 1α (Hif1α) pathway regulate polarization and maturation of post-mitotic cerebellar granule neurons (CGNs). At early postnatal stages with low GZ vascularization, Hif1α restrains CGN-progenitor cell-cycle exit. Unexpectedly, cell-intrinsic VHL-Hif1α pathway activation also delays the timing of CGN differentiation, germinal zone exit, and migration initiation through transcriptional repression of the partitioning-defective (Pard) complex. As vascularization proceeds, these inhibitory mechanisms are downregulated, implicating increasing oxygen tension as a critical switch for neuronal polarization and cerebellar GZ exit.

10ß,17α-Dihydroxyestra-1,4-dien-3-one: A Bioprecursor Prodrug Preferentially Producing 17α-Estradiol in the Brain for Targeted Neurotherapy.

Uterotrophic effect of 17α-estradiol, the C17 epimer of the main human estrogen 17ß-estradiol, was shown to manifest in animal models at doses lower than those necessary for central outcome raising concerns about its potential to treat maladies of the central nervous system. We introduce here 10ß,17α-dihydroxyestra-1,4-dien-3-one (α-DHED) that acts as a bioprecursor prodrug producing 17α-estradiol with remarkable selectivity to the brain and, therefore, without appreciable exposure of the periphery to the parent steroid. This distinguishing feature of α-DHED is shown by using an estrogen-responsive mouse model with complementary LC-MS/MS measurement of drug contents in target tissues. Our data warrant further research to fully establish the potential of α-DHED for a safe and efficacious 17α-estradiol-based neurotherapy.

Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury.

The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.

Treatment with an orally bioavailable prodrug of 17ß-estradiol alleviates hot flushes without hormonal effects in the periphery.

Estrogen deprivation has a profound effect on the female brain. One of the most obvious examples of this condition is hot flushes. Although estrogens relieve these typical climacteric symptoms, many women do not want to take them owing to unwanted side-effects impacting, for example, the uterus, breast and blood. Therefore, there is a need for developing safer estrogen therapies. We show here that treatment with 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), a novel brain-targeting bioprecursor prodrug of the main human estrogen, 17ß-estradiol, alleviates hot flushes in rat models of thermoregulatory dysfunction of the brain. Oral administration of DHED elicits a significant reduction of tail skin temperature (TST) rise representing hot flushes in the morphine-dependent ovariectomized rat model and results in the restoration of estrogen deprivation-induced loss of diurnal rhythm in TST. These beneficial effects occur without detrimental peripheral hormonal exposure; thus, the treatment avoids potentially harmful stimulation of estrogen-sensitive peripheral organs, including the uterus and the anterior pituitary, or the proliferation of MCF-7a breast cancer cell xenografts. Our promising preclinical assessments warrant further considerations of DHED for the development of a brain-selective 17ß-estradiol therapy to relieve hot flushes without undesirable peripheral side-effects.

Technique for office-based, ultrasonography-guided percutaneous biopsy of renal cortical neoplasms using a novel transducer for facilitated ultrasound targeting.

OBJECTIVES: To help clarify which small renal cortical neoplasms (RCNs) require surgery by using office-based, ultrasonography-guided percutaneous renal biopsy. PATIENTS AND METHODS: Biopsies were performed using facilitated ultrasound targeting (FUT) technology, which incorporates a needle guide and onscreen beam-steered technology to permit highly precise needle deployment. Patient and tumour characteristics, procedure time, complications and biopsy efficacy were documented. Wong-Baker pain levels were obtained before, during and 1 h after the procedure. RESULTS: Seven patients underwent biopsy, six for RCNs and one for medical renal disease. The mean (range) patient age was 68.5 (54-79) years, and the mean (range) tumour diameter was 2.55 (2.0-2.9) cm. Mean pain levels before, during and 1 h after the procedure were 0, 1.6 and 0.5, respectively. There were no intra- or post-procedural complications. Biopsy results were diagnostic in five of the six RCN cases and in the single case of medical renal disease. CONCLUSIONS: Our preliminary experience shows that office-based percutaneous renal biopsy using a novel transducer for FUT is safe and effective. An international multicentre study is planned to confirm these preliminary results.

The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

HIGD1A Regulates Oxygen Consumption, ROS Production, and AMPK Activity during Glucose Deprivation to Modulate Cell Survival and Tumor Growth.

Hypoxia-inducible gene domain family member 1A (HIGD1A) is a survival factor induced by hypoxia-inducible factor 1 (HIF-1). HIF-1 regulates many responses to oxygen deprivation, but viable cells within hypoxic perinecrotic solid tumor regions frequently lack HIF-1α. HIGD1A is induced in these HIF-deficient extreme environments and interacts with the mitochondrial electron transport chain to repress oxygen consumption, enhance AMPK activity, and lower cellular ROS levels. Importantly, HIGD1A decreases tumor growth but promotes tumor cell survival in vivo. The human Higd1a gene is located on chromosome 3p22.1, where many tumor suppressor genes reside. Consistent with this, the Higd1a gene promoter is differentially methylated in human cancers, preventing its hypoxic induction. However, when hypoxic tumor cells are confronted with glucose deprivation, DNA methyltransferase activity is inhibited, enabling HIGD1A expression, metabolic adaptation, and possible dormancy induction. Our findings therefore reveal important new roles for this family of mitochondrial proteins in cancer biology.

Arhgap36-dependent activation of Gli transcription factors.

Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are up-regulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.

Modelling the fate of pesticides in paddy rice-fish pond farming systems in northern Vietnam.

BACKGROUND: In Vietnam, paddy rice fields have been identified as a major non-point source of pesticide pollution of surface- and groundwater which is often directly used for domestic purposes. One strategy to assess the risk of pesticide pollution is to use process-based models. Here, we present a new model developed for simulating short-term pesticide dynamics in combined paddy rice field-fish pond farming systems. The model was calibrated using the Gauss-Marquardt-Levenberg algorithm and validated against measured pesticide concentrations of a paddy field-fish pond system typical for northern Vietnam. RESULTS: In the calibration period, model efficiencies were 0.82 for dimethoate and 0.87 for fenitrothion. In the validation period, modelling efficiencies slightly decreased to 0.42 and 0.76 for dimethoate and fenitrothion, respectively. Scenario simulations revealed that a field closure period of 1 day after pesticide application considerably reduces the risk of pond and surface water pollution. CONCLUSION: These results indicate that the proposed model is an effective tool to assess and evaluate management strategies, such as extended field closure periods, aiming to reduce the loss of pesticides from paddy fields.

PET/CT imaging of abdominal aorta with intramural hematomas, penetrating ulcer, and saccular pseudoaneurysm.

Acute aortic syndromes, encompassing intramural hematoma, penetrating ulcer, and pseudoaneurysm, are best demonstrated by angiographic CT and magnetic resonance imaging. These imaging modalities provide an accurate evaluation and allow timely therapies of these frequently symptomatic lesions, thus reducing their morbidity and mortality. The inflammatory pathogenesis of these acute aortic syndromes may exhibit positive PET findings predictive of prognosis and outcomes of these vascular events. The authors present a case of PET/CT imaging showing asymptomatic intramural hematomas with penetrating ulcer and saccular pseudoaneurysm of the proximal abdominal aorta.

In vitro comparison of a novel facilitated ultrasound targeting technology vs standard technique for percutaneous renal biopsy.

OBJECTIVE: To improve the understanding of the epidemiology of renal cortical neoplasms through pretreatment biopsy, we evaluated a facilitated ultrasound targeting (FUT) technology. The technology allows a needle to be passed through the transducer probe and guided along a virtual dotted line on the monitor. We compared the FUT with standard percutaneous biopsy (PB) technique. MATERIALS AND METHODS: Forty-eight participants with various levels of training were recruited. Participants performed ultrasound-guided biopsies on phantom models using FUT and the standard biopsy technique in a randomized sequence. The phantom models consisted of pimento olives embedded in an opaque mold of Metamucil and Knox gelatin. Patients were given up to 10 attempts to achieve 3 complete specimens from the olives. Patients rated each biopsy technique. Results were stratified by level of experience. RESULTS: The mean time to obtain 3 complete biopsy specimens was significantly faster for FUT compared with the standard technique (140 seconds vs 246 seconds, P = .0001). The mean number of attempts needed to obtain 3 specimens was significantly less with FUT compared with the standard technique (4.3 vs 5.6 attempts, P = .0007). Patients reported that FUT was significantly easier to use compared with the standard technique (P = .0005). No significant order effect was observed. CONCLUSION: In this in vitro comparison, FUT increased the efficiency and efficacy of PB for users of all experience levels. FUT may allow urologists with limited PB experience to perform the procedure reliably and easily. Clinical evaluation of this technology is actively in progress.

17ß-estradiol eye drops protect the retinal ganglion cell layer and preserve visual function in an in vivo model of glaucoma.

Neuroprotection in glaucoma as a curative strategy complementary to current therapies to lower intraocular pressure (IOP) is highly desirable. This study was designed to investigate neuroprotection by 17ß-estradiol (E2) to prevent retinal ganglion cell (RGC) death in a glaucoma model of surgically elevated IOP in rats. We found that daily treatment with E2-containing eye drops resulted in significant E2 concentration in the retina with concomitant profound neuroprotective therapeutic benefits, even in the presence of continually elevated IOP. The number of apoptotic cells in the RGC layer was significantly decreased in the E2-treated group, when compared to the vehicle-treated controls. Deterioration in visual acuity in these animals was also markedly prevented. Using mass spectrometry-based proteomics, beneficial changes in the expression of several proteins implicated in the maintenance of retinal health were also found in the retina of E2-treated animals. On the other hand, systemic side effects could not be avoided with the eye drops, as confirmed by the measured high circulating estrogen levels and through the assessment of the uterus representing a typical hormone-sensitive peripheral organ. Collectively, the demonstrated significant neuroprotective effect of topical E2 in the selected animal model of glaucoma provides a clear rationale for further studies aiming at targeting E2 into the eye while avoiding systemic E2 exposure to diminish undesirable off-target side effects.