Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.

Patterns of Neointima Formation After Coil or Stent Treatment in a Rat Saccular Sidewall Aneurysm Model.

BACKGROUND AND PURPOSE: Endovascular aneurysm treatment relies on a biological process, including cell migration for thrombus organization and growth of a neointima. To better understand aneurysm healing, our study explores the origin of neointima-forming and thrombus-organizing cells in a rat saccular sidewall aneurysm model. METHODS: Saccular aneurysms were transplanted onto the abdominal aorta of male Lewis rats and endovascularly treated with coils (n=28) or stents (n=26). In 34 cases, GFP+ (green fluorescent protein)-expressing vital aneurysms were sutured on wild-type rats, and in 23 cases, decellularized wild-type aneurysms were sutured on GFP+ rats. Follow-up at 3, 7, 14, 21, and 28 days evaluated aneurysms by fluorescence angiography, macroscopic inspection, and microscopy for healing and inflammation status. Furthermore, the origin of cells was tracked with fluorescence histology. RESULTS: In animals with successful functional healing, histological studies showed a gradually advancing thrombus organization over time characterized by progressively growing neointima from the periphery of the aneurysm toward the center. Cell counts revealed similar distributions of GFP+ cells for coil or stent treatment in the aneurysm wall (54.4% versus 48.7%) and inside the thrombus (20.5% versus 20.2%) but significantly more GFP+ cells in the neointima of coiled (27.2 %) than stented aneurysms (10.4%; P=0.008). CONCLUSIONS: Neointima formation and thrombus organization are concurrent processes during aneurysm healing. Thrombus-organizing cells originate predominantly in the parent artery. Neointima formation relies more on cell migration from the aneurysm wall in coiled aneurysms but receives greater contributions from cells originating in the parent artery in stent-treated aneurysms. Cell migration, which allows for a continuous endothelial lining along the parent artery's lumen, may be a prerequisite for complete aneurysm healing after endovascular therapy. In terms of translation into clinical practice, these findings may explain the variability in achieving complete aneurysm healing after coil treatment and the improved healing rate in stent-assisted coiling.

Total Severity Score (TSS) comparison in vaccinated and unvaccinated patients during the fourth wave (December 2021 - January 2022) of COVID-19 in Italy.

OBJECTIVE: This study aims at comparing the severity score assessed using high-resolution computed tomography (HRCT) in vaccinated and unvaccinated COVID-19 patients. PATIENTS AND METHODS: From the first of December 2021 to first of February 2022, we conducted a single-center retrospective analysis on COVID-19 patients who accessed ED services. The hospital in question is a level II facility with a catchment area of around 200,000 people. According to the Italian recommendations, patients were divided into four groups based on the CT score of Micheal Chung. The sum of acute inflammatory lung lesions involving each lobe was scored as 1 (0-25%), 2 (26-50%), 3 (51-75%) or 4 (76-100%) on a visual quantitative assessment of CT. The total severity score (TSS) was determined by summing the five lobe scores. RESULTS: The study included 134 patients divided into two groups: 67 vaccinated and 67 unvaccinated people. 53 people had incomplete (single dose/double dose) immunization, while 14 people completed the vaccination cycle. It was discovered that the mean CT severity score was lower in fully vaccinated patients compared to partially vaccinated or unvaccinated patients. The mean CT score was significantly lower in fully vaccinated patients aged 60 compared to older patients. The mean CT score was higher in unvaccinated patients compared to fully vaccinated patients. CONCLUSIONS: Individuals who received three doses of COVID-19 vaccination had lower CT severity scores than patients who received only one dose of vaccine or no vaccines at all.

Pediatric Sleep Questionnaire Predicts Moderate-to-Severe Obstructive Sleep Apnea in Children and Adolescents with Obesity.

Pediatric obesity is associated with an increased risk of morbidity during childhood. Alongside the well-known metabolic syndrome, during the last decades scientific research has deeply investigated the risk of sleep breathing disorders. Among them, obstructive sleep apnea (OSA) commonly affects children with obesity. The presence of OSA heightens the risk of metabolic impairment and weight gain. Therefore, it deserves specific treatment. However, polysomnography (PSG) is not always available in clinical settings, and alternative diagnostic tools are needed. This study aimed to investigate the predictivity of the pediatric sleep questionnaire (PSQ) for moderate-to-severe OSA diagnosis. Children and adolescents with obesity and suspected OSA with available full-night cardiorespiratory PSG were retrospectively enrolled. Receiver operating curve analysis was performed to test the ability of PSQ in predicting moderate-to-severe OSA (AHI > 5 episode/h). The final sample included 60 children and adolescents. The PSQ showed a good area under the curve (AUC) of 0.88 (95% CI 0.78−0.98, p < 0.0001). Moreover, a value above or equal to 0.65 showed an 80% sensitivity and 100% specificity for moderate and severe OSA. These findings suggest that PSQ might be used in clinical settings with limited access to PSG for stratifying disease severity and for selecting children with urgent need of sleep study.

Secondary Complications in COVID-19 Patients: A Case Series.

INTRODUCTION: Coronavirus SARS-CoV-2, the causative agent of COVID-19, primarily causes a respiratory tract infection that is not limited to respiratory distress syndrome, but it is also implicated in other body systems. Systemic complications were reported due to an exaggerated inflammatory response, which involves severe alveolar damage in the lungs and exacerbates the hypercoagulation that leads to venous thrombosis, ischemic attack, vascular dysfunction and infarction of visceral abdominal organs. Some complications are related to anticoagulant drugs that are administrated to stabilize hypercoagulability, but increase the risk of bleeding, hematoma and hemorrhage. The aim of this study is to report the diagnostic role of CT in the early diagnosis and management of patients with severe COVID-19 complications through the most interesting cases in our experience. MATERIAL AND METHODS: The retrospective analysis of patients studied for COVID-19 in our institution and hospitals, which are part of the university training network, was performed. CASES: Pneumomediastinum, cortical kidney necrosis, splenic infarction, cerebral ischemic stroke, thrombosis of the lower limb and hematomas are the most major complications that are reviewed in this study. CONCLUSIONS: Since the onset of the COVID-19 pandemic, the CT imaging modality with its high sensitivity and specificity remains the preferred imaging choice to diagnose early the different complications associated with COVID-19, such as thrombosis, ischemic stroke, infarction and pneumomediastinum, and their management, which significantly improved the outcomes.

Co-Expression of Nogo-A in Dopaminergic Neurons of the Human Substantia Nigra Pars Compacta Is Reduced in Parkinson's Disease.

Parkinson's disease is mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Together with the small number, the high vulnerability of the dopaminergic neurons is a major pathogenic culprit of Parkinson's disease. Our previous findings of a higher survival of dopaminergic neurons in the substantia nigra co-expressing Nogo-A in an animal model of Parkinson's disease suggested that Nogo-A may be associated with dopaminergic neurons resilience against Parkinson's disease neurodegeneration. In the present study, we have addressed the expression of Nogo-A in the dopaminergic neurons in the substantia nigra in postmortem specimens of diseased and non-diseased subjects of different ages. For this purpose, in a collaborative effort we developed a tissue micro array (TMA) that allows for simultaneous staining of many samples in a single run. Interestingly, and in contrast to the observations gathered during normal aging and in the animal model of Parkinson's disease, increasing age was significantly associated with a lower co-expression of Nogo-A in nigral dopaminergic neurons of patients with Parkinson's disease. In sum, while Nogo-A expression in dopaminergic neurons is higher with increasing age, the opposite is the case in Parkinson's disease. These observations suggest that Nogo-A might play a substantial role in the vulnerability of dopaminergic neurons in Parkinson's disease.

Tocilizumab Reduces Vasospasms, Neuronal Cell Death, and Microclot Formation in a Rabbit Model of Subarachnoid Hemorrhage.

Early brain injury (EBI), delayed cerebral vasospasm (DCVS), and delayed cerebral ischemia (DCI) are common complications of subarachnoid hemorrhage (SAH). Inflammatory processes in the cerebrospinal fluid (CSF) are one of the causes for such complications. Our aim to study the effects of an IL-6 receptor antagonist (Tocilizumab) examines the occurrence of DCVS, neuronal cell death, and microclot formation in an acute SAH rabbit model. Twenty-nine New Zealand white rabbits were randomized into one of three groups as the SAH, SAH + Tocilizumab, and sham groups. In SAH groups, hemorrhage was induced by extracranial-intracranial arterial blood shunting from the subclavian artery into the cisterna magna under intracranial pressure (ICP) monitoring. In the second group, Tocilizumab was given once intravenously 1 h after SAH induction. Digital subtraction angiography was performed, and CSF and blood were sampled before and after (day 3) SAH induction. IL-6 plasma and CSF levels were measured. TUNEL, FJB, NeuN, and caspase-3 immunostaining were used to assess cell apoptosis, neurodegeneration, and neuronal cell death, respectively. Microclot formation was detected by fibrinogen immunostaining. Between baseline and follow-up, there was a significant reduction of angiographic DCVS (p < 0.0001) in the Tocilizumab compared with the SAH group. Tocilizumab treatment resulted in decreased neuronal cell death in the hippocampus (p = 0.006), basal cortex (p = 0.001), and decreased microclot formation (p = 0.02). Tocilizumab reduced DCVS, neuronal cell death, and microclot formation in a rabbit SAH model, and could be a potential treatment to prevent DCVS and DCI in SAH patients.

Tumor Treating Fields - Behind and Beyond Inhibiting the Cancer Cell Cycle.

The unmet need for a safe treatment that significantly improves the overall survival, as well as the quality of life of patients with brain tumors, has urged researchers to work out new treatment modalities. About 15 years ago, it was shown that alternating electric fields significantly impair the growth of cancer cells. Recently, this potentially revolutionary approach called Tumor Treating Fields (TTFs) has been FDA-approved for the treatment of glioblastoma as well as mesothelioma. However, despite the promising reports on the potential of TTFs, the precise knowledge of the mechanisms of action is still lacking. The purpose of this review is, thus, to present the current state of research and to highlight the variety of ultrastructural effects of TTFs. Moreover, the aim is to bring to the foreground less discussed mechanisms of action of TTFs, which might develop into novel therapeutic approaches. Therefore, a systematic literature search in Ovid Medline and Embase was performed on clinical and preclinical data concerning TTFs. The alternating electric fields force cellular components to aberrant dynamics, among which the most evident is the inhibition of the mitotic spindle assembly leading to impaired cancer cell division and cell death. However, a variety of other microstructural events induced by TTFs, such as inhibition of DNA repair and cell migration, as well as an enhancement of anti- tumor immune response and membrane permeability, have been reported. In addition, apart from a suggested interference with angiogenesis, no TTF-induced effects on normal cells have been described so far.

The Role of Sartans in the Treatment of Stroke and Subarachnoid Hemorrhage: A Narrative Review of Preclinical and Clinical Studies.

Background: Delayed cerebral vasospasm (DCVS) due to aneurysmal subarachnoid hemorrhage (aSAH) and its sequela, delayed cerebral ischemia (DCI), are associated with poor functional outcome. Endothelin-1 (ET-1) is known to play a major role in mediating cerebral vasoconstriction. Angiotensin-II-type-1-receptor antagonists such as Sartans may have a beneficial effect after aSAH by reducing DCVS due to crosstalk with the endothelin system. In this review, we discuss the role of Sartans in the treatment of stroke and their potential impact in aSAH. Methods: We conducted a literature research of the MEDLINE PubMed database in accordance with PRISMA criteria on articles published between 1980 to 2019 reviewing: "Sartans AND ischemic stroke". Of 227 studies, 64 preclinical and 19 clinical trials fulfilled the eligibility criteria. Results: There was a positive effect of Sartans on ischemic stroke in both preclinical and clinical settings (attenuating ischemic brain damage, reducing cerebral inflammation and infarct size, increasing cerebral blood flow). In addition, Sartans reduced DCVS after aSAH in animal models by diminishing the effect of ET-1 mediated vasoconstriction (including cerebral inflammation and cerebral epileptogenic activity reduction, cerebral blood flow autoregulation restoration as well as pressure-dependent cerebral vasoconstriction). Conclusion: Thus, Sartans might play a key role in the treatment of patients with aSAH.

Sex differences in implantable cardiac defibrillator therapy according to arrhythmia detection times.

OBJECTIVE: In implantable cardiac defibrillators (ICDs), long-detection times safely reduce unnecessary and inappropriate therapies. We aimed to evaluate ICD treatment of ventricular arrhythmias in women, compared with men, also taking into account ICD detection. METHODS: The Advance III trial randomised patients implanted with an ICD for primary or secondary prevention in two arms-long and nominal ventricular arrhythmias detection times before therapy delivering (number of intervals needed to detect (NID) 30/40 and 18/24, respectively). The main endpoint of this post hoc analysis was the incidence of ICD therapies evaluated through Kaplan-Meier method and univariate Cox regression models. RESULTS: Overall, 1902 patients (304 women, 65±11 years) were randomised. Women showed a lower risk of ICD therapy (HR 0.63, 95% CI 0.43 to 0.93, p=0.022); this difference was observed only in the long-detection arm (HR 0.37, p=0.013) and not in the short detection arm (HR 0.82, p=0.414). No significant sex differences were observed concerning inappropriate therapies and mortality rate. Long-detection settings significantly reduced overall ICD therapies and appropriate ICD therapies, both in women (overall HR 0.31, p=0.007; appropriate HR 0.33, p=0.033) and in men (overall HR 0.69, p=0.006; appropriate HR 0.73, p=0.048). CONCLUSIONS: In patients with ICDs, the strategy of setting a long-detection time to treat ventricular arrhythmias (NID 30/40) reduces overall delivered therapies, both in women and men, when compared with nominal setting (NID 18/24). The reduction was significantly higher in women. Overall, women were less likely to experience ICD therapies than men; this result was only observed in the long-detection arm. CLINICAL TRIAL REGISTRATION: NCT00617175.

Another win for endothelial progenitor cells: Endothelial progenitor cell-derived conditioned medium promotes proliferation and exerts neuroprotection in cultured neuronal progenitor cells.

Progress in stem cell research demonstrates stem cells' potential for treating neurodegenerative diseases. Stem cells have proliferative/differentiative properties and produce a variety of paracrine factors that can potentially be used to regenerate nervous tissue. Previous studies have shown the positive regenerative effects of endothelial progenitor cells (EPCs), and thus, they may be used as a tool for regeneration. A study by Di Santo et al. explored whether EPC-derived conditioned medium (EPC-CM) promotes the survival of cultured striatal progenitor cells and attempted to find the paracrine factors and signaling pathways involved with EPC-CM's effects. The neuronal progenitor cells that were cultured with EPC-CM had much higher densities of GABA-immunoreactive (GABA-ir) neurons. It was shown that phosphatidylinositol-3-kinase/AKT and mitogen-activated protein kinase/ERK signaling pathways are involved in the proliferation of GABAergic neurons, as inhibition of these pathways decreased GABAergic densities. In addition, the results suggest that paracrine factors from EPC, both proteinaceous and lipidic, significantly elevated the viability and/or differentiation in the cultures. Importantly, it was found that EPC-CM provided neuroprotection against toxins from 3-nitropropionic acid. In sum, EPC-CM engendered proliferation and regeneration of the cultured striatal cells through paracrine factors and imparted neuroprotection. Furthermore, the effects of EPC-CM may generate a cell-free therapeutic strategy to address neurodegeneration.

Conditioned medium from Endothelial Progenitor Cells promotes number of dopaminergic neurons and exerts neuroprotection in cultured ventral mesencephalic neuronal progenitor cells.

Transplantation of stem and progenitor cells offers a promising tool for brain repair in the context of neuropathological disorders including Parkinson's disease. There is growing proof that the capacity of adult stem and progenitor cells for tissue regeneration relies rather on the release of paracrine factors than on their cell replacement properties. In line with this notion, we have previously reported that conditioned medium (CM) collected from cultured Endothelial Progenitor Cells (EPC) stimulated survival of striatal neurons. In the present study we investigated whether EPC-CM promotes survival of cultured midbrain progenitor cells. For that purpose primary cultures from fetal rat embryonic ventral mesencephalon (VM) were prepared and grown for 7 days in vitro (DIV). EPC-CM was administered from DIV5-7. First, we found that EPC-CM treatment resulted in significantly increased cell densities of TH-ir neurons. Interestingly, this effect was no longer seen after proteolytic digestion of the EPC-CM. EPC-CM also significantly increased densities of beta-III-tubulin positive neurons and lba-1-ir microglial cells. The effect on dopaminergic neurons was not due to higher cell proliferation as no incorporation of EdU was observed in TH-ir cells. Importantly, EPC-CM exerted neuroprotection against MPP+ induced toxicity as in vitro model of Parkinson's disease. Taken together, our findings identified EPC-CM as a powerful tool to promote survival of cultured VM neurons and further support the importance of paracrine factors in the actions of stem and progenitor cells for brain repair.

Systemic and CSF Interleukin-1α Expression in a Rabbit Closed Cranium Subarachnoid Hemorrhage Model: An Exploratory Study.

BACKGROUND: The inflammatory pathway in cerebrospinal fluid (CSF) leads to delayed cerebral vasospasm (DCVS) and delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH). The role of IL-1α has never been evaluated in a rabbit SAH model. The aim of our study is to analyze systemic and CSF changes of IL-1α, and to evaluate potential associations with the onset of DCVS in a rabbit closed cranium SAH model. Methods: 17 New Zealand white rabbits were randomized into two groups, SAH (n = 12) and sham (n = 5). In the first group, SAH was induced by extracranial-intracranial shunting from the subclavian artery into the cerebral cistern of magna under intracranial pressure (ICP) monitoring. The sham group served as a control. The CSF and blood samples for IL-1α measurement were taken at day zero before SAH induction and at day three. RESULTS: There was a significant increase of ICP (p = 0.00009) and a decrease of cerebral perfusion pressure (CPP) (p = 0.00089) during SAH induction. At follow up, there was a significant increase of systemic IL-1α in the SAH as compared with the sham group (p = 0.042). There was no statistically significant difference in the CSF values in both groups. The CSF IL-1α values showed a correlation trend of DCVS. CONCLUSIONS: Systemic IL-1α levels are elevated after SAH induction in a rabbit SAH model.

Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5.

BACKGROUND: Oxidized low density lipoprotein (oxLDL) has been shown to induce apoptosis and senescence of endothelial progenitor cells (EPC). In the present study, we hypothesized that even sub-apoptotic concentrations of oxLDL impair the angiogenic potential of EPC and investigated if this effect is mediated by affecting adhesion and incorporation. METHODS: A co-culture system of human microvascular endothelial cells and EPC was used to study the effect of sub-apoptotic concentrations of native (nLDL) and oxLDL on cell-cell interaction. The expression and the functional role of angiogenic adhesion molecules and integrins was monitored by FACS and neutralizing assay, respectively. RESULTS: We observed an inhibition of tube formation and impairment of EPC integration into the vascular network of mature endothelial cells by oxLDL. In contrast, nLDL did not affect angiogenic properties of EPC. Incubation of EPC with sub-apoptotic oxLDL concentrations significantly decreased E-selectin and integrin alpha(v)beta(5) expression (37.6% positive events vs. 71.5% and 24.3% vs. 49.9% compared to control culture media without oxLDL). Interestingly, expression of alpha(v)beta(3), VE-cadherin and CD31 remained unchanged. Blocking of E-selectin and integrin alpha(v)beta(5) by neutralizing antibody effectively inhibited adhesion of EPC to differentiated endothelial cells (56.5% and 41.9% of control; p<0.001). CONCLUSION: In conclusion, oxidative alteration of LDL impairs angiogenic properties of EPC at sub-apoptotic levels by downregulation of E-selectin and integrin alpha(v)beta(5), both substantial mediators of EPC-endothelial cell interaction.

Severe structural damage of the seemingly non-diseased infrarenal aortic aneurysm neck.

OBJECTIVE: The success of open and endovascular repair of abdominal aortic aneurysms (AAA) is hampered by postoperative dilatation of the anatomical neck of the AAA, which is used for graft attachment. The purpose of this study was to determine whether the macroscopically non-diseased infrarenal aortic neck of AAA is histologically and biochemically altered at the time of operative repair. METHODS: We harvested full-thickness aortic wall samples as longitudinal stripes spanning from AAA neck to aneurysmal sac in 22 consecutive patients undergoing open surgical AAA repair. Control tissue was obtained from five organ donors and five deceased subjects undergoing autopsy without evidence of aneurysmal disease. We assessed aortic media thickness, number of intact elastic lamellar units, media destruction, and neovascularization grade and performed immunohistochemistry for matrix metalloproteinase (MMP)-9 and phosphorylated c-Jun N-terminal kinase (p-JNK). MMP-9 and p-JNK protein expressions were quantified using Western Blots. RESULTS: The median thickness of the aortic media was 1150 mum in control tissue (range, 1000-1300), 510 mum in aortic necks (250-900), and 200 mum in aortic sacs (50-500, P from nonparametric test for trend <.001). The number of intact elastic lamellar units was 33 in controls (range, 33-55), 12 in aortic necks (0-31) and three in aortic sacs (0-10, P < .001). The expression of MMP-9 and p-JNK as assessed by Western Blots (P = .007 and .061, respectively) and zymography (P for trend <.001) were up regulated in both the AAA neck and sac compared with controls. Except for p-JNK expression, differences between tissues were similar after the adjustment for age, gender, and type of sampling. CONCLUSION: The seemingly non-diseased infrarenal AAA neck in patients with AAA undergoing surgical repair shows histological signs of destruction and upregulation of potential drug targets.

Neurotrophic factor-based strategies to enhance survival and differentiation of neural progenitor cells toward the dopaminergic phenotype.

Parkinson's disease (PD) is a neurodegenerative disorder that presents with hallmark clinical symptoms of tremor at rest, bradykinesia, and muscle rigidity. Stem cell therapy has emerged as an experimental treatment for PD. However, optimizing the cell culture condition that allows enhanced survival and differentiation of cells toward the dopaminergic phenotype remains a logistical challenge. Here, we discuss the utility of a combination of neurotrophin-4/5 (NT-4/5) and glial cell line-derived neurotrophic factor (GDNF) in increasing the dopaminergic phenotypic expression of rat ventral mesencephalic (VM) tissue. Using organotypic explant cultures of fetal human ventral mesencephalon, we observed that NT-4/5 and GDNF as single factors, or in combination on DAergic neurons, increased survival and number of tyrosine hydroxylase immunoreactive neurons as well as the dopamine content in the culture medium. The application of specific neurotrophic factors, such as NT-4/5 and GDNF, as cell culture supplements or as adjunctive therapy to cell transplantation may achieve improved functional outcomes when contemplating cell-based regenerative medicine for PD.

A Combination of NT-4/5 and GDNF Is Favorable for Cultured Human Nigral Neural Progenitor Cells.

Idiopathic Parkinson's disease (PD) is a progressive neurodegenerative disorder, clinically manifested by cardinal motor symptoms including tremor at rest, bradykinesia, and muscle rigidity. Transplantation of dopaminergic (DAergic) neurons is an experimental therapy for PD, however, it is limited by suboptimal integration and low survival of grafts. Pretreatment of donor tissue may offer a strategy to improve properties of transplanted DAergic neurons and thereby clinical outcome. We have previously shown that a combination of neurotrophin-4/5 (NT-4/5) and glial cell line-derived neurotrophic factor (GDNF) demonstrated additive effects on rat ventral mesencephalic (VM) tissue. The present study investigated the effects of NT-4/5 and GDNF as single factors, or in combination on DAergic neurons, in organotypic explant cultures of fetal human ventral mesencephalon. For that purpose, free-floating roller-tube cultures were prepared from VM and the equally sized pieces grown for 1 week in the presence or absence of neurotrophic factors. Both neurotrophic factors increased dopamine content in the culture medium and in the number of tyrosine hydroxylase immunoreactive neurons, most prominently after combined GDNF + NT-4/5 treatment. Culture volumes did not differ between groups while content of lactate dehydrogenase in the culture medium was moderately reduced in all treated groups. In conclusion, we identified that a combination of GDNF and NT-4/5 robustly promoted differentiation and survival of human fetal VM DAergic neurons, an observation with potential promising impact for cell replacement approaches in PD.

Nogo-receptor 1 antagonization in combination with neurotrophin-4/5 is not superior to single factor treatment in promoting survival and morphological complexity of cultured dopaminergic neurons.

Cell transplantation using ventral mesencephalic tissue is an experimental approach to treat Parkinson's disease. This approach is limited by poor survival of the transplants and the high number of dopaminergic neurons needed for grafting. Increasing the yield of dopaminergic neurons in donor tissue is of great importance. We have previously shown that antagonization of the Nogo-receptor 1 by NEP1-40 promoted survival of cultured dopaminergic neurons and exposure to neurotrophin-4/5 increased dopaminergic cell densities in organotypic midbrain cultures. We investigated whether a combination of both treatments offers a novel tool to further improve dopaminergic neuron survival. Rat embryonic ventral mesencephalic neurons grown as organotypic free-floating roller tube or primary dissociated cultures were exposed to neurotrophin-4/5 and NEP1-40. The combined and single factor treatment resulted in significantly higher numbers of tyrosine hydroxylase positive neurons compared to controls. Significantly stronger tyrosine hydroxylase signal intensity was detected by Western blotting in the combination-treated cultures compared to controls but not compared to single factor treatments. Neurotrophin-4/5 and the combined treatment showed significantly higher signals for the neuronal marker microtubule-associated protein 2 in Western blots compared to control while no effects were observed for the astroglial marker glial fibrillary acidic protein between groups, suggesting that neurotrophin-4/5 targets mainly neuronal cells. Finally, NEP1-40 and the combined treatment significantly augmented tyrosine hydroxylase positive neurite length. Summarizing, our findings substantiate that antagonization of the Nogo-receptor 1 promotes dopaminergic neurons but does not further increase the yield of dopaminergic neurons and their morphological complexity when combined with neurotrophin-4/5 hinting to the idea that these treatments might exert their effects by activating common downstream pathways.

Antagonization of the Nogo-Receptor 1 Enhances Dopaminergic Fiber Outgrowth of Transplants in a Rat Model of Parkinson's Disease.

Intrastriatal transplantation of fetal human ventral mesencephalic dopaminergic neurons is an experimental therapy for patients suffering from Parkinson's disease. The success of this approach depends on several host brain parameters including neurotrophic factors and growth inhibitors that guide survival and integration of transplanted neurons. While the potential of neurotrophic factors has been extensively investigated, repression of growth inhibitors has been neglected, despite the significant effects reported in various CNS injury models. Recently, we demonstrated that infusion of neutralizing antibodies against Nogo-A into the lateral ventricles of hemi-parkinsonian rats significantly enhanced graft function. Since the Nogo-receptor 1 also interacts with other neurite growth inhibitors, we investigated whether a direct antagonization of the receptor would result in more robust effects. Therefore, rats with unilateral striatal 6-hydroxydopamine lesions were grafted with ventral mesencephalic tissue in combination with intraventricular infusions of the Nogo-receptor 1 antagonist NEP1-40. Transplanted rats receiving saline infusions served as controls. To test whether NEP1-40 treatment alone affects the remaining dopaminergic striatal fibers, rats with unilateral striatal 6-hydroxydopamine lesions were infused with NEP1-40 or saline without receiving a transplant. Motor behavior was assessed prior to the lesion as well as prior and 1, 3, and 5 weeks after the transplantations. At the end of the experimental period the number of graft-derived dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons and graft volume were analyzed. In rats without a transplant, the density of dopaminergic fibers in the striatum was analyzed. We detected that NEP1-40 treatment significantly enhanced graft-derived dopaminergic fiber outgrowth as compared to controls while no effects were detected for graft volume and survival of grafted dopaminergic neurons. Notably, the enhanced dopaminergic fiber outgrowth was not sufficient to improve the functional recovery as compared to controls. Moreover, NEP1-40 infusions in hemi-parkinsonian rats without a transplant did not result in enhanced striatal dopaminergic fiber densities and consequently did not improve behavior. In sum, our findings demonstrate that antagonization of the Nogo-receptor 1 has the capacity to support the engraftment of transplanted mesencephalic tissue in an animal model of Parkinson's disease.