Aneurysm healing following treatment with biodegradable embolization materials: assessment in a rat sidewall aneurysm model.

BACKGROUND: Biodegradable materials that dissolve after aneurysm healing are promising techniques in the field of neurointerventional surgery. We investigated the effects of various bioabsorable materials in combination with degradable magnesium alloy stents and evaluated aneurysm healing in a rat aneurysm model. METHODS: Saccular aneurysms were created by end-to-side anastomosis in the abdominal aorta of Wistar rats. Untreated arterial grafts were immediately transplanted (vital aneurysms) whereas aneurysms with loss of mural cells were chemically decellularized before implantation. All aneurysms were treated with biodegradable magnesium stents. The animals were assigned to vital aneurysms treated with stent alone or decellularized aneurysms treated with stent alone, detachable coil, or long-term or short-term biodegradable thread. Aneurysm healing, rated microscopically and macroscopically at follow-up days 7 and 21, was defined by both neointima formation and absence of aneurysm volume increase over time. RESULTS: Of 56 animals included, significant increases in aneurysm volume 7 days after surgery were observed in aneurysms with vital and decellularized walls treated with a stent only (P=0.043 each group). Twenty-one days after surgery an increase in aneurysm volume was observed in decellularized aneurysms treated with long- and short-term biodegradable threads (P=0.027 and P=0.028, respectively). Histological changes associated with an increase in aneurysm volume were seen for aneurysm wall inflammation, periadventitial fibrosis, and luminal thrombus. CONCLUSIONS: An increase in aneurysm volume was associated with an absence of intrasaccular embolization material (early phase) and the breakdown of intrasaccular biodegradable material over time (late phase). Thrombus remnant and aneurysm wall inflammation promote aneurysm volume increase.

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.

Topographic distribution of inflammation factors in a healing aneurysm.

BACKGROUND: Healing of intracranial aneurysms following endovascular treatment relies on the organization of early thrombus into mature scar tissue and neointima formation. Activation and deactivation of the inflammation cascade plays an important role in this process. In addition to timely evolution, its topographic distribution is hypothesized to be crucial for successful aneurysm healing. METHODS: Decellularized saccular sidewall aneurysms were created in Lewis rats and coiled. At follow-up (after 3 days (n = 16); 7 days (n = 19); 21 days (n = 8)), aneurysms were harvested and assessed for healing status. In situ hybridization was performed for soluble inflammatory markers (IL6, MMP2, MMP9, TNF-α, FGF23, VEGF), and immunohistochemical analysis to visualize inflammatory cells (CD45, CD3, CD20, CD31, CD163, HLA-DR). These markers were specifically documented for five regions of interest: aneurysm neck, dome, neointima, thrombus, and adjacent vessel wall. RESULTS: Coiled aneurysms showed enhanced patterns of thrombus organization and neointima formation, whereas those without treatment demonstrated heterogeneous patterns of thrombosis, thrombus recanalization, and aneurysm growth (p = 0.02). In coiled aneurysms, inflammation markers tended to accumulate inside the thrombus and in the neointima (p < 0.001). Endothelial cells accumulated directly in the neointima (p < 0.0001), and their presence was associated with complete aneurysm healing. CONCLUSION: The presence of proinflammatory cells plays a crucial role in aneurysm remodeling after coiling. Whereas thrombus organization is hallmarked by a pronounced intra-thrombotic inflammatory reaction, neointima maturation is characterized by direct invasion of endothelial cells. Knowledge concerning topographic distribution of regenerative inflammatory processes may pave the way for future treatment modalities which enhance aneurysm healing after endovascular therapy.

Human pluripotent stem cells-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 (iPSCs) 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 human iPSC-derived 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 critical markers at these equivalent stages in human embryos. We show that the placode derived in vitro (days 8-12) has similar marker expression to the developing otic placode of Carnegie Stage (CS) 11 embryos and subsequently (days 20-40) this gives rise to otic epithelia and neuroblasts comparable to the CS13 embryonic stage. Differentiation of sensory epithelia, including supporting cells and hair cells starts in vitro at days 50-60 of culture. The maturity of these cells is equivalent to vestibular sensory epithelia at week 10 or cochlear tissue at week 12 of development, before functional onset. 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.

Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model.

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and thrombus formation. The source of endothelial cells covering the endoluminal layer of the neointima remains unclear. Therefore, the aim of the present study was to investigate the origin of neointima-forming cells after cell-tracer injection in the already well-established Helsinki rat microsurgical sidewall aneurysm model. Sidewall aneurysms were created by suturing decellularized or vital arterial pouches end-to-side to the aorta in male Lewis rats. Before arteriotomy with aneurysm suture, a cell-tracer injection containing CM-Dil dye was performed into the clamped aorta to label endothelial cells in the adjacent vessel and track their proliferation during follow-up (FU). Treatment followed by coiling (n = 16) or stenting (n = 15). At FU (7 days or 21 days), all rats underwent fluorescence angiography, followed by aneurysm harvesting and macroscopic and histological evaluation with immunohistological cell counts for specific regions of interest. None of the 31 aneurysms had ruptured upon follow-up. Four animals died prematurely. Macroscopically residual perfusion was observed in 75.0% coiled and 7.0% of stented rats. The amount of cell-tracer-positive cells was significantly elevated in decellularized stented compared to coiled aneurysms with respect to thrombus on day 7 (p = 0.01) and neointima on day 21 (p = 0.04). No significant differences were found in thrombus or neointima in vital aneurysms. These findings confirm worse healing patterns in coiled compared to stented aneurysms. Neointima formation seems particularly dependent on the parent artery in decellularized aneurysms, whereas it is supported by the recruitment from aneurysm wall cells in vital cell-rich walls. In terms of translation, stent treatment might be more appropriate for highly degenerated aneurysms, whereas coiling alone might be adequate for aneurysms with mostly healthy vessel walls.

Parent artery-initiated and stent-mediated neointima formation in a rat saccular side wall model.

BACKGROUND: Unlike clipping that forms an immediate barrier of blood flow into intracranial aneurysms, endovascular treatments rely on thrombus organization and neointima formation. Therefore, a continuous endothelial cell layer is crucial to prevent blood flow in the former aneurysm. This study investigates the origin of endothelial cells in the neointima of endovascular treated aneurysms, specifically whether cells from the parent artery play a role in neointima formation. METHODS: In male rats, decellularized and vital side wall aneurysms were treated by coil (n=16) or stent embolization (n=15). The cell tracer CM-Dil dye was injected into the clamped aorta before aneurysm suture to mark initial endothelial cells in the parent artery and enable tracking of their proliferation during follow-up. Aneurysms were analyzed for growth, thrombus formation, and recurrence. Histological evaluation followed with cell counts for specific regions-of-interest. RESULTS: During follow-up, none of the 31 aneurysms ruptured. Macroscopic residual perfusion was observed in 12/16 rats after coiling and in 1/15 after stenting. Amounts of CM-Dil +cells in coiled versus stented decellularized aneurysms significantly decreased in the thrombus on day 7 (p=0.01) and neointima on day 21 (p=0.04). For vital aneurysms, the number of CM-Dil +cells in the neointima on day 21 showed no significant difference. CONCLUSIONS: Healing patterns were worse in coil-treated than stent-treated aneurysms. Cell migration forming a neointima seemed mainly dependent on the adjacent vessel in decellularized aneurysms, but appeared buoyed by recruitment from aneurysm wall cells in vital aneurysms. Therefore, a cell-rich parent artery might be crucial.

Aspirin treatment prevents inflammation in experimental bifurcation aneurysms in New Zealand White rabbits.

BACKGROUND: Aneurysm wall degeneration is linked to growth and rupture. To address the effect of aspirin (ASA) on aneurysm formation under various wall conditions, this issue was analyzed in a novel rabbit bifurcation model. METHODS: Bifurcation aneurysms created in 45 New Zealand White rabbits were randomized to vital (n=15), decellularized (n=13), or elastase-degraded (n=17) wall groups; each group was assigned to a study arm with or without ASA. At follow-up 28 days later, aneurysms were evaluated for patency, growth, and wall inflammation at macroscopic and histological levels. RESULTS: 36 rabbits survived to follow-up at the end of the trial. None of the aneurysms had ruptured. Patency was visualized in all aneurysms by intraoperative fluorescence angiography and confirmed in 33 (92%) of 36 aneurysms by MRI/MRA. Aneurysm size was significantly increased in the vital (without ASA) and elastase-degraded (with and without ASA) groups. Aneurysm thrombosis was considered complete in three (50%) of six decellularized aneurysms without ASA by MRI/MRA. Locoregional inflammation of the aneurysm complex was significantly reduced in histological analysis among all groups treated with ASA. CONCLUSION: ASA intake prevented inflammation of both the periadventitial tissue and aneurysm wall, irrespective of initial wall condition. Although ASA prevented significant growth in aneurysms with vital walls, this preventive effect did not have an important role in elastase-degraded pouches. In possible translation to the clinical situation, ASA might exert a potential preventive effect during early phases of aneurysm formation in patients with healthy vessels but not in those with highly degenerative aneurysm walls.

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.

Persistent bone impairment despite long-term control of hyperprolactinemia and hypogonadism in men and women with prolactinomas.

While prolactinoma patients have high bone turnover, current data are inconclusive when it comes to determining whether correction of hyperprolactinemia and associated hypogandism improves osteodensitometric data in men and women over the long term. In a large cohort of including 40 men and 60 women, we studied the long-term impact of prolactinoma treatment on bone mineral density (BMD) in men versus women, assessed adverse effects of a primary surgical or medical approach, and evaluated data for risk factors for impaired BMD at last follow-up using multivariate regression analyses. Median duration of follow-up was 79 months (range 13-408 months). Our data indicate that the prevalence of impaired BMD remained significantly higher in men (37%) than in women (7%, p < 0.001), despite the fact that hyperprolactinemia and hypogonadism are under control in the majority of men. We found that persistent hyperprolactinemia and male sex were independent risk factors for long-term bone impairment. Currently, osteoporosis prevention and treatment focus primarily on women, yet special attention to bone loss in men with prolactinomas is advised. Bone impairment as "end organ" reflects the full range of the disease and could become a surrogate marker for the severity of long-lasting hyperprolactinemia and associated hypogonadism.