Intraperitoneal delivery of acetate-encapsulated liposomal nanoparticles for neuroprotection of the penumbra in a rat model of ischemic stroke.

BACKGROUND: Ischemic stroke is a devastating condition, with metabolic derangement and persistent inflammation enhancing the initial insult of ischaemia. Recombinant tissue plasminogen remains the only effective treatment but limited as therapy must commence soon after the onset of symptoms. PURPOSE: We investigated whether acetate, which modulates many pathways including inflammation, may attenuate brain injury in stroke. As acetate has a short blood half-life and high amounts irritate the gastrointestinal tract, acetate was administered encapsulated in a liposomal nanoparticle (liposomal-encapsulated acetate, LITA). METHODS: Transient ischemia was induced by 90 mins middle-cerebral artery occlusion (MCAO) in Sprague-Dawley rats, and LITA or control liposomes given intraperitoneally at occlusion and daily for up to two weeks post-MCAO. Magnetic resonance imaging (MRI) was used to estimate lesion volume at 24 h, 1 and 2 weeks post-MCAO and anterior lateral ventricular volume (ALVv) at 2 weeks post-MCAO. Locomotive behaviour was tested prior to the final MRI scan. After the final scan, brains were collected, and immunohistochemistry was performed. RESULTS: Lesion volumes were decreased by ~80% from 24 h to one-week post-MCAO, in both control and LITA groups (P⩽0.05). However, the lesion was increased by ~50% over the subsequent 1 to 2 weeks after MCAO in the control group (from 24.1±10.0 to 58.7±28.6 mm3; P⩽0.05) but remained unchanged in the LITA group. ALVv were also attenuated by LITA treatment at 2 weeks post-MCAO (177.2±11.9% and 135.3±10.9% of contralateral ALVv for control and LITA groups, respectively; P⩽0.05). LITA-treated animals also appeared to have improved motor activity, moving with greater average velocity than control animals. Microglial immunoreactivity was ~40% lower in the LITA group compared to the control group (P⩽0.05), but LITA did not modulate neurogenesis, apoptosis, histone acetylation and lipid peroxidation. CONCLUSION: LITA appears to attenuate the harmful chronic neuroinflammation observed during brain remodeling after a focal ischemic insult.

Regional Distributions of Iron, Copper and Zinc and Their Relationships With Glia in a Normal Aging Mouse Model.

Microglia and astrocytes can quench metal toxicity to maintain tissue homeostasis, but with age, increasing glial dystrophy alongside metal dyshomeostasis may predispose the aged brain to acquire neurodegenerative diseases. The aim of the present study was to investigate age-related changes in brain metal deposition along with glial distribution in normal C57Bl/6J mice aged 2-, 6-, 19- and 27-months (n = 4/age). Using synchrotron-based X-ray fluorescence elemental mapping, we demonstrated age-related increases in iron, copper, and zinc in the basal ganglia (p < 0.05). Qualitative assessments revealed age-associated increases in iron, particularly in the basal ganglia and zinc in the white matter tracts, while copper showed overt enrichment in the choroid plexus/ventricles. Immunohistochemical staining showed augmented numbers of microglia and astrocytes, as a function of aging, in the basal ganglia (p < 0.05). Moreover, qualitative analysis of the glial immunostaining at the level of the fimbria and ventral commissure, revealed increments in the number of microglia but decrements in astroglia, in older aged mice. Upon morphological evaluation, aged microglia and astroglia displayed enlarged soma and thickened processes, reminiscent of dystrophy. Since glial cells have major roles in metal metabolism, we performed linear regression analysis and found a positive association between iron (R 2 = 0.57, p = 0.0008), copper (R 2 = 0.43, p = 0.0057), and zinc (R 2 = 0.37, p = 0.0132) with microglia in the basal ganglia. Also, higher levels of iron (R 2 = 0.49, p = 0.0025) and zinc (R 2 = 0.27, p = 0.040) were correlated to higher astroglia numbers. Aging was accompanied by a dissociation between metal and glial levels, as we found through the formulation of metal to glia ratios, with regions of basal ganglia being differentially affected. For example, iron to astroglia ratio showed age-related increases in the substantia nigra and globus pallidus, while the ratio was decreased in the striatum. Meanwhile, copper and zinc to astroglia ratios showed a similar regional decline. Our findings suggest that inflammation at the choroid plexus, part of the blood-cerebrospinal-fluid barrier, prompts accumulation of, particularly, copper and iron in the ventricles, implying a compromised barrier system. Moreover, age-related glial dystrophy/senescence appears to disrupt metal homeostasis, likely due to induced oxidative stress, and hence increase the risk of neurodegenerative diseases.

Importance of Proactive Treatment of Depression in Lewy Body Dementias: The Impact on Hippocampal Neurogenesis and Cognition in a Post-Mortem Study.

OBJECTIVE: To examine the impact of selective serotonin reuptake inhibitors (SSRIs) and depression on neurogenesis and cognition in dementia with Lewy bodies (DLB) and Parkinson disease dementia (PDD). METHODS: Late-stage progenitor cells were quantified in the subgranular zone (SGZ) of the hippocampal dentate gyrus of DLB/PDD patients (n = 41) and controls without dementia (n = 15) and compared between treatment groups (unmedicated, SSRIs, acetyl cholinesterase inhibitors [AChEIs], combined SSRIs and AChEIs). RESULTS: DLB/PDD patients had more doublecortin-positive cells in the SGZ compared to controls. The doublecortin-positive cell count was higher in the SGZ of patients treated with SSRIs and correlated to higher cognitive scores. CONCLUSION: SSRI treatment was associated with increased hippocampal neurogenesis and preservation of cognition in DLB/PDD patients.

Dissociation between iron accumulation and ferritin upregulation in the aged substantia nigra: attenuation by dietary restriction.

Despite regulation, brain iron increases with aging and may enhance aging processes including neuroinflammation. Increases in magnetic resonance imaging transverse relaxation rates, R2 and R2*, in the brain have been observed during aging. We show R2 and R2* correlate well with iron content via direct correlation to semi-quantitative synchrotron-based X-ray fluorescence iron mapping, with age-associated R2 and R2* increases reflecting iron accumulation. Iron accumulation was concomitant with increased ferritin immunoreactivity in basal ganglia regions except in the substantia nigra (SN). The unexpected dissociation of iron accumulation from ferritin-upregulation in the SN suggests iron dyshomeostasis in the SN. Occurring alongside microgliosis and astrogliosis, iron dyshomeotasis may contribute to the particular vulnerability of the SN. Dietary restriction (DR) has long been touted to ameliorate brain aging and we show DR attenuated age-related in vivo R2 increases in the SN over ages 7 - 19 months, concomitant with normal iron-induction of ferritin expression and decreased microgliosis. Iron is known to induce microgliosis and conversely, microgliosis can induce iron accumulation, which of these may be the initial pathological aging event warrants further investigation. We suggest iron chelation therapies and anti-inflammatory treatments may be putative 'anti-brain aging' therapies and combining these strategies may be synergistic.

Stage-specific changes in neurogenic and glial markers in Alzheimer's disease.

BACKGROUND: Reports of altered endogenous neurogenesis in people with Alzheimer's disease (AD) and transgenic AD models have suggested that endogenous neurogenesis may be an important treatment target, but there is considerable discrepancy among studies. We examined endogenous neurogenesis and glia changes across the range of pathologic severity of AD in people with and without dementia to address this key question. METHODS: Endogenous neurogenesis and glia in the subventricular zone and dentate gyrus neurogenic niches were evaluated using single and double immunohistochemistry and a validated antibody selection for stage-specific and type-specific markers in autopsy tissue from a representative cohort of 28 participants in the Medical Research Council Cognitive Function and Ageing Study. Immunopositive cells were measured blinded to diagnosis using bright-field and fluorescent microscopy. RESULTS: The number of newly generated neurons significantly declined only in the dentate gyrus of patients with severe tau pathology. No other changes in other neurogenic markers were observed in either of the neurogenic niches. Alterations in astrocytes and microglia were also observed in the dentate gyrus across the different stages of tau pathology. No change in any of the markers was observed in individuals who died with dementia compared with individuals who did not die with dementia. CONCLUSIONS: Alterations in endogenous neurogenesis appeared to be confined to a reduction in the generation of new neurons in the dentate gyrus of patients with AD and severe neurofibrillary tangle pathology and were accompanied by changes in the glia load. These data suggest that intervention enhancing endogenous neurogenesis may be a potential therapeutic target in AD.

Neurogenic abnormalities in Alzheimer's disease differ between stages of neurogenesis and are partly related to cholinergic pathology.

Neurogenesis occurs in the subventricular zone and the sub-granular layer of the hippocampus and is thought to take place in 5 stages, including proliferation, differentiation, migration, targeting, and integration phases, respectively. In Alzheimer's disease (AD) both increased and decreased neurogenesis has been reported and cholinergic activity is assumed to be involved in neurogenesis. The aim of this study was to systematically assess different phases of neurogenesis and their relation to AD and cholinergic pathology. We investigated post-mortem brain tissue from 20 AD patients and 21 non-demented controls that was neuropathologically characterized according to standardized criteria. Hippocampal sections were stained with antibodies against neurogenic markers Musashi-1, nestin, PSA-NCAM, doublecortin, and ß-III-tubulin as well as ChAT (choline-acetyltransferase). Using image analysis immunoreactivity was assessed in the subventricular zone, the sub-granular layer, and the granule cell layer by determining the integrated optical density. In the sub-granular layer and the granule cell layer Musashi-1 and ChAT immunoreactivities were significantly lower in AD and decreased with increasing Braak stages. Conversely, immunorreactivities of both nestin and PSA-NCAM were significantly higher in AD and increased with increasing Braak stages while no changes were seen for doublecortin and ß-III-tubulin, except for significantly higher doublecortin levels in the granule cell layer of AD cases. Of note, Musashi-1 immunoreactivity significantly correlated with ChAT immuonoreactivity across different Braak stages. In the subventricular zone only nestin immunoreactivity was significantly higher in AD and significantly increased with increasing Braak stages, while no significant differences were seen for all other markers. Our finding of a reduction of ChAT and Musashi-1 levels in AD is compatible with the assumption that cholinergic pathology per se has a detrimental influence on neurogenesis. We conclude that neurogenic abnormalities in AD differ between phases and areas of neurogenesis and stages of AD; while hippocampal stem cells (Musashi-1) decrease, proliferation (nestin) increases and differentiation/migration phase as well as axonal/dendritic targeting (doublecortin and ß-III-tubulin) remains virtually unchanged. This suggests an attenuation of stem cells together with compensatory increased proliferation that, however, does not result in an increased number of migratory neuroblasts and differentiated neurons in AD.

Increased neural progenitors in vascular dementia.

Since groundbreaking studies demonstrated the presence of progenitor cells in the adult human brain, there have been intense interests in their potential therapeutic application, but to date only limited data has been obtained in man. An immunohistological study was performed in order to examine neurogenesis in both the subventricular and peri-infarct zones of vascular dementia patients compared to age-matched controls. The results were striking, showing a significant increase of progenitor cells in both the subventricular zone and in peri-infarct area in patients with vascular dementia compared to controls, which was sustained even in patients with infarcts occurring more than three months prior to autopsy. Moreover, the peri-infarct response appeared to be unified with that of the subventricular zone via a stream of cells, with some of them differentiating into immature neurons. We conclude that neurogenesis is stimulated in vascular dementia patients and, specifically, in patients with visible infarcts. Progenitors may migrate from the neurogenic niche to areas of infarction and differentiate into neurons, even three months after cerebrovascular damage, thus implicating the feasibility of enhancing neurogenesis as a novel treatment approach.

Neurogenic marker abnormalities in the hippocampus in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is associated with alpha synuclein pathology and slowly progressive dementia. Progenitor abnormalities have previously been reported in the subventricular zone (SVZ) adjacent to the lateral ventricle. To evaluate changes in neural stem cells and progenitors in the hippocampal neurogenic niche, immunohistochemistry (IHC) using the neural stem cell markers Musashi 1, nestin, proliferating cell nuclear antigen (PCNA), doublecortin, and glial fibrillary acidic protein (GFAP) were examined in age-matched control and DLB groups. Staining was quantified in the hippocampal SVZ, subgranular layer (SGL) and ependymal cell layer (EPL). There was a significant loss in DLB of Musashi 1 (P < 0.01) in all areas, an increase in PCNA in hippocampal SVZ (P = 0.01) and SGL (P = 0.05), and an increase in doublecortin in the hippocampal SVZ (P = 0.04) and EPL (P = 0.02). This is the first report of the changes in neurogenic markers in the hippocampal SVZ and EPL in DLB and may offer the potential for understanding disease pathology and in the devising of treatment.

Endogenous neurogenesis in the human brain following cerebral infarction.

Increased endogenous neurogenesis has a significant regenerative role in many experimental models of cerebrovascular diseases, but there have been very few studies in humans. We therefore examined whether there was evidence of altered endogenous neurogenesis in an 84-year-old patient who suffered a cerebrovascular accident 1 week prior to death. Using antibodies that specifically label neural stem/neural progenitor cells, we examined the presence of immunopositive cells around and distant from the infarcted area, and compared this with a control, age-matched individual. Interestingly, a large number of neural stem cells, vascular endothelial growth factor-immunopositive cells and new blood vessels were observed only around the region of infarction, and none in the corresponding brain areas of the healthy control. In addition, an increased number of neural stem cells was observed in the neurogenic region of the lateral ventricle wall. Our results suggest increased endogenous neurogenesis associated with neovascularization and migration of newly-formed cells towards a region of cerebrovascular damage in the adult human brain and highlight possible mechanisms underlying this process.

Altered neurogenesis in Alzheimer's disease.

BACKGROUND: Exciting preliminary work indicates an increase in progenitor activity in the subgranular zone of the dentate gyrus of people with Alzheimer's disease (AD) compared to that of controls. We examine progenitor activity in the other main progenitor niche, the subventricular zone (SVZ), as well as potential associations with key pathological and neurochemical substrates. METHOD: Immunocytochemistry techniques utilizing nestin and Musashi1 antibodies were used to examine progenitor activity in the SVZ and to enable comparisons between seven patients with AD and seven controls, based upon the quantification of the percentage area covered, using the Image Pro Plus v.4.1 image analysis system. AD pathology was staged using the Consortium to Establish a Registry for Alzheimer's Disease and Braak criteria. Choline acetyl transferase (ChAT) was measured in the temporal cortex as an indication of the severity of cortical cholinergic deficits. Glial fibrillary acidic protein (GFAP) was used to label astrocytes. RESULTS: There was a significant ninefold decrease (Z = 2.2, P = .046) of Musashi1 immunoreactivity in the SVZ of patients with AD in comparison with that of controls, but there was a significant increase in nestin immunoreactivity in the same region (Z = 2.2, P = .028) without any significant change in GFAP immunoreactivity. Reduced ChAT enzymatic activity was the main association of Musashi immunoreactivity (R = -.90, P = .03). DISCUSSION: The current results indicate a significant reduction of progenitor cells (as labeled by Musashi1) in the SVZ of patients with AD, but an increase in GFAP-negative astrocyte-like cells with progenitor characteristics. Cortical cholinergic loss was strongly associated with the reduction of progenitors, with potential implications of important treatment targets.

Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1.

Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing beta-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuronal identity and migration, but this is accomplished in postmitotic cells, which require the continued presence of SOX1. Our data also suggest that other SOXB1 members showing expression in specific neuronal populations are likely to play continuous roles from the establishment of precursors to their final differentiation.

Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis.

Human embryonic stem (hES) cells are pluripotent cells isolated from early human embryos. They can be grown in vitro and made to differentiate into many different cell types. These properties have suggested that they may be useful in cell replacement therapy for many degenerative diseases. However, if hES cells could also be manufactured with mutations significant in human disease, they could provide a powerful in-vitro tool for modelling disease processes and progression in a number of different cell types, as well as providing an ideal system for studying in-vitro toxicity and efficacy of drugs and other therapeutic systems such as gene therapy. Embryos with such mutations are generated as part of routine genetic testing during preimplantation genetic diagnosis, providing the opportunity to generate cell lines with significant mutations. A human embryonic stem cell line homozygous for the most common mutation leading to cystic fibrosis in humans (delta F508) has been generated and characterized. This cell line has the same morphology and expresses proteins typical of other unaffected hES cell lines. This cell line represents an important in-vitro tool for understanding the pathophysiology of cystic fibrosis, and presents exciting opportunities to test the efficacy and toxicity of new therapies relevant to CF.