Improved DiOlistic labelling technique for neurons in situ: Detailed visualisation of dendritic spines and concurrent histochemical-detection in fixed tissue.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

Assessment of Lab4P Probiotic Effects on Cognition in 3xTg-AD Alzheimer's Disease Model Mice and the SH-SY5Y Neuronal Cell Line.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer's disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with ß-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

Plasticity in Adult Mouse Visual Cortex Following Optic Nerve Injury.

Optic nerve (ON) injury is an established model of axonal injury which results in retrograde degeneration and death of retinal ganglion cells as well anterograde loss of transmission and Wallerian degeneration of the injured axons. While the local impact of ON crush has been extensively documented we know comparatively little about the functional changes that occur in higher visual structures such as primary visual cortex (V1). We explored the extent of adult cortical plasticity using ON crush in aged mice. V1 function of the contralateral hemisphere was assessed longitudinally by intrinsic signal imaging and 2-photon calcium imaging before and after ON crush. Functional imaging demonstrated an immediate shift in V1 ocular dominance towards the ipsilateral, intact eye, due to the expected almost complete loss of responses to contralateral eye stimulation. Surprisingly, within 2 weeks we observed a delayed increase in ipsilateral eye responses. Additionally, spontaneous activity in V1 was reduced, similar to the lesion projection zone after retinal lesions. The observed changes in V1 activity indicate that severe ON injury in adulthood evokes cortical plasticity not only cross-modally but also within the visual cortex; this plasticity may be best compared with that seen after retinal lesions.

Brain-derived neurotrophic factor prevents dendritic retraction of adult mouse retinal ganglion cells.

We used cultured adult mouse retinae as a model system to follow and quantify the retraction of dendrites using diolistic labelling of retinal ganglion cells (RGCs) following explantation. Cell death was monitored in parallel by nuclear staining as 'labelling' with RGC and apoptotic markers was inconsistent and exceedingly difficult to quantify reliably. Nuclear staining allowed us to delineate a lengthy time window during which dendrite retraction can be monitored in the absence of RGC death. The addition of brain-derived neurotrophic factor (BDNF) produced a marked reduction in dendritic degeneration, even when application was delayed for 3 days after retinal explantation. These results suggest that the delayed addition of trophic factors may be functionally beneficial before the loss of cell bodies in the course of conditions such as glaucoma.

Use of magnetic nanoparticles and oscillating magnetic field for non-viral gene transfer into mouse cornea.

BACKGROUND: Inherited, iatrogenic, and metabolic corneal disease could be potentially treated by supplying a functional gene or changing the expression levels of specific genes. Viral-based gene therapy is efficient, but restricted by evoking immune responses and inflammation. This study aimed to transfect mouse cornea with a non-viral based (oscillating magnetofection) method. METHODS: Cultured mouse corneas were treated with magnetic nanoparticles (MNP) tethered to CAG promoter and green fluorescent protein (GFP) reporter plasmids exposed to a 1 Hz, 2 Hz, and 4 Hz oscillating magnetic field for 30 min and 60 min and in three DNA:MNP ratios (1:2, 1:1, 2:3). Corneas were cultured for up to 3 days and their green fluorescent channel intensity and number of GFP-positive cells were recorded. Transfection efficiency was estimated as the percentage of GFP-positive cells per total cells in a microscopic field. FINDINGS: Control experiments with absent magnetic exposure showed no GFP-positive cells. The optimum condition was recorded at 3:2 DNA:MNP ratio, 1 Hz magnetic oscillation, and 30 min duration of magnetic exposure (mean GFP-positive endothelial cell count 191·7 [SD 54·5], p=0·009; mean green fluorescent intensity 85·3 [SD 48·5]; and average transfection efficiency 23·3% [range 10·6-30·9]). INTERPRETATION: A novel non-viral method of transfecting cornea, magnetofection, is demonstrated and gives proof of principle for its translation into corneal gene therapy. FUNDING: Welsh Clinical Academic Training Scheme.

Associations with Intraocular Pressure in a Large Cohort: Results from the UK Biobank.

PURPOSE: To describe the associations of physical and demographic factors with Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) in a British cohort. DESIGN: Cross-sectional study within the UK Biobank, a large-scale multisite cohort study in the United Kingdom. PARTICIPANTS: We included 110 573 participants from the UK Biobank with intraocular pressure (IOP) measurements available. Their mean age was 57 years (range, 40-69 years); 54% were women, and 90% were white. METHODS: Participants had 1 IOP measurement made on each eye using the Ocular Response Analyzer noncontact tonometer. Linear regression models were used to assess the associations of IOP with physical and demographic factors. MAIN OUTCOME MEASURES: The IOPg and IOPcc. RESULTS: The mean IOPg was 15.72 mmHg (95% confidence interval [CI], 15.70-15.74 mmHg), and the mean IOPcc was 15.95 mmHg (15.92-15.97 mmHg). After adjusting for covariates, IOPg and IOPcc were both significantly associated with older age, male sex, higher systolic blood pressure (SBP), faster heart rate, greater myopia, self-reported glaucoma, and colder season (all P < 0.001). The strongest determinants of both IOPg and IOPcc were SBP (partial R(2): IOPg 2.30%, IOPcc 2.26%), followed by refractive error (IOPg 0.60%, IOPcc 1.04%). The following variables had different directions of association with IOPg and IOPcc: height (-0.77 mmHg/m IOPg; 1.03 mmHg/m IOPcc), smoking (0.19 mmHg IOPg, -0.35 mmHg IOPcc), self-reported diabetes (0.41 mmHg IOPg, -0.05 mmHg IOPcc), and black ethnicity (-0.80 mmHg IOPg, 0.77 mmHg IOPcc). This suggests that height, smoking, diabetes, and ethnicity are related to corneal biomechanical properties. The increase in both IOPg and IOPcc with age was greatest among those of mixed ethnicities, followed by blacks and whites. The same set of covariates explained 7.4% of the variability of IOPcc but only 5.3% of the variability of IOPg. CONCLUSIONS: This analysis of associations with IOP in a large cohort demonstrated that some variables clearly have different associations with IOPg and IOPcc, and that these 2 measurements may reflect different biological characteristics.

Aged complement factor H knockout mice kept in a clean barriered environment have reduced retinal pathology.

Age-related macular degeneration (AMD) is the largest cause of visual loss in those over 60 years in the West and is a condition increasing in prevalence. Many diseases result from genetic/environmental interactions and 50% of AMD cases have an association with polymorphisms of the complement system including complement factor H. Here we explore interactions between genetic predisposition and environmental conditions in triggering retinal pathology in two groups of aged complement factor H knock out (Cfh(-/-)) mice. Mice were maintained over 9 months in either a conventional open environment or a barriered pathogen free environment. Open environment Cfh(-/-) mice had significant increases in subretinal macrophage numbers, inflammatory and stress responses and reduced photoreceptor numbers over mice kept in a pathogen free environment. Hence, environmental factors can drive retinal disease in these mice when linked to complement deficits impairing immune function. Both groups of mice had similar levels of retinal amyloid beta accumulation. Consequently there is no direct link between this and inflammation in Cfh(-/-) mice.

Microbead models in glaucoma.

The sustained and moderate elevation of intraocular pressure, which can be initiated at precise time points, remains the cornerstone of research into the mechanisms of glaucomatous retinal damage. We focus on the use of microbeads to block the outflow of aqueous following anterior chamber injection in a range of animals (mouse, rat and primate). We describe some of the most commonly used parameters and present guidance on injection technique and bead manipulation to facilitate the successful generation of experimental glaucoma.

VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.

Vascular endothelial growth factor A (VEGF-A) is a validated therapeutic target in several angiogenic- and vascular permeability-related pathological conditions, including certain cancers and potentially blinding diseases, such as age-related macular degeneration and diabetic retinopathy. We and others have shown that VEGF-A also plays an important role in neuronal development and neuroprotection, including in the neural retina. Antagonism of VEGF-A function might therefore present a risk to neuronal survival as a significant adverse effect. Herein, we demonstrate that VEGF-A acts directly on retinal ganglion cells (RGCs) to promote survival. VEGF receptor-2 signaling via the phosphoinositide-3-kinase/Akt pathway was required for the survival response in isolated RGCs. These results were confirmed in animal models of staurosporine-induced RGC death and experimental hypertensive glaucoma. Importantly, we observed that VEGF-A blockade significantly exacerbated neuronal cell death in the hypertensive glaucoma model. Our findings highlight the need to better define the risks associated with use of VEGF-A antagonists in the ocular setting.

A novel system for the classification of diseased retinal ganglion cells.

Retinal ganglion cell (RGC) dendritic atrophy is an early feature of many forms of retinal degeneration, providing a challenge to RGC classification. The characterization of these changes is complicated by the possibility that selective labeling of any particular class can confound the estimation of dendritic remodeling. To address this issue we have developed a novel, robust, and quantitative RGC classification based on proximal dendritic features which are resistant to early degeneration. RGCs were labeled through the ballistic delivery of DiO and DiI coated tungsten particles to whole retinal explants of 20 adult Brown Norway rats. RGCs were grouped according to the Sun classification system. A comprehensive set of primary and secondary dendrite features were quantified and a new classification model derived using principal component (PCA) and discriminant analyses, to estimate the likelihood that a cell belonged to any given class. One-hundred and thirty one imaged RGCs were analyzed; according to the Sun classification, 24% (n = 31) were RGCA, 29% (n = 38) RGCB, 32% (n = 42) RGCC, and 15% (n = 20) RGCD. PCA gave a 3 component solution, separating RGCs based on descriptors of soma size and primary dendrite thickness, proximal dendritic field size and dendritic tree asymmetry. The new variables correctly classified 73.3% (n = 74) of RGCs from a training sample and 63.3% (n = 19) from a hold out sample indicating an effective model. Soma and proximal dendritic tree morphological features provide a useful surrogate measurement for the classification of RGCs in disease. While a definitive classification is not possible in every case, the technique provides a useful safeguard against sample bias where the normal criteria for cell classification may not be reliable.

Oral nicotinamide provides robust, dose-dependent structural and metabolic neuroprotection of retinal ganglion cells in experimental glaucoma.

A compromised capacity to maintain NAD pools is recognized as a key underlying pathophysiological feature of neurodegenerative diseases. NAD acts as a substrate in major cell functions including mitochondrial homeostasis, cell signalling, axonal transport, axon/Wallerian degeneration, and neuronal energy supply. Dendritic degeneration is an early marker of neuronal stress and precedes cell loss. However, little is known about dendritic structural preservation in pathologic environments and remodelling in mature neurons. Retinal ganglion cell dendritic atrophy is an early pathological feature in animal models of the disease and has been demonstrated in port-mortem human glaucoma samples. Here we report that a nicotinamide (a precursor to NAD through the NAD salvage pathway) enriched diet provides robust retinal ganglion cell dendritic protection and preserves dendritic structure in a rat model of experimental glaucoma. Metabolomic analysis of optic nerve samples from the same animals demonstrates that nicotinamide provides robust metabolic neuroprotection in glaucoma. Advances in our understanding of retinal ganglion cell metabolic profiles shed light on the energetic shift that triggers early neuronal changes in neurodegenerative diseases. As nicotinamide can improve visual function short term in existing glaucoma patients, we hypothesize that a portion of this visual recovery may be due to dendritic preservation in stressed, but not yet fully degenerated, retinal ganglion cells.

Patient preferences for ocular hypertension monitoring: a discrete choice experiment.

BACKGROUND/AIMS: To elicit the preferences and calculate the willingness to pay (WTP) of patients with ocular hypertension (OHT) for eye monitoring services in the UK. METHODS: Patients with OHT aged at least 18 years recruited from four NHS ophthalmology departments were included in the study. Patients' preferences and WTP for an OHT monitoring service in the National Health Service were elicited using a discrete choice experiment (DCE) within a postal survey based on six attributes: (1) how OHT monitoring is organised, (2) monitoring frequency, (3) travel time from home, (4) use of a risk calculator for conversion to glaucoma, (5) risk of developing glaucoma in the next 10 years and (6) cost of monitoring. We used a sequential mixed-methods approach to design the survey. RESULTS: 360 patients diagnosed with OHT were recruited with a mean age of 69 years. In the DCE, reducing the risk of conversion to glaucoma was the most important factor influencing respondents' choice of monitoring service. Respondents preferred hospital-based monitoring services to community optometrist monitoring, and annual monitoring compared with more frequent (every 6 months) and less frequent (every 18 or 24 months) monitoring. These results can be monetised using WTP. Results of heterogeneity analysis suggest that patients with prior experience in community optometrist monitoring preferred this to hospital-based monitoring. CONCLUSIONS: Although hospital-based monitoring is generally preferred, patients with prior experience in community services have a different opinion, suggesting that patients who are unfamiliar with community optometry services may need additional support to accept monitoring in this setting.

Extracellular vesicle encapsulated nicotinamide delivered via a trans-scleral route provides retinal ganglion cell neuroprotection.

The progressive and irreversible degeneration of retinal ganglion cells (RGCs) and their axons is the major characteristic of glaucoma, a leading cause of irreversible blindness worldwide. Nicotinamide adenine dinucleotide (NAD) is a cofactor and metabolite of redox reaction critical for neuronal survival. Supplementation with nicotinamide (NAM), a precursor of NAD, can confer neuroprotective effects against glaucomatous damage caused by an age-related decline of NAD or mitochondrial dysfunction, reflecting the high metabolic activity of RGCs. However, oral supplementation of drug is relatively less efficient in terms of transmissibility to RGCs compared to direct delivery methods such as intraocular injection or delivery using subconjunctival depots. Neither method is ideal, given the risks of infection and subconjunctival scarring without novel techniques. By contrast, extracellular vesicles (EVs) have advantages as a drug delivery system with low immunogeneity and tissue interactions. We have evaluated the EV delivery of NAM as an RGC protective agent using a quantitative assessment of dendritic integrity using DiOlistics, which is confirmed to be a more sensitive measure of neuronal health in our mouse glaucoma model than the evaluation of somatic loss via the immunostaining method. NAM or NAM-loaded EVs showed a significant neuroprotective effect in the mouse retinal explant model. Furthermore, NAM-loaded EVs can penetrate the sclera once deployed in the subconjunctival space. These results confirm the feasibility of using subconjunctival injection of EVs to deliver NAM to intraocular targets.

Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity.

Retinal ganglion cell dendritic pruning has been reported in association with a 50% reduction in Opa1 transcript and protein in retinal and neural tissue, which manifests as visual dysfunction in the heterozygous mutant mouse, B6;C3-Opa1(Q285STOP). Here we report a marked reduction in retinal ganglion cell synaptic connectivity in the absence of soma loss and explore the mechanism and relationship between mitochondrial integrity and synaptic connectivity. We observed decreased levels of postsynaptic density protein 95 in Opa1(+/-) mutant mice consistent with synaptic loss in the inner plexiform layer. Glutamatergic but not γ-aminobutyric acid-ergic synaptic sites were reduced in Opa1(+/-) mice. We observed increased synaptic vesicle number in bipolar cell terminal arbours assessed by immunohistochemistry, electron microscopy and western blot analysis. These changes occur without significant loss of mitochondrial membrane potential in retina and optic nerve. Analysis of biolistically transfected retinal ganglion cells shows the retraction of mitochondria towards the soma, and mitochondrial fragmentation, preceding dendritic loss. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina.

Brain-derived neurotrophic factor measurements in mouse serum and plasma using a sensitive and specific enzyme-linked immunosorbent assay.

This study is about the quantification and validation of BDNF levels in mouse serum and plasma using a sensitive immunoassay. While BDNF levels are readily detectable in human serum, the functional implications of these measurements are unclear as BDNF released from human blood platelets is the main contributor to the serum levels of BDNF. As mouse platelets do not contain BDNF, this confounding factor is absent in the mouse. Accordingly, BDNF levels in mouse serum and plasma were found to be indistinguishable at 9.92 ± 1.97 pg/mL for serum and 10.58 ± 2.43 pg/mL for plasma (p = 0.473). These levels are approximately a thousand times lower than those measured in human serum and pre-adsorption with anti-BDNF, but not with anti-NGF or anti-NT3 monoclonal antibodies, markedly reduced the BDNF signal. These results open the possibility to explore the relevance of BDNF levels as a biomarker in accessible body fluids using existing mouse models mimicking human pathological conditions.

Brain-derived neurotrophic factor released from blood platelets prevents dendritic atrophy of lesioned adult central nervous system neurons.

In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using 'humanized' mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group (P ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

Gene Gun DiOlistic Labelling of Retinal Ganglion Cells.

Gene gun DiOlistic labelling enables the detailed visualization of retinal ganglion cells (RGCs) dendritic structure. Since the level of labelling is independent of cellular health, it is useful for the characterization of neuronal structure in degenerating neurons where expressed reporters may be inadequate. The method uses compressed helium gas to fire tungsten or gold microparticles coated in carbocyanine dyes (DiD, DiI, DiO) into flat mounted retinas. Here we describe the methods to optimize labelling and ensure a high yield of adequately labelled cells, with a focus on retinal ganglion cells.

The accuracy of the inferior>superior>nasal>temporal neuroretinal rim area rule for diagnosing glaucomatous optic disc damage.

PURPOSE: To determine the accuracy with which the optic disc can be diagnosed as normal or glaucomatous according to the ISNT rule, whereby, in the normal eye, the neuroretinal rim area follows the order inferior (I) > superior (S) > nasal (N) > temporal (T). DESIGN: Prospective, cross-sectional, observational, case series. PARTICIPANTS: Fifty-one normal individuals and 78 individuals with open-angle glaucoma exhibiting field loss (median mean deviation, -4.37 dB; interquartile range [IQR], -2.10 to -7.96 dB; median pattern standard deviation, 5.65 dB; IQR, 2.94 to 8.56 dB). The reference diagnosis was made by 2 experts on the basis of the appearance of the optic disc and of the corresponding visual field. METHODS: Stereoscopic optic disc photographs, acquired for each individual, were digitized at high resolution and analyzed using a digital, quad-buffered, stereoscopic viewing system in which a Z screen was used to dissociate the images to the 2 eyes of the observer. Three expert observers, trained to fellowship standard in glaucoma, independently undertook planimetry of the neuroretinal rim and of the disc margin from 1 eye of each individual, using a cursor moving in stereoscopic space to minimize parallax errors. Software automatically calculated the neuroretinal rim area in 10°, 30°, 40°, and 90° segments. For the ISNT rule to be obeyed, the 3 Boolean comparisons of the neuroretinal rim area, I>S, S>N, and N>T, had to be true. If any of the comparisons returned false, the rule was considered not to have been obeyed. Values were compared at a precision of 0.0001 mm(2). MAIN OUTCOME MEASURES: The outcome of the ISNT rule in terms of the 3 Boolean comparisons of the neuroretinal rim area was specified in terms of the sensitivity, specificity, and hence, the positive and negative likelihood ratios. RESULTS: Based on the ISNT rule being obeyed for 10° segments, the positive likelihood ratio among the 3 observers was 1.11 (95% confidence interval [CI], 0.99-1.25), 1.07 (95% CI, 0.94-1.21), and 1.06 (95% CI, 0.96-1.18), respectively. It was similar for the other segment sizes. Variants of the rule were not appreciably better. CONCLUSIONS: The ISNT rule has limited utility in the diagnosis of open-angle glaucoma.

Retina ganglion cell degeneration in glaucoma: an opportunity missed? A review.

Retinal ganglion cell degeneration has been reported in a range of experimental models of glaucoma. Manifest as pruning of retinal ganglion cell dendrites, it is likely to influence both the function and viability of affected cells. Electrophysiological studies in primate glaucoma have shown that affected cells retain some function and could therefore form a neural substrate for the recovery of visual function in glaucoma. Clinical studies in which the intraocular pressure is reduced have suggested that some improvement in retinal function may be possible in hypotensive eyes. These experimental studies highlight the importance of establishing the extent to which retinal ganglion cell degeneration occurs in human glaucoma. If substantial numbers of degenerating retinal ganglion cells are present in glaucoma, they could present an ideal target for the recovery of vision.

The Impact of Probiotic Supplementation on Cognitive, Pathological and Metabolic Markers in a Transgenic Mouse Model of Alzheimer's Disease.

Brain degenerative disorders such as Alzheimer's disease (AD) can be exacerbated by aberrant metabolism. Supplementation with probiotic bacteria is emerging as a promising preventative strategy for both neurodegeneration and metabolic syndrome. In this study, we assess the impact of the Lab4b probiotic consortium on (i) cognitive and pathological markers of AD progression and (ii) metabolic status in 3xTg-AD mice subjected to metabolic challenge with a high fat diet. The group receiving the probiotic performed better in the novel object recognition test and displayed higher hippocampal neuronal spine density than the control group at the end of the 12 weeks intervention period. These changes were accompanied by differences in localised (brain) and systemic anti-inflammatory responses that favoured the Probiotic group together with the prevention of diet induced weight gain and hypercholesterolaemia and the modulation of liver function. Compositional differences between the faecal microbiotas of the study groups included a lower Firmicutes:Bacteroidetes ratio and less numbers of viable yeast in the Probiotic group compared to the Control. The results illustrate the potential of the Lab4b probiotic as a neuroprotective agent and encourage further studies with human participants.