Partial endothelial nitric oxide synthase deficiency exacerbates Alzheimer’s pathology in a murine model

HC Mein,[[1]] S Ahmed,[[1]] Y Jing,[[1]] BG Mockett,[[2]] WC Abraham,[[2]] P Liu.[[1]] [[1]]Department of Anatomy, [[2]]Department of Psychology, Brain Health Research Centre, Brain Research New Zealand, University of Otago, Dunedin.

Increasing evidence implicates endothelial nitric oxide synthase (eNOS) dysfunction in the pathogenesis of Alzheimer’s disease (AD). Complete eNOS deficient (eNOS[[-/-]]) mice have increased amyloid beta (Aβ) levels and tau phosphorylation in the brain and cognitive decline. APPswe/PSdE1 (APP/PS1) mice display age-related Aβ accumulation and memory deficits. In this study, APP/PS1 and eNOS[[-/-]] mice were crossed to produce APP/PS1/eNOS[[+/-]] mice in order to add an element of eNOS dysfunction to the model. At eight months of age, APP/PS1/eNOS[[+/-]] mice performed poorly relative to their age-matched APP/PS1 mice in a water maze test. This study aimed to compare AD-like pathology in the hippocampus of APP/PS1 and APP/PS1/eNOS[[+/-]] mice using immunofluorescence.

Male and female eight-months-old wild-type (WT), eNOS[[+/-]], APP/PS1 and APP/PS1/eNOS[[+/-]] mice (n = 3–5/genotype/sex) were perfused transcardially and the brains were fixed by 4% paraformaldehyde. Coronal sections of anterior and posterior hippocampus from each animal were double-labelled with 6E10 (for Aβ) and Iba-1 (for microglia) antibodies using immunofluorescence. The Aβ load (the percentage area covered by plaques) in the hippocampus was quantified using the ImageJ under blind conditions.

Aβ plaques (6E10 immunoreactivity) were evident in the anterior and posterior hippocampus in both APP/PS1 and APP/PS1/eNOS[[+/-]] mice, but not in WT and eNOS[[+/-]] mice. While Iba-1 immunoreactivity was seen in the hippocampus in all four genotype groups, there were intensely stained microglia forming clusters in APP/PS1 and APP/PS1/eNOS[[+/-]] mice. The ImageJ analysis and one sample t-test revealed that the APP/PS1/eNOS[[+/-]] group had significantly higher plaque load in the hippocampus at both the anterior and posterior levels (all p < 0.01; 1.7-fold increases) relative to the APP/PS1 group.

These preliminary findings demonstrate that partial eNOS deficiency exacerbates AD pathology in APP/PS1 mice, which may account for poor spatial learning observed in APP/PS1/eNOS+/- mice in previous studies. The underlying mechanisms remain to be explored in the future.

Biodistribution and toxicological assessment of carbon nano-onions in female BALB/c mice

LR Thomsen,[[1]] N Shrestha,[[1]] S Giordani,[[2]] RJ Rosengren.[[1]] [[1]]Department of Pharmacology and Toxicology, School of Biomedical Sciences, University of Otago, Dunedin. [[2]]Department of Chemical Sciences, Dublin City University, Ireland.

Carbon nano-onions (CNOs) are multi-layered fullerenes comprising of concentric, quasi-spherical, carbon shells. In order for these nanomaterials to be used as biosensors, imaging tools or drug-delivery vehicles, the systemic safety of CNOs must first be confirmed. Previous toxicological assessment has only been conducted in freshwater polyps and zebrafish. Therefore, this study investigated the biodistribution and toxicity of CNOs following intravenous administration in mice.

CNOs were dispersed in a biologically compatible dispersant (50:50 ddH2O: PEG-200) and injected intravenously into female BALB/c mice. Mice were administered a single dose of CNO (125, 250 or 500 µg/mouse) or vehicle (n=5 per group) and monitored for seven days. Mice were then euthanised by CO{{2}} inhalation, with blood and organs collected for analysis. Changes to physiological parameters were analysed using a one-way ANOVA coupled with Tukey’s multiple comparisons post hoc.

Daily monitoring of body weight showed no significant differences between treatment groups across the seven days. There were also no significant differences observed in organ weight between treatments. Furthermore, evaluation of hepatotoxicity showed no significant differences between treatments, as plasma ALT activity was within the normal range (0–80 IU/L). Organs from each treatment group (n=3) were examined to evaluate the biodistribution of CNOs. As is typically seen with other nanomaterials, organ histology revealed dose-dependent accumulation of CNOs within the liver, lung and spleen. Specifically, in the liver, total CNO aggregates were 2.5- and 3.3-fold higher following 250 and 500 µg does, respectively, compared to 125 µg (P < 0.05). Significant dose-dependent CNO accumulation was also observed in the lung and spleen (P < 0.05).

Acute systemic CNO administration appeared to be non-toxic to mice. However, due to the observed organ accumulation, further chronic studies are needed to definitively determine safety.

Supported by a University of Otago Scholarship in Biomedical Sciences.

Inflammation and scarring in an ex vivo model of spinal cord injury

HRT Stent, LM Wise. Department of Pharmacology and Toxicology, School of Biomedical Sciences, University of Otago, Dunedin.

Traumatic spinal cord injury (SCI) is followed by a marked inflammatory response that greatly exacerbates damage and generates scar tissue which prevents axonal regeneration. Recently, an ex vivo SCI model was developed to reduce highly invasive and costly in vivo testing. However, it is unknown whether this model can mimic processes that occur following SCI. This study investigated whether the ex vivo SCI model can generate an inflammatory response and fibrotic and glial scarring following SCI.

Spinal cords obtained from euthanised male Sprague Dawley rats (6–7 weeks post-natal) were dissected into six segments then cultured for 24 hours. Segments received a compression injury and then were cultured for another 24 hours or seven days (n = 3 rats per group). Tissue viability was assessed using a TUNEL assay and quantitative PCR was used to measure mRNA levels of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF). Immunohistochemistry was used to detect glial fibrillary acidic protein (GFAP) and collagen IV, markers of glial and fibrotic scarring, respectively.

Compression injury induced a 2.5-fold increase in cell death at 24 hours post-injury compared to uninjured controls (P > 0.05, Wilcoxon test), but no increase in cell death was detectable at seven days. No significant differences in expression of IL-6, TNF or IL-1β were identified between uninjured and injured groups at either time-point. GFAP-staining suggested the initiation of glial scar formation, while collagen IV-staining indicated no fibrotic scarring.

These findings suggest that SCI was unable to trigger inflammation or fibrotic scarring in the ex vivo model. Optimisation of model parameters, such as longer culture duration and co-culturing with lymphocytes, may be needed to more accurately emulate the inflammatory and scarring responses seen in live animals. These steps will be required before the ex vivo model can be used for preclinical testing of SCI therapies.

Supported by a Maurice and Phyllis Paykel Trust Student Scholarship.

Oleoylethanolamide cubosomes exert potent anti-inflammatory effects on lipopolysaccharide-induced dendritic cells and human brain endothelial cells

PWL Goh, B Gibson, SB Rizwan. School of Pharmacy, University of Otago, Dunedin.

Oleoylethanolamide (OEA) is an endogenous lipid found in vertebrates with reported anti-inflammatory effects in animal models of brain disorders but is currently unavailable as a therapy due to poor solubility and in vivo enzymatic degradation. To circumvent these issues, OEA has been successfully formulated into lipid nanoparticles (cubosomes). However, their biological effects on viable cells have yet to be investigated. This study aimed to determine whether OEA cubosomes exert anti-inflammatory effects on bone-marrow derived dendritic cells (DCs), the primary mediators of inflammation, and human cerebral microvascular endothelial cells (hCMEC/D3) that line the blood–brain barrier.

OEA cubosomes were prepared by solvent precursor dilution and their size was determined using dynamic light scattering (DLS). Inflammation in murine DCs and hCMEC/D3 cells was stimulated with lipopolysaccharide (LPS) for one hour and incubated with a range of OEA cubosome concentrations for 12 hours. Flow cytometry was used to quantify cytotoxicity and co-stimulatory molecule expression by DCs as well as cytokine concentrations produced by DCs and hCMEC/D3 cells (via Cytometric Bead Array) in response to LPS-induced inflammation.

Cubosomes produced were 120 to 140 nm. OEA cubosomes were non-toxic to DC and hCMEC/D3 cells up to a total lipid concentration of 15 µg/mL. A marked dose-dependent reduction of CD40 and CD86 expression was observed in DCs. In contrast, an elevation in CD80 expression across all total lipid concentrations was observed. Compared to untreated controls, TNF-α and IL-6 concentrations decreased by 10-fold and 1,000-fold, respectively, for LPS-induced DCs, while IL-6 levels decreased by 10-fold for hCMEC/D3 cells.

These preliminary results show, for the first time, that OEA cubosomes exert anti-inflammatory effects on DCs and hCMEC/D3 cells by modulating co-stimulatory molecule expression and cytokine production. This study highlights the potential of OEA cubosomes as an anti-inflammatory agent and warrants further investigation into mechanisms involved in its cellular uptake and anti-inflammatory effects.

Supported by a University of Otago, School of Pharmacy Summer Scholarship.

Secreted amyloid precursor protein alpha (sAPPα) regulates surface expression of GluA1-containing glutamate receptors in primary hippocampal neurons

CM Westlake,[[1]] RW Livingstone,[[1]] K Peppercorn,[[2]] WP Tate,[[2]] JM Williams.[[1]] [[1]]Department of Anatomy, [[2]]Department of Biochemistry, School of Biomedical Sciences, Brain Research New Zealand, University of Otago, Dunedin

Precise molecular changes at synapses underlie the processes of learning and memory. A central component of synapse strengthening is synthesis and trafficking of excitatory glutamate receptors. GluA1-containing receptors are plasticity-promoting through the ability to permit calcium influx, a key event in initiation of many intracellular mechanisms that underpin synaptic plasticity. Recently, we have shown that secreted amyloid precursor protein-alpha (sAPPα), a neuroprotective and memory-enhancing molecule, promotes synthesis of GluA1 specifically. We hypothesised that these receptors are rapidly trafficked to the cell surface and contribute to the enhancement of synaptic plasticity. We aimed to determine the level of GluA1 surface expression in response to sAPPα.

Primary hippocampal cell cultures were prepared from postnatal day 0–1 Sprague Dawley rats. Neurons (DIV20-22) were treated with sAPPα (1 nM) for 30 minutes, two hours or four hours. Utilising a primary antibody (Merck; #MAB2263) targeted to an extracellular epitope of GluA1 and detergent-free conditions allowed for detection of solely surface expressed receptors. Fluorophore conjugated secondary antibodies (Alexa Fluor 647nm; Invitrogen; #A21236) allowed for signal visualisation and measurement of total cell fluorescence for ten cells/dish. Values were averaged and converted to fold changes relative to no treatment controls. Significance was determined by two-tailed Mann–Whitney t-tests.

We found that sAPPα (1 nM) significantly enhanced cell surface expression of GluA1 in the dendrites at 30 mins (2.37 ± 0.37 SEM; P = 0.0141, N = 3 experiments; N = 31 cells), two hrs (3.01 ± 0.44 SEM; P =0.0003, N = 3 experiments; N = 31 cells) and four hrs (2.51 ± 0.40 SEM; P = 0.0043; P = 0.0043; N = 3 experiments; N = 28 cells).

These findings suggest that sAPPα potentially exerts memory-enhancing effects through mechanisms involving insertion of GluA1-containing receptors into the synaptic membrane, occurring as early as 30 minutes after treatment. As Alzheimer’s disease is initially characterised by memory impairment, understanding the biological mechanisms harnessed by sAPPα presents an opportunity to develop novel memory-enhancing therapies.

Can carbon monoxide protect hypertrophic hearts against ischaemia-reperfusion injury?

SH Thwaite, MI Read, DS Larsen, JC Harrison, IA Sammut. Department of Pharmacology and Toxicology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.

The imposition of ischaemia-reperfusion episodes in frequently used cardiac surgery interventions involving cardiopulmonary bypass can induce peri-operative complications, such as myocardial injury, arrhythmias and end-organ injury. The presence of pre-existing cardiac pathologies, such as hypertrophic cardiomyopathy, potentiates these peri-operative complications and results in reduced outcome benefits. A new class of carbon monoxide delivery molecules (oCOms) have been developed as potential anti-ischaemic agents. The present study investigated the cardioprotective potential of oCOm-21 in hypertrophic hearts subjected to an acute ischaemia-reperfusion episode.

Hypertension induced in male, 10-week-old Cyp1a1-Ren2 rats fed indole-3-carbinol (0.167%; eight-weeks) resulted in larger heart weights (P < 0.001) and increased myocardial fibrosis (P < 0.001) against control littermates. Hearts were isolated and perfused using the Langendorff technique. oCOm-21 (1–10 µM) or vehicle control was infused (10 minutes) prior to a 30-minute warm global ischaemic episode followed by a 60-minute reperfusion period. In normotrophic hearts (N = 4–5/group), oCOm-21 (1 and 3 µM) improved left ventricular developed pressure (LVDP) recovery (P < 0.01 and P < 0.001 respectively against vehicle control). In hypertrophic hearts (N = 8–10/group), LVDP recovery to pre-ischaemic baselines was only significantly improved when higher concentrations (3 and 10 µM) of oCOm-21 were applied. Furthermore, oCOm-21 (3 and 10 µM) decreased myocardial injury as seen by the reduction of lactate dehydrogenase leakage upon reperfusion in the hypertrophic hearts compared to control, and a reduction in apoptotic cell death at 60 minutes of reperfusion with oCOm-21 (1 and 3 µM; P < 0.01).

This study provides valuable evidence supporting oCOm-21 use as a pre-conditioning agent in acute cardiovascular interventions in hearts burdened with hypertrophic cardiomyopathy.

Investigating the wound healing capacity of subcutaneous adipose tissue

H Cao,[[1]] M Stitely,[[2]] L Wise.[[1]] [[1]]Department of Pharmacology and Toxicology, School of Biomedical Sciences, [[2]]Department of Women’s & Children’s Health, Dunedin School of Medicine, University of Otago, Dunedin.

Surgical wound complications, such as seroma formation, dehiscence and infection, that occur during recovery represent a significant health burden occurring in 5–10% of patients. As obese patients have the greatest incidence of wound breakdown, we hypothesised that ineffective healing of the subcutaneous adipose tissue may be a predisposing factor. Therefore, this study investigated the healing capacity of cells derived from adipose tissue, in conditions simulating the ‘normal’ and ‘obese’ microenvironment of surgical wounds.

The healing capacities of pre-adipocyte and mature adipocyte cells derived from human subcutaneous adipose tissue were investigated using in vitro wound assays. To mimic different wound conditions, cells were pre-treated with standard growth media, pro-fibrotic cytokine transforming growth factor-ß (TGFß), or conditioned media from a human macrophage cell-line stimulated with interferon-γ and lipopolysaccharide, containing pro-inflammatory cytokines. To mimic a wound, cell monolayers were scratched to create a wound area, and the migration of cells was monitored over time using image capture. Results are reported as mean percentage reduction in wound area after 20 hours (SD) (N = 3).

Pre-adipocytes showed significantly greater healing capacity than mature adipocytes, with 86 ± 2% and 38 ± 7% reductions in wound area, respectively (P < 0.01, 2-way ANOVA). In the presence of macrophage-conditioned media, pre-adipocyte wound area only reduced by 42 ± 11% (P < 0.05), indicating an impaired healing response. A similar inhibitory effect was also seen in the presence of TGFα (20ng/ml), with pre-adipocyte wound area reducing by 42  6% (P < 0.01).

The inhibitory effects of pro-inflammatory and fibrotic cytokines on pre-adipocyte healing aligns with clear clinical evidence showing an increased risk of surgical wound complications with obesity or prolonged inflammation. Biological therapies targeting these cytokines may therefore improve the adipose tissue microenvironment, leading to a state that is more conducive to healing for the patient.

Supported by a Phyllis Paykel Memorial Scholarship in Medicine for BMedSci(Hons).

Patient perspectives of a neuroscience-informed physiotherapy programme for persistent rotator cuff-related shoulder pain

R Acker,[[1]] N Swain,[[2]] M Perry,[[1]] C Wassinger,[[3]] G Sole.[[1]] [[1]]Centre for Health, Activity and Rehabilitation Research, School of Physiotherapy, University of Otago, Dunedin, [[2]]Department of Psychological Medicine, Otago Medical School, University of Otago, Dunedin, [[3]]Department of Physical Therapy, College of Clinical and Rehabilitative Health Sciences, University of East Tennessee, Johnson City, Tennessee, USA.

Rotator cuff-related shoulder pain (RCRSP) is the most common shoulder disorder in middle- to older-aged people. Health costs for this persistent disorder are rising and can lead to decreasing quality of life and increased care dependency. Physiotherapy is the primary treatment, and in individuals with persistent pain, a holistic biopsychosocial approach is warranted. Besides directing treatment to the shoulder, patient pain education and general health issues also need to be considered. Pain neuroscience education (PNE) is such an approach. The Otago Shoulder Health Study developed such a programme, providing patient pain education resources. This study explored the perspective of patients with persistent RCRSP of this programme of PNE integrated with pragmatic physiotherapy.

Five men and five women from Christchurch and Dunedin, aged 43–75 years, with persistent RCRSP (<3 months) underwent a three-month PNE physiotherapy programme. Individual semi-structured interviews were recorded and transcribed verbatim and analysed using the general inductive approach.

Four themes emerged: (1) ‘Patient beliefs’ related to the pathoanatomic cause of their pain and influenced their expectations of the programme. (2) Patients’ experiences were enhanced by a strong, supportive ‘rapport and relationship’ with their physiotherapist. (3) ‘Perspective and understanding of the programme’ described the diverse levels of patients’ acceptability and application of the programme. (4) Despite diverse perspectives, ‘empowerment: my shoulder into the future’ reflected reassurance, hope and decreased fear of their pain, with enhanced self-management strategies.

Most patients were able to change their pain beliefs, enhanced by an individualised treatment delivery and a strong therapeutic relationship. Discussions emanating from the provided resources allowed patients to be partners with the physiotherapists and apply new understanding of pain to their lives. Physiotherapists integrating PNE into treatment should invest time in understanding patients’ pain beliefs and embrace their role as facilitators to help patients reconceptualise persistent RCRSP and to enhance self-efficacy and confidence.

Altered architecture of the cerebellar cortex circuitry in mouse models of human spinocerebellar ataxia type 2

ZD Tonacao,[[1]] K Potapov,[[1]] MF Ibrahim,[[1]] SM Pulst,[[2]] RM Empson.[[1]] [[1]]Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin. [[2]]Department of Neurology, University of Utah, Utah, USA.

Spinocerebellar ataxias (SCA) are a family of human neurodegenerative movement disorders that impair the cerebellar circuitry and results from dysfunction of the Purkinje neuron, the sole output of the cerebellar cortex. Ataxias are progressive with severe consequences, and there is no cure. SCA2 is one of many types of SCAs, but how the cerebellar circuitry is impaired in SCA2 remains unknown. Using a mouse model that recapitulates the human condition, we aimed to investigate the nature of changes to the cerebellar cortex architecture and synaptic circuitry both early and late in the disease.

In sagittal brain slices from eight- and twenty-week-old wild-type (WT) and SCA2 mice (N = 5 per group), we performed fluorescence immunohistochemistry and confocal imaging to identify Purkinje neuron shape and the location and morphology of their synaptic circuitry. Analysis used ImageJ and statistical comparison by one-way ANOVA.

Purkinje neurons from SCA2 mice displayed atrophy of their dendrites and soma in all regions of the cerebellum at both early and late stages of disease progression (P < 0.0001, ANOVA). Interestingly, we identified normal synaptic circuitry in the early stages of the disease in the anterior regions of the cerebellum, but not the posterior regions (P < 0.0001, ANOVA). At the later disease stages, we observed disrupted synaptic circuitry (P < 0.0001, ANOVA) in all cerebellar areas.

Understanding why the anterior cerebellar circuitry remains robust for longer during disease progression, or why the posterior regions are more vulnerable, could help identify and guide new approaches to effectively treat SCA2 and other SCAs.