Invited Review - Understanding cause and effect in Alzheimer's pathophysiology: Implications for clinical trials.

Alzheimer's disease (AD) pathology is multi-faceted, including extracellular accumulation of amyloid-ß (Aß), accumulation of tau within neurons, glial activation and loss of neurons and synapses. From a neuropathological perspective, usually at a single time-point and often at the end-stage of the disease, it is challenging to understand the cause and effect relationships between these components. There are at least four ways of trying to unravel these relationships. First, genetic studies demonstrate mutations that influence Aß production, but not tau, can initiate AD; whereas genetic variants influencing AD risk are related to innate immunity and lipid metabolism. Second, studies at early time points show that pathology begins decades before the onset of dementia and indicate different anatomical locations for initiation of Aß and tau accumulation. Third, cause and effect can be studied in experimental models, but most animal models do not fully replicate AD pathology. However, induced pluripotent stem cells (iPSCs) to study live human neurons has introduced a new perspective. Fourth, clinical trials may alter AD pathology giving insights into cause and effect relationships. Therefore, a sequence of (i) neocortical Aß accumulation followed by (ii) a microglial inflammatory reaction to Aß, causing neuritic dystrophy which promotes (iii) spread of tau from the limbic system to the neocortex with (iv) progressive tau accumulation and spread resulting in (v) neurodegeneration, explains the evidence. It is proposed that different therapeutic targets are required for different stages of the disease process: Aß for primary prevention, microglia for secondary prevention, and tau for established disease.

A novel method to visualise the three-dimensional organisation of the human cerebral cortical vasculature.

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aß42 were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Systems proteomic analysis reveals that clusterin and tissue inhibitor of metalloproteinases 3 increase in leptomeningeal arteries affected by cerebral amyloid angiopathy.

AIMS: Amyloid beta (Aß) accumulation in the walls of leptomeningeal arteries as cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease. In this study, we used global quantitative proteomic analysis to examine the hypothesis that the leptomeningeal arteries derived from patients with CAA have a distinct endophenotypic profile compared to those from young and elderly controls. METHODS: Freshly dissected leptomeningeal arteries from the Newcastle Brain Tissue Resource and Edinburgh Sudden Death Brain Bank from seven elderly (82.9 ± 7.5 years) females with severe capillary and arterial CAA, as well as seven elderly (88.3 ± 8.6 years) and five young (45.4 ± 3.9 years) females without CAA were used in this study. Arteries from four patients with CAA, two young and two elderly controls were individually analysed using quantitative proteomics. Key proteomic findings were then validated using immunohistochemistry. RESULTS: Bioinformatics interpretation of the results showed a significant enrichment of the immune response/classical complement and extracellular matrix remodelling pathways (P < 0.05) in arteries affected by CAA vs. those from young and elderly controls. Clusterin (apolipoprotein J) and tissue inhibitor of metalloproteinases-3 (TIMP3), validated using immunohistochemistry, were shown to co-localize with Aß and to be up-regulated in leptomeningeal arteries from CAA patients compared to young and elderly controls. CONCLUSIONS: Global proteomic profiling of brain leptomeningeal arteries revealed that clusterin and TIMP3 increase in leptomeningeal arteries affected by CAA. We propose that clusterin and TIMP3 could facilitate perivascular clearance and may serve as novel candidate therapeutic targets for CAA.

CD169(+) macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169(+) macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68(+) macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune-competent mouse model, CD169(+) macrophage ablation significantly inhibited prostate cancer-induced woven bone formation, suggesting that CD169(+) macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast, pan-phagocytic cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Contact radiotherapy boost in association with 'watch and wait' for rectal cancer: initial experience and outcomes from a shared programme between a district general hospital network and a regional oncology centre.

AIM: Recent data have highlighted the potential of more intensive neoadjuvant protocols to increase and sustain the rate of complete response in rectal cancer managed nonoperatively. This study aimed to review the outcome of all patients from our district general hospitals network who had received standard neoadjuvant therapy and were additionally referred to a centre of excellence for contact X-ray brachytherapy or high-dose-rate brachytherapy boost. METHOD: A retrospective, chart-based review of all patients co-managed in this manner was performed. Patient details were retrieved from a prospectively maintained departmental database. Indications for treatment, patient outcome and serial data from follow-up clinical and radiological assessment were analysed. RESULTS: Seventeen patients treated over a 6-year period were identified. Median follow-up was 20 (5-54) months. Fourteen patients were clinically staged as T2 or T3 and eight were clinically node positive. Three patients died, of whom only one was initially a surgical candidate but refused an exenteration. Of the 14 patients who remain alive, 11 (79%) have a sustained complete (n = 8) or partial (n = 3) response. Two patients had an incomplete response, one is being palliated and the other awaits salvage surgery. One patient underwent abdominoperineal excision for suspected local recurrence. Currently 13 (93%) surviving patients are stoma free. CONCLUSIONS: This series shows that the addition of a radiotherapy boost offered sustained responses and stoma-free survival even in advanced disease and adverse patient populations whilst providing the majority of care closer to home.

Subetta Enhances Sensitivity of Human Muscle Cells to Insulin.

Addition of Subetta to insulin (10 nM) increased insulin-stimulated glucose uptake 43% (p<0.001). Moreover, glucose uptake stimulated by insulin (10 nM) in the presence of Subetta was similar to that stimulated by 300 nM insulin. These findings suggest that Subetta significantly enhanced insulin sensitivity of tissues through stimulation of glucose transport to myocytes mediated by glucose transporter 4.

Subetta increases phosphorylation of insulin receptor ß-subunit alone and in the presence of insulin.

It has been previously shown that Subetta (a drug containing released-active forms of antibodies to the insulin receptor ß-subunit and antibodies to endothelial nitric oxide synthase) stimulated insulin-induced adiponectin production by mature human adipocytes in the absence of insulin. Therefore, it was assumed that Subetta could activate the insulin receptor. To confirm this hypothesis, the capacity of Subetta to activate the insulin receptor in mature human adipocytes in the absence or presence of the insulin was investigated. Cells were incubated either with Subetta or with vehicle, or with basal medium for 3 days. Then, adipocytes were treated with water or insulin (100 nm) for 15 min. Following treatment, lysates were prepared and phosphorylation of insulin receptor ß-subunits was analyzed by western blot analysis. It was shown that Subetta significantly increased (P<0.001) the 'phosphorylated-insulin receptor ß-subunit/total insulin receptor ß-subunit' ratios in both the presence and the absence of insulin. These results support previously published data and indicate that Subetta could activate the insulin receptor through the effect on its ß-subunits, whose conformational state is essential for insulin receptor activation. This action might serve as one of the primary mechanisms of the drug's antidiabetic effect.

Haemoglobin scavenging after subarachnoid haemorrhage.

Rapid and effective clearance of cell-free haemoglobin after subarachnoid haemorrhage (SAH) is important to prevent vasospasm and neurotoxicity and improve long-term outcome. Haemoglobin is avidly bound by haptoglobin, and the complex is cleared by CD163 expressed on the membrane surface of macrophages. We studied the kinetics of haemoglobin and haptoglobin in cerebrospinal fluid after SAH. We show that haemoglobin levels rise gradually after SAH. Haptoglobin levels rise acutely with aneurysmal rupture as a result of injection of blood into the subarachnoid space. Although levels decline as haemoglobin scavenging occurs, complete depletion of haptoglobin does not occur and levels start rising again, indicating saturation of CD163 sites available for haptoglobin-haemoglobin clearance. In a preliminary neuropathological study we demonstrate that meningeal CD163 expression is upregulated after SAH, in keeping with a proinflammatory state. However, loss of CD163 occurs in meningeal areas with overlying blood compared with areas without overlying blood. Becauses ADAM17 is the enzyme responsible for shedding membrane-bound CD163, its inhibition may be a potential therapeutic strategy after SAH.

Effect of cardiac output changes on exhaled carbon dioxide in newborn piglets.

INTRODUCTION: International neonatal resuscitation guidelines recommend that correct tube placement should be confirmed by clinical assessment and exhaled CO2 detection. Absence of exhaled CO2 after intubation suggests oesophageal intubation, non-aerated lungs, low tidal volume delivery, or low cardiac output. The relationship between changes in cardiac output and exhaled CO2 in neonates is unknown. The aim of the study was to determine if changes in cardiac output affect exhaled carbon dioxide in a porcine model of neonatal resuscitation. METHOD: Term piglets (n=5) aged 3-4 days were anesthetised, intubated, instrumented and exposed to normocapnic hypoxia. Exhaled CO2 was continuously measured using a flow sensor (Respironics NM3(®)). Pulmonary artery blood flow, a surrogate for cardiac output was measured using an ultrasonic flow probe (Transonic(®)). A semi-quantitative CO2-detector (Pedi-Cap(®)) was placed between the tracheal tube and flow sensor to assess colour change at changing levels of cardiac output. RESULTS: Median (IQR) pulmonary artery blood flow significantly decreased from 177 (147-177)mL/kg/min at baseline to 4 (3-26)mL/kg/min during hypoxia (p=0.02). Exhaled CO2 remained similar throughout the experiment, 47 (41-47)mmHg at baseline vs. 40 (38-41)mmHg at the end of the hypoxia (p=1.00). Additionally, at each time point, colour change at the Pedi-Cap(®) was observed. CONCLUSION: A significant decrease in cardiac output was not associated with changes in exhaled CO2 or failure to achieve a Pedi-Cap(®) colour change.

Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model.

AIM: Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas. METHODS: Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added. Over the following 7 days, infiltration and cell death were studied using fluorescent C6 tumour cells and confocal microscopy; immunophenotyping of microglia was performed using CD68 (phagocytosis), MHCII (antigen-presentation) and Iba1 (microglial marker regardless of functional state). Cell proliferation was assessed using Ki67 and qPCR to detect cytokine expression. Sham and control groups were included. RESULTS: Microscopy showed proliferation of C6 tumour cells with both infiltration of tumour cells into the hippocampal tissue and of microglia among the tumour cells. Confocal experiments confirmed increasing tumour cell infiltration into the hippocampal slice with time (P<0.001), associated with cell death (σ=0.313, P=0.022). Ki67 showed increased proliferation (P<0.001), of both tumour cells and Iba1+ microglia and increased microglial phagocytosis (CD68: P<0.001). Expression of pro-inflammatory cytokines IL1, IL6 and TNFα were downregulated with expression of the anti-inflammatory cytokine TGFß1 maintained. CONCLUSION: This model allows study of the proliferation and infiltration of astrocytic tumour cells in central nervous system tissue and their interaction with microglia. Our data suggest that microglial function is altered in the presence of tumour cells, putatively facilitating tumour progression. Manipulation of the microglial functional state may have therapeutic value for astrocytic tumours.

The neuroinflammatory response in humans after traumatic brain injury.

AIMS: Traumatic brain injury is a significant cause of morbidity and mortality worldwide. An epidemiological association between head injury and long-term cognitive decline has been described for many years and recent clinical studies have highlighted functional impairment within 12 months of a mild head injury. In addition chronic traumatic encephalopathy is a recently described condition in cases of repetitive head injury. There are shared mechanisms between traumatic brain injury and Alzheimer's disease, and it has been hypothesized that neuroinflammation, in the form of microglial activation, may be a mechanism underlying chronic neurodegenerative processes after traumatic brain injury. METHODS: This study assessed the microglial reaction after head injury in a range of ages and survival periods, from <24-h survival through to 47-year survival. Immunohistochemistry for reactive microglia (CD68 and CR3/43) was performed on human autopsy brain tissue and assessed 'blind' by quantitative image analysis. Head injury cases were compared with age matched controls, and within the traumatic brain injury group cases with diffuse traumatic axonal injury were compared with cases without diffuse traumatic axonal injury. RESULTS: A major finding was a neuroinflammatory response that develops within the first week and persists for several months after traumatic brain injury, but has returned to control levels after several years. In cases with diffuse traumatic axonal injury the microglial reaction is particularly pronounced in the white matter. CONCLUSIONS: These results demonstrate that prolonged microglial activation is a feature of traumatic brain injury, but that the neuroinflammatory response returns to control levels after several years.

Review: activation patterns of microglia and their identification in the human brain.

Microglia in the central nervous system are usually maintained in a quiescent state. When activated, they can perform many diverse functions which may be either beneficial or harmful depending on the situation. Although microglial activation may be accompanied by changes in morphology, morphological changes cannot accurately predict the function being undertaken by a microglial cell. Studies of peripheral macrophages and in vitro and animal studies of microglia have resulted in the definition of specific activation states: M1 (classical activation) and M2 (sometimes subdivided into alternative activation and acquired deactivation). Some authors have suggested that these might be an overlapping continuum of functions rather than discrete categories. In this review, we consider translational aspects of our knowledge of microglia: specifically, we discuss the question as to what extent different activation states of microglia exist in the human central nervous system, which tools can be used to identify them and emerging evidence for such changes in ageing and in Alzheimer's disease.

Comparison of complete scaling and a field-theoretic treatment of asymmetric fluid criticality.

We investigate the connection between the theory of complete scaling and a field-theoretic (FT) treatment of asymmetric fluid criticality. To facilitate the comparison, we develop an equation of state from a simplified form of the complete scaling transformations and systematically compare this equation of state with the equation of state generated by a FT treatment of an asymmetric Landau-Ginzburg-Wilson Hamiltonian. We find, with care in interpretation, that these two approaches may be read as equivalent up to terms involving an independent higher-order asymmetric correction-to-scaling exponent.

Association between APOE genotype, neuropathology and dementia in the older population of England and Wales.

AIMS: Apolipoprotein E (APOE) genotype is the major genetic risk factor for sporadic Alzheimer's disease (AD) but it is unclear how this is mediated. Most studies of APOE genotype have used case-control design to compare groups differing by two variables: i.e. dementia and AD pathology, so it is unclear to which of these variables APOE genotype is more strongly related. The prospective Medical Research Council Cognitive Function and Ageing Study neuropathology cohort is population-based sample in which donations are unbiased by dementia status. METHODS: We investigated the association between APOE genotypes and neuropathological and cognitive data in this cohort (n = 310). RESULTS: APOEε4 was associated with an increased risk of diffuse plaques, neuritic plaques, tangles and cerebral amyloid angiopathy. APOEε4 was not associated with infarcts, lacunes, haemorrhages or small vessel disease. APOEε2 appeared to have a protective effect on AD pathology and also on the risk of cortical atrophy. APOE genotype had a non-significant effect on the presence of dementia after adjusting for AD pathology. CONCLUSIONS: APOE genotype is associated with each of the key features of AD pathology but not with cerebrovascular disease other than cerebral amyloid angiopathy. The excess risk of dementia in those with an APOEε4 allele is explained by the pathological features of AD. However, it remains unclear to what extent cognitive dysfunction is caused by these specific pathological features or more directly by closely related APOE-associated mechanisms.

Microglial alterations in human Alzheimer's disease following Aß42 immunization.

AIMS: In Alzheimer's disease (AD), microglial activation prompted by the presence of amyloid has been proposed as an important contributor to the neurodegenerative process. Conversely following Aß immunization, phagocytic microglia have been implicated in plaque removal, potentially a beneficial effect. We have investigated the effects of Aß42 immunization on microglial activation and the relationship with Aß42 load in human AD. METHODS: Immunostaining against Aß42 and microglia (CD68 and HLA-DR) was performed in nine immunized AD cases (iAD - AN1792, Elan Pharmaceuticals) and eight unimmunized AD (cAD) cases. RESULTS: Although the Aß42 load (% area stained of total area examined) was lower in the iAD than the cAD cases (P=0.036), the CD68 load was higher (P=0.046). In addition, in the iAD group, the CD68 level correlated with the Aß42 load, consistent with the immunization upregulating microglial phagocytosis when plaques are present. However, in two long-surviving iAD patients in whom plaques had been extensively cleared, the CD68 load was less than in controls. HLA-DR quantification did not show significant difference implying that the microglial activation may have related specifically to their phagocytic function. CD68 and HLA-DR loads in the pons were similar in both groups, suggesting that the differences in microglial activation in the cortex were due to the presence of AD pathology. CONCLUSION: Our findings suggest that Aß42 immunization modifies the function of microglia by increasing their phagocytic activity and when plaques have been cleared, the level of phagocytosis is decreased below that seen in unimmunized AD.

Decompressive craniectomy for acute disseminated encephalomyelitis.

BACKGROUND: Acute disseminated encephalomyelitis (ADEM) is a rare, acute demyelinating condition. Although it usually presents in an acute or subacute manner over days, its clinical course may be rapid with symptoms and signs of severe intracerebral mass effect secondary to cerebral oedema. METHODS: Case report and literature review. RESULTS: We report a case of a patient presenting with a hyperacute course manifested by rapid loss of consciousness and focal neurological signs. Management with emergency hemicraniectomy and steroids resulted in rapid neurological improvement and minimal long-term deficit. CONCLUSIONS: We believe that only surgical decompression is likely to be life saving in similar cases of hyperacute cerebral oedema due to ADEM. The wide decompression performed was concordant with that indicated for traumatic brain swelling. Such aggressive management is vindicated by the rapid recovery shown by our patient within days of surgery and the finding of minimal neurological sequelae at 3 months.

Population variation in glial fibrillary acidic protein levels in brain ageing: relationship to Alzheimer-type pathology and dementia.

BACKGROUND: The cellular pathology of astrocytes in brain ageing and their role in modulating the brain's response to neurodegenerative pathology remain incompletely understood. METHODS: Using quantitative ELISA, we have investigated glial fibrillary acidic protein (GFAP) expression in the population-based neuropathology cohort of the Medical Research Council Cognitive Function and Ageing Study to determine: (1) the population variation in the astroglial hypertrophic response, (2) its relationship to the presence of Alzheimer-type pathology, and (3) its association with cognition. RESULTS: Increasing GFAP was found with increasing Braak stage, levels increasing even at early stages. Within Braak stages, GFAP did not differ between demented and non-demented individuals, but there was greater variance in GFAP in the demented. Possession of ApoE epsilon4 was associated with slightly increased GFAP levels (not significant) for given amyloid beta protein loads. CONCLUSION: In a population-based sample, increasing gliosis precedes development of Alzheimer lesions. Population variation in GFAP with varying Alzheimer lesion burdens suggests that they are not the only driver for astrogliosis. GFAP was not independently predictive of dementia, but the variation in astrocytic responses may be a factor modulating brain responses to neurodegenerative pathology.

Consequence of Abeta immunization on the vasculature of human Alzheimer's disease brain.

A major feature of Alzheimer's disease is the accumulation of amyloid-beta peptide (Abeta) in the brain both in the form of plaques in the cerebral cortex and in blood vessel as cerebral amyloid angiopathy (CAA). Experimental models and human clinical trials have shown that accumulation of Abeta plaques can be reversed by immunotherapy. In this study, we hypothesized that Abeta in plaques is solubilized by antibodies generated by immunization and drains via the perivascular pathway, detectable as an increase in cerebrovascular Abeta. We have performed a follow up study of Alzheimer's disease patients immunized against Abeta42. Neuropathological examination was performed on nine patients who died between four months and five years after their first immunization. Immunostaining for Abeta40 and Abeta42 was quantified and compared with that in unimmunized Alzheimer's disease controls (n = 11). Overall, compared with these controls, the group of immunized patients had approximately 14 times as many blood vessels containing Abeta42 in the cerebral cortex (P<0.001) and seven times more in the leptomeninges (P = 0.013); among the affected blood vessels in the immunized cases, most of them had full thickness and full circumference involvement of the vessel wall in the cortex (P = 0.001), and in the leptomeninges (P = 0.015). There was also a significantly higher level of cerebrovascular Abeta40 in the immunized cases than in the unimmunized cases (cortex: P = 0.009 and leptomeninges: P = 0.002). In addition, the immunized patients showed a higher density of cortical microhaemorrhages and microvascular lesions than the unimmunized controls, though none had major CAA-related intracerebral haemorrhages. The changes in cerebral vascular Abeta load did not appear to substantially influence the structural proteins of the blood vessels. Unlike most of the immunized patients, two of the longest survivors, four to five years after first immunization, had virtually complete absence of both plaques and CAA, raising the possibility that, given time, Abeta is eventually cleared from the cerebral vasculature. The findings are consistent with the hypothesis that Abeta immunization results in solubilization of plaque Abeta42 which, at least in part, exits the brain via the perivascular pathway, causing a transient increase in the severity of CAA. The extent to which these vascular alterations following Abeta immunization in Alzheimer's disease are reflected in changes in cognitive function remains to be determined.