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.

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.

Exposure of veterinary personnel to ionising radiation during bone scanning of horses by nuclear scintigraphy with 99mtechnetium methylene diphosphonate.

The aim of this study was to compare the radiation doses received by the personnel drawing up and injecting the radiopharmaceutical and operating the nuclear scintigraphy equipment, and those restraining nine horses while they were being scanned during scintigraphic investigations of lameness. Sensitive electronic dosimeters were worn by the personnel and the doses they received during the administration of the radiopharmaceutical and during the period of image acquisition were recorded at intervals. On average, 90 per cent of the total doses were received during the period of image acquisition. There was no significant difference between the total dose received by the person who drew up and injected the radiopharmaceutical, and the person restraining the horse during its administration. However, the person holding the horse received approximately twice the dose received by the person operating the equipment during the period of image acquisition.

Application of the 51Cr-EDTA urinary recovery test for assessment of intestinal permeability in the horse.

Altered intestinal permeability is implicated in the pathogenesis of diverse equine medical conditions including alimentary laminitis and protein-losing enteropathies associated with parasitic infection. The aims of this study were to assess the feasibility of applying the 51Cr-EDTA absorption test for the assessment of intestinal permeability in the horse, and to apply this test in horses with experimentally induced alterations in gastrointestinal function. Four healthy ponies were administered 36 MBq of 51Cr-EDTA via naso-gastric tube, and urine samples were collected into polythene bags strapped to the pony's abdomen. Total urine voided every 6 h was collected during each test, and 1 ml samples were taken for measurement of gamma-radiation. Urinary recovery of 51Cr-EDTA was measured following intravenous atropine sulphate or bethanecol, and following 22 and 46 days of administration of 250,000 third-stage cyathostome larvae. There was no significant difference in urinary 51Cr-EDTA recovery following the control treatment, and following atropine or bethanecol administration, but significant increases were detected in the animals with experimental cyathostome infection consistent with increased permeability of the intestinal membrane. Motility modifying agents (bethanecol and atropine) did not affect absorption of 51Cr-EDTA, suggesting that subtle changes in motility might not affect the ability of this test to detect altered intestinal permeability. The finding of increased urinary recovery of 51Cr-EDTA in ponies with cyathostome infection suggests that 51Cr-EDTA may be a useful marker for assessment of intestinal permeability in the horse.

Somatostatin- and neuropeptide Y-synthesizing neurones in the fascia dentata of humans with temporal lobe epilepsy.

We used in situ hybridization techniques to study the distribution of neurones synthesizing somatostatin mRNA and neuropeptide Y mRNA in the hilar region of the hippocampal formation of patients with temporal lobe epilepsy. In the dentate gyrus, somatostatin mRNA- and neuropeptide Y mRNA-synthesizing neurones were found to be exclusively located within the hilar region. Unlike animal models, no ectopic expression of either peptide was found in principal cells. The numbers of hilar interneurones expressing somatostatin mRNA and neuropeptide Y mRNA were compared with the degree of hilar cell loss determined by immunohistochemistry against neuronal nuclear antigen. The numbers of somatostatin and neuropeptide Y mRNA-synthesizing neurones varied considerably between patients, but both were found to be highly correlated to the total number of neuronal nuclear antigen-immunoreactive hilar neurones. These results suggest that loss of somatostatin and neuropeptide Y interneurones occurs in proportion to overall hilar cell loss, and therefore the hypothesis of a selective loss of these interneurones in temporal lobe epilepsy seems unlikely.

Mossy fibre innervation is not required for the development of kainic acid toxicity in organotypic hippocampal slice cultures.

The glutamate analogue kainic acid (KA) generates convulsions when applied systemically or directly into the brain and produces lesions comparable to those seen in Ammon's horn sclerosis, observed in many patients with temporal lobe epilepsy. The neurotoxic actions of KA in-vivo appear to be mediated by a combination of direct effects on neurons and indirect effects mediated by seizures. Understanding the contribution of both direct and indirect effects of KA towards neuronal cell death is important for elucidating excitotoxic mechanisms, which may represent a common final pathway in a variety of neurodegenerative disorders including stroke, traumatic brain injury and epilepsy. We have investigated the effects of mossy fibre innervation on the development of KA toxicity in organotypic hippocampal slice cultures in order to assess the role of this input pathway on the specific toxicity of KA toward CA3 pyramidal neurones in vitro.

Aberrant Timm-stained fibres in the dentate gyrus following tetanus toxin-induced seizures in the rat.

The present study addresses whether seizures, which result from the chronic block of inhibition caused by an intrahippocampal injection of tetanus toxin, induce axonal sprouting of the hippocampal mossy fibres. Timm stain was used to identify the mossy fibre terminals. In nine of 15 animals killed at 1 month or later after an injection of tetanus toxin, Timm-stained terminals were observed bilaterally in the inner molecular layer and in seven animals a meshwork of Timm-stained fibres/ terminals was also observed bilaterally in the outer molecular layer of the fascia dentata. None of these changes were observed in any of the 12 saline-injected controls. There was no obvious correlation between the number of motor fits an animal exhibited and the amount of Timm-stained fibre sprouting present in either the inner or outer molecular layer. The Timm-stained axonal sprouting into the outer molecular layer of the fascia dentata may simply reflect the reinnervation of sites on the granule cell dendrites, previously occupied by the terminals of the hilar somatostatin-containing cells. These hilar somatostatin-containing cells which are believed to project to the outer molecular layer are known to succumb to the seizure activity in this animal model of epilepsy.

Hippocampal NPY neurons project to the fascia dentata in organotypic cultures.

The distribution of neuropeptide Y immunoreactive (NPY-ir) neurons in organotypic cultures of hippocampi from neonates was compared to that seen in adult rats. In addition to the known NPY-ir neurons in the hippocampus proper and in the hilus of the fascia dentata, isolated, large, multipolar, NPY-ir neurons were observed in the subiculum and in areas CA1 and CA3. Their axons projected into stratum radiatum of the hippocampus proper and into the molecular layers and hilus of the fascia dentata where they branched profusely. These NPY-ir neurons were regularly distributed throughout the septo temporal extent of the hippocampus and were present in both neonates and adult hippocampi. The hilar NPY-ir neurons have always been considered the source of the NPY-ir plexus in the outer molecular layer of the dentate gyrus. However, our results show that there is also a contribution from the NPY-ir neurons in the hippocampus proper.

Loss of hilar somatostatin neurons following tetanus toxin-induced seizures.

A loss of inhibitory interneurons has been reported in the hippocampus following seizure activity in various animal models of epilepsy and in human epileptic tissue. The question of whether particular populations of inhibitory neurons are similarly affected by the chronic block of inhibition that results after tetanus toxin injections directly into the brain has not previously been addressed. In the present study a unilateral intrahippocampal injection of tetanus toxin into the ventral hippocampus was used to produce a chronic epileptic syndrome characterised by brief seizures that recurred intermittently for 6-8 weeks. The results reveal, for the first time, the morphological changes in somatostatin interneurons following tetanus toxin-induced seizures in the rat. A bilateral short-term increase in immunoreactivity of somatostatin neurons is present 1 week after injection. This is accompanied by an increased intensity of somatostatin-immunoreactive axon terminals in the outer molecular layer of the dentate gyrus, which is more marked on the contralateral side. A chronic and significant loss of somatostatin-immunoreactive neurons was noted in the hilus of the dentate gyrus 2 months later. The significance of the chronic loss of the hilar somatostatin neurons in the control of excitatory activity in the dentate gyrus and whether the acute morphological changes are due to a direct action of the toxin on release mechanisms or as a result of seizure activity are discussed.