Cognitive Decline and Modulation of Alzheimer's Disease-Related Genes After Inhibition of MicroRNA-101 in Mouse Hippocampal Neurons.

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4-5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aß) 42 levels, the more toxic species of Aß peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.

Eruptive syringomas with calcium deposits in a young woman with Down's syndrome.

Eruptive syringomas are uncommon in the general population. We describe here an 18-year-old female, affected by Down's syndrome, who presented with an abrupt eruption of small skin-colored or reddish papules on the face, neck and limbs. Light microscopy allowed us to diagnose syringomas, whereas the study of the ultrastructural features revealed calcium deposits in many lumina and also in the mitochondria. This observation confirms the hypothesis that the syringeal structure plays a role in the pathogenesis of calcinosis cutis.

Primary adenocarcinoma of the arytenoid: an ultrastructural study.

Primary adenocarcinoma of the larynx is a rare neoplasm that tends to spread to both regional lymph nodes and distant sites. A case of primary adenocarcinoma of the arytenoid in a 74-year-old man is presented. The tumor was evaluated by light and electron microscopy. A high percentage of intranuclear pseudoinclusions (more than 20% of the neoplastic cells) was a peculiar characteristic of the tumor. To the best of our knowledge, such a feature has not been reported previously and should be considered a hallmark of more aggressive behavior.

Chronic heat-induced skin lesions (erythema ab Igne): ultrastructural studies.

Erythema ab igne (EI) is an uncommon skin lesion caused by mild and repeated exposure to infrared sources. The aim of this study was to investigate the ultrastructural alterations in this condition. The ultrastructural study was carried out on 5-outpatients who presented typical EI of their exposed sites. Skin punch biopsies were processed for standard electron microscopy. The epidermis was hyperpigmented, with focal regressive changes of basal keratinocytes. An apparent functional activation of melanocytes with numerical increase of dendritic processes was also observed. The dermis showed abundant melanophages and occasional elastic fiber alterations similar to actinic elastosis. No alterations consistent with preneoplastic skin conditions were observed. The ultrastructural findings associated with EI seem to be nonspecific and consistent with moderate regressive changes of keratinocytes as well as a consensual melanocytic activation and elastic fiber modifications. Similar alterations can be observed in chronic actinic skin damage. This condition is presumably more benign than the ultraviolet exposure. The association of EI and premalignant skin lesions, though occasionally described, seems relatively infrequent.

Morphological characteristics by SEM observations and regulatory volume decrease (RVD) of tentacular nematocytes isolated by heat dissociation from Aiptasia diaphana (Cnidaria: Anthozoa).

This paper examines the morphological characteristics and response to hyposmotic shock of nematocytes isolated by heat dissociation at 45 degrees C for 20 min from the tentacles of Aiptasia diaphana, an anthozoan living in the brackish water of Lake Faro (Messina, Italy). Morphological characteristics were evaluated by scanning electron microscopy (SEM) observations and cytological test, functional characteristics by exposure to 35% hyposmotic stress. 81% of the population of isolated nematocytes had ciliary protrusions. Microbasic-mastigophore and amastigophore nematocytes had a hair bundle at the crown shaped apex composed of ciliary protrusions of different heights and diameters. In basitrichs, instead, a single ciliary protrusion was observed. Following exposure to 35% hyposmotic shock, nematocytes isolated by heat dissociation did not show RVD, while, following treatment with 1 microM and 2 microM gramicidin-S, activation of volume regulation in conditions of hyposmoticity was observed. The effect of gramicidin was concentration-dependent and confirmed the relevant role of conductance to K+ in volume regulation, as previously described (17) on nematocytes isolated by chemical methods.

Primary macular amyloidosis: an ultrastructural approach to diagnosis.

Seven cases of primary macular amyloidosis were studied on skin biopsies. The Congo red stain was positive only in three cases, whereas the ultrastructural observation allowed for the detection of amyloid deposits in all biopsies. Fibrillary degeneration of basal keratynocytes was occasionally observed, and regressive changes of keratynocytes and dermal nerve bundles presumably related to the intensity of the scratch trauma were detected in one case. In six biopsies mast cell profiles exhibiting various degrees of degranulation were detected in the dermis. Melanosome aggregates were also observed consistently in dermal macrophages and occasionally in Schwann cells. A variable degree of structural alteration was observed in dermal unmyelinated nerve fibers. Even if the intimate mechanism of amyloid deposition was not explained by the ultrastructural study, this approach is a useful instrument in the differential diagnosis of cutaneous macular hyperpigmented lesions.

Time-related ultrastructural changes in an experimental model of whole brain irradiation.

To stimulate therapeutic irradiation, we exposed rats to conventional fractionation (200 +/- 4 cGy/d, 5 d/wk; total dose, 4000 cGy). The effects of this regimen were assessed by electron microscopic examinations of brain microvascular and parenchymal cells 15 and 90 days after irradiation. Studies of the transendothelial passage of horseradish peroxidase provided information about the functional status of the blood-brain barrier. At 15 days after irradiation, there was an increased vesicular transport of horseradish peroxidase across the intact endothelium without opening of the tight junctions, and without evidence of structural alterations of neuropil, neuronal bodies, and astrocytes. Ninety days after irradiation, well-defined ultrastructural alterations were observed, involving the microvasculature, the neuropil, the neuronal bodies, and astrocytes. The main ultrastructural feature of cortical microvessels was their collapsed aspect, associated with perivascular edema containing cell debris. Altered neurons and reactive activated astrocytes were also noticeable. These data suggest a possible association, not necessarily causal, between damage of the microvascular/glial unit of tissue injury and development of radiation-induced brain toxicity.

Brain energy metabolism in the acute stage of experimental subarachnoid haemorrhage: local changes in cerebral glucose utilization.

An experimental model was used to investigate acute alterations of cerebral metabolic activity in rats subjected to subarachnoid haemorrhage (SAH). Haemorrhages were produced in anaesthetized animals by injecting 0.3 ml of autologous, arterial nonheparinized blood into the cisterna magna. Control rats received subarachnoid injections of mock-cerebrospinal fluid to study the effect of sudden raised intracranial pressure, or underwent sham operation. Three hours after SAH rats were given an intravenous injection of [14C]-2-deoxyglucose. Experiments were terminated by decapitation, and the brains were removed and frozen. Regional brain metabolic activity was studied by quantitative autoradiography. In comparison with sham-operated controls, cerebral metabolic activity was diffusely decreased after SAH. Statistically significant decreases in metabolic rate were observed in 23 of 27 brain regions studied. Subarachnoid injections of mock-cerebrospinal fluid also produced depression of cerebral metabolic activity, but quantitatively these changes were not as pronounced and diffuse as in SAH rats. The present study shows that a widespread depression of brain metabolism occurs in the acute stage after experimental SAH and is probably secondary to the subarachnoid presence of blood itself and/or blood products.

Progressive perturbations in cerebral energy metabolism after experimental whole-brain radiation in the therapeutic range.

Basic mechanisms underlying the tolerance and reaction of the central nervous system to ionizing radiation have not been fully elucidated in the literature. The authors employed the [14C]-2-deoxy-D-glucose autoradiography method to investigate the effect of whole-brain x-irradiation on local cerebral glucose utilization in the rat brain. The animals were exposed to conventional fractionation (200 +/- 4 cGy/day, 5 days/week for a total dose of 4000 cGy), and the effects of this regimen were assessed at 2 weeks and 3 months postirradiation. In rats evaluated 2 weeks after treatment, statistically significant decreases in cerebral metabolic activity were found in 13 of the 27 regions studied, compared to control animals. In rats studied 3 months after treatment, additional metabolic suppression and statistically significant decreases in cerebral metabolic activity were found in 11 of the 27 regions, compared to rats studied 2 weeks after treatment. A weighted-average rate for the brain as a whole was approximately 15% and approximately 25% below that of control animals 2 weeks and 3 months after exposure, respectively. Although the difference in species is significant enough so that direct extrapolation to humans may not be appropriate, the data reported here may have potential clinical implications for the evaluation of the risk-benefit ratio for radiotherapy. This model can be used reproducibly for further investigations, including evaluation of therapies that may reduce irradiation-induced brain injury.

Morphological, phenotypic and karyotypic characterization of a novel natural-killer-cell target - evidence of involvement of MHC class-I molecules in NK cell recognition.

A new cell line from a neoplastic ascites was established. This strain, designated PAT-206, was characterized by plastic adherence and a high proliferative potential without any specific growth factor requirement. Karyotype analysis showed that the line was of human chromosomal constitution and aneuploid. Surface marker analysis showed that CD45, CD33 and CD15 were positive. In addition, the presence of human cytokeratins was detected by cytoplasmic immunofluorescence. Interestingly, the cell line did not express major hystocompatibility complex (MHC) class I and II, and was more sensitive than the 'classic' K562 cell line, to killing mediated by fresh uncultured peripheral blood lymphocytes. Following differentiation with interferon-gamma, the cell line expressed MHC class I antigens and resulted resistant to natural killing mediated lysis. This novel NK cell target seems to be suitable for further studies on NK cell specificities.

Circadian rhythm of glucose utilization in the pineal body of rats of different ages.

Employing quantitative autoradiography, pineal body glucose utilization (GU) was measured in daytime or at night in prepubertal (aged 1 month), adult (aged 3 months), and mature (over 12 months old) rats. In prepubertal and adult rats, in daytime, GU values within the pineal tissue were homogeneously distributed around 65 mumol glucose/100 g per min. In prepubertal animals no significant variations in GU were observed between daytime and nocturnal measurements. A circadian metabolic rhythmicity was evident in adult rats, with a GU peak measured at 2 a.m. In mature animals, GU also varied between day and night, with an increment in the relative difference between the two values. The present investigation is the first to demonstrate that circadian metabolic rhythmicity is absent before sexual maturation while it is enhanced in 12-month-old rats. These changes in pineal energy metabolism with advancing age are intriguing in view of the concept that the pineal gland may be involved in functional changes occurring during the process of aging.

Quantitative study of blood-brain barrier permeability changes after experimental whole-brain radiation.

Basic mechanisms underlying the tolerance and reaction of the central nervous system to ionizing radiation are not known precisely. We investigated the possibility of a change in blood-brain barrier (BBB) function as a causative factor for early delayed whole-brain radiation-induced cerebral dysfunction. Rats were exposed to conventional fractionation (200 cGy/d, 5 d/wk; total dose, 4000 cGy). BBB changes were assessed by means of the quantitative 14C-alpha-aminoisobutyric acid technique and electron microscopy. Studies of the passage of horseradish peroxidase across the BBB permitted comparative quantitative isotopical and qualitative morphological data. Experiments were carried out 2 to 3 weeks after the completion of the radiation exposure. The transport of 14C-alpha-aminoisobutyric acid across the BBB increased significantly in cerebral cortex and cerebellar gray matter, averaging 1.3 to 1.5 times over the normal values. Electron microscopy disclosed an intense vesicular response of the cortical microvascular endothelium that occurred without the opening of the tight junctions and resulted in an intense transport of HRP across the intact endothelium. The present data indicate that moderate doses of whole-brain radiation induce well-defined changes in BBB function, which possibly are involved in the pathogenesis of radiation-induced cerebral dysfunction in humans.

Effect of whole brain radiation on local cerebral glucose utilization in the rat.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of whole-brain x-radiation on local cerebral glucose utilization in the rat brain. Animals were exposed to conventional fractionation (200 +/- 4 cGy/day, 5 days/week; total dose, 4000 cGy). Metabolic experiments were made 2 to 3 weeks after completion of the radiation exposure. In comparison with control and sham-irradiated animals, cerebral metabolic activity was diffusely decreased after irradiation. Statistically significant decreases in metabolic activity were observed in 13 of 27 brain regions studied. In general, the brain areas with the highest basal metabolic rates showed the greatest percentage of decrease in glucose utilization. The concept that radiation suppresses glucose utilization before any morphological change takes place in the cell structures was the basis of this study. Metabolic alterations after irradiation may explain the syndrome of early delayed deterioration observed in humans after whole-brain radiotherapy. These studies have applications to observations made with the [18F]-fluorodeoxyglucose method in conjunction with positron emission tomographic scans in patients receiving radiation therapy for intracranial malignancies. The data reported here also have potential clinical implications for the evaluation of a risk/benefit ratio for radiotherapy in patients with benign neurosurgical diseases or children undergoing prophylactic treatment of the central nervous system.

[Cerebral metabolism and permeability of the hemato-encephalic barrier in an experimental model for brain radiotherapy]. / Metabolismo cerebrale e permeabilità della barriera ematoencefalica in un modello sperimentale di trattamento radioterapico cerebrale.

Whole brain irradiation (WBR) can produce acute and chronic neurological adverse effects, which are usually divided into acute, early delayed and late delayed reactions according to the time of onset. To assess the impact of WBR on brain functional parameters during the early-delayed phase, we employed the [14C]-2-deoxyglucose (2-DG) and the [14C]-alfa-aminoisobutyric (AIB) acid quantitative autoradiographic techniques to study local cerebral glucose utilization and blood-brain barrier permeability, respectively. Sprague-Dowley albino rats were exposed to conventional fractionation (200 Gy/day 5 days a week) for a total dose of 4000 Gy. Experiments were made 3 weeks after completion of the radiation exposure. In comparison with control and sham-irradiated animals, cerebral metabolic activity was diffusely decreased following irradiation. As a rule, brain areas with the highest basal metabolic rates showed the highest percentage drop in glucose utilization. Changes in blood-brain barrier function, as assessed by an increased transcapillary transport of AIB, were also demonstrated in specific brain regions. This study illustrates how moderate doses of WBR induce well-defined changes in brain metabolism and BBB function, which are possibly involved in the pathogenesis of the early-delayed radiation-induced cerebral dysfunction in humans.

Effect of x-radiation to brain on cerebral glucose utilization in the rat.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of whole-brain x-radiation on local cerebral glucose utilization in the rat brain. Animals were exposed to conventional fractionation (200 +/- cGy/day given 5 days a week) to a total dose of 4000 cGy. Metabolic experiments were made 2 weeks after completion of the radiation exposure. In comparison with control and sham-irradiated animals, cerebral metabolic activity was diffusely decreased following irradiation. Statistically significant decreases in metabolic activity were observed in 13 of 27 brain regions studied. In general, brain areas with the highest basal metabolic rates showed the greatest percentage drop of glucose utilization. Post-irradiation metabolic alterations possibly provide an explanation for the syndrome of early delayed deterioration observed in humans after whole-brain radiotherapy.

Autoradiographic assessment of pineal gland glucose utilization and capillary permeability in the unanesthetized rat.

Pineal gland glucose utilization (GU) and capillary permeability (CP) were measured in unanesthetized rats, using complementary quantitative autoradiographic techniques. GU values within the pineal tissue were homogeneously distributed around 70 mumol of glucose/100 g each min, i.e., they were approximately 30% lower than in the cortical gray structures. The blood-to-brain transfer constant of [14C]-alpha-aminoisobutyric acid, as an index of CP, was up to ten orders of magnitude higher than that for the rest of the brain. These measurements were carried out at that point in the circadian rhythm that corresponds to the minimum level of neurosecretory activity of the pineal gland.

Effect of injections of contrast media on regional uptake of (14C)-2-deoxyglucose by the rat brain.

Mechanisms contributing to the rare but consistent neurotoxicity of contrast media currently in clinical use for the radiological examination of the subarachnoid space remain to be isolated. We assessed, by means of the (14C)-2-deoxy-D-glucose (2-DG) autoradiographic method, the effect of three non-ionic, low-osmolar contrast media, namely metrizamide, iopamidol and iohexol, on the local cerebral glucose utilization in the rat brain after intracisternal application. A significant (-30%) global reduction of the brain's metabolic activity occurred following intracisternal metrizamide injection. When compared with the mock-CSF control group the greater relative changes were observed in the supratentorial grey matter structures. In contrast, no significant changes were observed in metabolic brain activity in rats treated intracisternally with iopamidol and iohexol. These findings were consistent with the hypothesis that metrizamide is a competitive inhibitor of human brain hexokinase. The apparent lack of interference on neural tissue metabolism makes the second generation contrast media less neurotoxic and more suitable for neuroradiological subarachnoid investigations in clinical settings. The present experimental work establishes the 2-DG method as a viable laboratory approach to investigate aspects of neuronal dysfunction induced by contrast media.

Effect of intracarotid injection of iopamidol on local cerebral glucose utilization in rat brain.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of intracarotid injection of a nonionic, low-osmolar contrast medium (iopamidol) on local cerebral glucose utilization in the rat brain. Contrast medium was injected at 20 degrees C and at 37 degrees C, and the relative changes in local cerebral glucose utilization were measured. At 20 degrees C the viscosity of the contrast agent was about twice that of the same solution at 37 degrees C, and resulted in a statistically significant increase in local cerebral glucose utilization in the hemisphere ipsilateral to the side of intracarotid infusion. Saline control studies showed that the metabolic change was not related to either the solution temperature or the osmolality. These findings suggest that increased viscosity of a contrast medium may contribute to its neurotoxic effects during cerebral angiography, hence emphasizing the importance of preheating contrast material to avoid adverse reactions.

Scanning electron microscope study of human arachnoid villi.

The functional morphology of human arachnoid villi obtained from surgical biopsy specimens has been studied by scanning electron microscopy (SEM). On SEM examination, the villi appeared to be distended, as if functioning normally. The endothelial cells constituting the cerebrospinal fluid (CSF)-blood interface were covered by numerous microvilli, uniformly oriented along the major axis of the villus. Examination for cell-to-cell contact revealed only occasional areas of tight adherence between adjacent endothelial cells, while widened intercellular spaces were frequently observed. Generally corresponding to the apex of the villus, points of emergence of endothelium-lined hollow structures were identified; these may represent apical openings of open pathways from the subarachnoid space to the venous system. Ultrastructural arrangements consistent with a closed system of CSF reabsorption were also observed. Large cells maximally distended and protruding into the sinus lumen were commonly seen; these were interpreted as the result of the formation of giant vacuoles within the endothelium covering the villus. This study has provided ultrastructural evidence for both closed and open systems of CSF reabsorption. Ultrastructural findings, such as gaps between endothelial cells and tubule-like endothelium-lined structures as previously identified in animals and observed in man by transmission electron microscopy, were demonstrated in human biopsy specimens by SEM.