Juan Arévalo-Serrano

Oligomers of beta-amyloid protein (Aβ1-42) induce the activation of cyclooxygenase-2 in astrocytes via an interaction with interleukin-1beta, tumour necrosis factor-alpha, and a nuclear factor kappa-B mechanism in the rat brain

Despite growing evidence indicating the effects of cytokines, including interleukin-1beta (IL-1β) and tumour necrosis factor-α (TNFα), and the enzyme cyclooxygenase-2 (COX-2) in Alzheimers diseases, little is known about the signalling mechanisms that mediate its activation in response to beta-amyloid protein (Aβ). The aim of this study was first to investigate whether Aβ1-42 peptide induced the up-regulation of COX-2. We then examined the expression of COX-2 and cytokines, such as IL-1β and TNFα, in reactive astrocytes. Finally, we analyzed the role of nuclear factor kappa-B (NF-κB) as a signalling pathway in early stages of Aβ-toxicity. In Wistar rats anaesthetised with equitesine, a single microinjection of Aβ1-42 oligomers was made in the left retrosplenial cortex. Control animals were injected with Aβ42-1 peptide into the corresponding region of the cerebral cortex. By COX-2 immunoblotting, we detected two immunopositive protein bands, at 70 and 50 kDa molecular mass. In the Aβ1-42-injected animals the 50 kDa fragment showed a significant increase at 3 and 14 days, as compared with that seen in control animals. The 70 kDa fragment showed a maximal increase at 14 days. In the Aβ1-42-injected animals immunoblot staining of NF-κB detected an active protein band at 50 kDa molecular mass, showing a maximal increase at the 72 h time point. Confocal analysis revealed that COX-2 protein co-localized with Aβ-IR material at the injection site and in endothelial blood vessels, increasing at 72 h. In the Aβ oligomer-treated animals, COX-2, IL-1β, and TNFα proteins were expressed in reactive astrocytes surrounding the injection site and blood vessels at early stages of Aβ toxicity. Double-labelling immunofluorescence studies also revealed that GFAP and COX-2 proteins co-localized with NF-κB-positive material at early time-points. In conclusion, our results suggest that in reactive astrocytes and in COX-2 positive cells NF-κB may mediate pro-, and/or inflammatory gene expression and that, develop strategies that target the GFAP/NF-κB and COX-2/NF-κB pathways might contribute to reducing Aβ-induced toxicity.

Environmental risk factors for essential tremor.

We conducted a case-control study searching for a possible role of environment in the risk of essential tremor (ET). We interviewed 142 ET patients and 284 age- and sex-matched controls about a family history of ET, exposure to environmental products containing lead, mercury, manganese, solvents and β-carbolines, and exposure to agricultural work, well water, pesticides, and cigarette smoking and alcohol drinking habits. In a univariate study, reported family history of ET and exposure to agricultural work, pesticides, smelting, frosted glass, paintings, wheat, corn, and barley were more frequent in the ET patient group. With a multivariate study, only reported family history of ET and exposure to agricultural work and frosted glass remained significant. Time of exposure to agricultural work, wheat and barley was significantly higher in ET patients. Age at onset of ET was significantly lower in patients with a family history of tremor and higher in patients exposed to iron-manganese alloys and alcohol. Time of exposure, but not total consumption of alcohol and cigarettes, was correlated with age at onset of ET. In conclusion, our study shows that the association between ET and reported family history of ET was robust, and that there were also associations between ET and exposure to some environmental factors (agricultural work and frosted glass).

Soluble oligomeric forms of beta-amyloid (Aβ) peptide stimulate Aβ production via astrogliosis in the rat brain

The aim of this study was to investigate the interaction between beta-amyloid (Abeta) peptide and astrogliosis in early stages of Abeta toxicity. In Wistar rats, anaesthetised with equitesine, a single microinjection of Abeta1-42 oligomers was placed into the retrosplenial cortex. Control animals were injected with Abeta42-1 peptide into the corresponding regions of cerebral cortex. Immunocytochemical analysis revealed an intense Abeta immunoreactivity (IR) at the level of Abeta1-42 injection site, increasing from the first 24 h to later (72 h) time point. Control injection showed a light staining surrounding the injection site. In Abeta oligomers-treated animals, Abeta-immunopositive product also accumulates in cortical cells, particularly in frontal and temporal cortices at an early (24 h) time point. Abeta-IR structures-like diffuse aggregates forms were also observed in hippocampus and in several cortical areas, increasing from the first 24 h to later (72 h) time point. In control animals no specific staining was seen neither in cortical cells nor in structures-like diffuse aggregates forms. Injections of Abeta oligomers also induce activation of astrocytes surrounding and infiltrating the injection site. Astrocyte activation is evidenced by morphological changes and upregulation of glial fibrillary acidic protein (GFAP). By GFAP immunoblotting we detected two immunopositive protein bands, at 50 and 48 kDa molecular mass. Confocal analysis also showed that GFAP co-localized with Abeta-IR material in a time-dependent manner. In conclusion, our results indicate that astrocyte activation might have a critical role in the mechanisms of Abeta-induced neurodegeneration, and that should be further studied as possible targets for therapeutic intervention in AD.

Functional assessment of the visual pathway with multifocal visual evoked potentials, and their relationship with disability in patients with multiple sclerosis.

Objective: To objectively evaluate the visual function, and the relationship between disability and optic nerve dysfunction, in patients with multiple sclerosis (MS) and optic neuritis (ON), using multifocal visual evoked potentials (mfVEP). Methods: This observational, cross-sectional study assessed 28 consecutive patients with clinically definite MS, according to the McDonald criteria, and 19 age-matched healthy subjects. Disability was recorded using the Expanded Disability Status Scale (EDSS) score. The patients’ mfVEP were compared to their clinical, psychophysical (Humphrey perimetry) and structural (optic coherence tomography (OCT)) diagnostic test data. Results: We observed a significant agreement between mfVEP amplitude and Humphrey perimetry/OCT in MS-ON eyes, and between mfVEP amplitude and OCT in MS but non-ON eyes. We found significant differences in EDSS score between patients with abnormal and normal mfVEP amplitudes. Abnormal mfVEP amplitude defects (from interocular and monocular probability analysis) were found in 67.9% and 73.7% of the MS-ON and MS-non-ON group eyes, respectively. Delayed mfVEP latencies (interocular and monocular probability analysis) were seen in 70.3% and 73.7% of the MS-ON and MS-non-ON groups, respectively. Conclusions: We found a significant relationship between mfVEP amplitude and disease severity, as measured by EDSS score, that suggested there is a role for mfVEP amplitude as a functional biomarker of axonal loss in MS.

Evaluation of visual structural and functional factors that predict the development of multiple sclerosis in clinically isolated syndrome patients.

PURPOSE To evaluate visual pathway structure and function in patients with clinical isolated syndrome (CIS) by using spectral-domain optical coherence tomography (OCT) and multifocal visual-evoked potentials (mfVEP), predicting CIS conversion to clinically definite multiple sclerosis (MS). METHODS This observational, longitudinal study assessed the eyes with no previous history of optic neuritis of 29 consecutive patients with CIS according to the McDonald criteria. The relationships of the mfVEP results with the clinical findings, and psychophysical (Humphrey perimetry) and structural (OCT) diagnostic test data were investigated. RESULTS The mfVEP amplitude responses (interocular and monocular probability analysis) showed abnormal cluster visual field defects in 48.3% of the CIS eyes, whereas mfVEP latency analysis showed significant delays in 20.7%. The OCT average retinal nerve fiber layer thickness (RNFLT) was significantly reduced compared with the control group (P = 0.02). Significant differences between CIS eyes with abnormal and normal mfVEP latencies were found for the OCT RNFLT (P < 0.001) with a longer latency being linked to more severe axonal damage. Using multivariate logistic regression analysis, OCT average RNFLT was found to be an independent predictor of clinically definitive MS diagnosis at 12 months. CONCLUSIONS The combined use of OCT and mfVEP is helpful to detect significant subclinical visual pathway abnormalities and axonal loss in CIS patients. Retinal axonal loss measured by OCT is an important prognostic factor of conversion to MS in patients with CIS in absence of symptomatic optic neuritis.

Effects of β‐amyloid peptide on the density of M2 muscarinic acetylcholine receptor protein in the hippocampus of the rat: relationship with GABA‐, calcium‐binding protein and somatostatin‐containing cells

Aims: The deposition of amyloid peptides (Aβ) in the cortex and hippocampus is the primary trigger of Alzheimers disease (AD). Recent studies also indicated that the M2 subtype of muscarinic acetylcholine receptors (M2mAChR) may be a key molecule involved in cognitive dysfunction. Thus, the purpose of this study was to determine the effects of extracellular deposition of Aβ on the density of M2mAChR in the hippocampus of the rat by M2mAChR‐immunohistochemistry. Methods: Special attention was paid to discerning any interaction between Aβ and M2mAChR in GABA‐, and calcium‐binding protein containing cells by double‐labelling immunohistochemistry. Densitometric analysis of M2mAChR‐immunoreactivity was performed using Scion Image Beta Software. Quantitative analysis of GABA‐, and calcium‐binding protein interneurones containing M2mAChR protein was performed using a NeuroLucida morphometric system. Results: Injections of Aβ into the retrosplenial cortex resulted in a significant reduction in M2mAChR‐immunoreactivity in the CA1 ipsilateral to the Aβ‐injected side as compared with the corresponding hemisphere of non‐treated control animals and with that in the corresponding region of the CA1 in the phosphate‐buffered saline‐injected side. Co‐localization studies showed that the M2mAChR is localized in a subset of GABA‐positive cells of the hippocampus, in cells that contain calcium‐binding proteins, and in a subpopulation of cells that contain the neuropeptide somatostatin. Conclusions: Our findings suggest that Aβ induces a significant reduction in M2mAChR‐immunoreactivity in the CA1 of the hippocampus and a reduction in GABAergic interneurones containing M2mAChR, which may contribute to impairment of GABAergic synaptic transmission in area CA1 of hippocampus.

Beta-amyloid peptide-induced modifications in α7 nicotinic acetylcholine receptor immunoreactivity in the hippocampus of the rat: Relationship with GABAergic and calcium-binding proteins perikarya

The effects of the injected beta-amyloid (Abeta) protein on the alpha7 subtype of nicotinic acetylcholine receptor protein (alpha7nAChR) in the hippocampus were studied in rats. Injections of Abeta into the retrosplenial cortex resulted in a decrease in alpha7nAChR-immunoreactivity in the hippocampus. Quantitative analysis revealed a significant reduction in alpha7nAChR-immunoreactivity in the dorsal part of the CA1 ipsilateral to the Abeta-injected side as compared to the corresponding hemisphere of non-treated control animals and with that seen in the contralateral hemisphere, which corresponds to the control (PBS)-injected side. A significant decrease in alpha7nAChR-immunoreactivity was also found in the dorsal part of the ipsilateral CA1 as compared with that in the ventral part of the CA1, in CA2, and in CA3 ipsilateral to the Abeta-injected side. The analysis also revealed a significant decrease in alpha7nAChR-immunoreactivity in the dentate gyrus ipsilateral to the Abeta-injected side as compared to the corresponding hemisphere of non-treated control animals and with that in the PBS-injected side co-localization studies showed that the alpha7nAChR protein is highly localized in GABA- and Parv-immunoreactive cells, while only few Calb-positive cells expressed immunoreactivity for alpha7nAChR. In addition, injections of Abeta protein resulted in a significant reduction in the number of GABA- and Parv-immunoreactive cells in the dorsal part of the ipsilateral CA1 as compared to the corresponding region of non-treated control animals and with that in the corresponding region of the PBS-injected side. Our findings suggest that Abeta induces a reduction in alpha7nAChR-containing cells, which may contribute to impairment of GABAergic synaptic transmission in the hippocampus.

Effects of β-amyloid protein on M1 and M2 subtypes of muscarinic acetylcholine receptors in the medial septum-diagonal band complex of the rat: relationship with cholinergic, GABAergic, and calcium-binding protein perikarya

Cortical cholinergic dysfunction has been correlated with the expression and processing of β-amyloid precursor protein. However, it remains unclear as to how cholinergic dysfunction and beta-amyloid (Aβ) formation and deposition might be related to one another. Since the M1- and M2 subtypes of muscarinic acetylcholine receptors (mAChRs) are considered key molecules that transduce the cholinergic message, the purpose of the present study was to assess the effects of the injected Aβ peptide on the number of M1mAchR- and M2mAChR-immunoreactive cells in the medial septum-diagonal band (MS-nDBB) complex of the rat. Injections of Aβ protein into the retrosplenial cortex resulted in a decrease in M1mAChR and M2mAChR immunoreactivity in the MS-nDBB complex. Quantitative analysis revealed a significant reduction in the number of M1mAChR- and M2mAChR-immunoreactive cells in the medial septum nucleus (MS) and in the horizontal nucleus of the diagonal band of Broca (HDB) as compared to the corresponding hemisphere in control animals and with that seen in the contralateral hemisphere, which corresponds to the PBS-injected side. Co-localization studies showed that the M1mAChR protein is localized in GABA-immunoreactive cells of the MS-nDBB complex, in particular those of the MS nucleus, while M2mAChR protein is localized in both the cholinergic and GABAergic cells. Moreover, GABAergic cells containing M2mAChR are mainly localized in the MS nucleus, while cholinergic cells containing M2mAChR are localized in the MS and the HDB nuclei. Our findings suggest that Aβ induces a reduction in M1mAChR- and M2mAChR-containing cells, which may contribute to impairments of cholinergic and GABAergic transmission in the MS-nDBB complex.

Evaluation of visual function and retinal structure in adult amblyopes.

Purpose To evaluate visual function and its relationship to structure in adult amblyopic subjects. Methods This observational, cross-sectional study included 24 adult amblyopes and 19 healthy subjects. The amblyopes were separated into three groups: anisometropic amblyopes (n = 15), strabismic amblyopes (n = 5), and strabismic amblyopes with anisometropia (n = 4). The relationships of the multifocal visual evoked potential (VEP) results with the clinical findings and psychophysical (Humphrey visual field) and structural (spectral domain optical coherence tomography) diagnostic test data were then investigated. Results Significant differences in the multifocal VEP amplitude responses (abnormal cluster defects), combining the interocular and monocular probability analysis, were observed between the anisometropic amblyopic (80%) and nonamblyopic eyes (13.3%) (p < 0.001), whereas in strabismic amblyopia, such defects were found in 100% of the amblyopic and nonamblyopic eyes. Delayed multifocal VEP interocular and monocular latencies were seen in 66.6 and 26.6% of the anisometropic amblyopic and nonamblyopic eyes, with no significant differences between eyes (p = 0.065). Likewise, latency delays were found in 40% of both strabismic amblyopic and nonamblyopic eyes. Multifocal VEP latency showed significant differences between anisometropic and strabismic amblyopic eyes (p = 0.036). Significant agreement was found between the Humphrey visual field and the multifocal VEP visual field defects in the central area of the visual field (p = 0.033). The average retinal nerve fiber layer thickness, foveal and macular thickness, and macular volume, as measured by spectral domain optical coherence tomography, did not show any significant differences between the amblyopic and nonamblyopic eyes and the control group. Conclusions Multifocal VEP amplitudes and latencies were significantly affected in amblyopic eyes and, to a lesser extent, in nonamblyopic eyes. Multifocal VEP response latencies were more delayed in anisometropic eyes than in strabismic eyes, suggesting that anisometropic and strabismic amblyopia may represent different neural abnormalities.

Treating amblyopia in adults with prosthetic occluding contact lenses

To investigate the feasibility, effectiveness and acceptability of using prosthetic occluding contact lenses (OCLs) to treat moderate amblyopia in adults and of the role of the multifocal visual evoked potential (mfVEP) as a predictor of postamblyopic therapy.