Caroline L. Speck

Subnormal Cytokine Profile in the Tear Fluid of Keratoconus Patients

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Response of the medial temporal lobe network in amnestic mild cognitive impairment to therapeutic intervention assessed by fMRI and memory task performance

Studies of individuals with amnestic mild cognitive impairment (aMCI) have detected hyperactivity in the hippocampus during task-related functional magnetic resonance imaging (fMRI). Such elevated activation has been localized to the hippocampal dentate gyrus/CA3 (DG/CA3) during performance of a task designed to detect the computational contributions of those hippocampal circuits to episodic memory. The current investigation was conducted to test the hypothesis that greater hippocampal activation in aMCI represents a dysfunctional shift in the normal computational balance of the DG/CA3 regions, augmenting CA3-driven pattern completion at the expense of pattern separation mediated by the dentate gyrus. We tested this hypothesis using an intervention based on animal research demonstrating a beneficial effect on cognition by reducing excess hippocampal neural activity with low doses of the atypical anti-epileptic levetiracetam. In a within-subject design we assessed the effects of levetiracetam in three cohorts of aMCI participants, each receiving a different dose of levetiracetam. Elevated activation in the DG/CA3 region, together with impaired task performance, was detected in each aMCI cohort relative to an aged control group. We observed significant improvement in memory task performance under drug treatment relative to placebo in the aMCI cohorts at the 62.5 and 125 mg BID doses of levetiracetam. Drug treatment in those cohorts increased accuracy dependent on pattern separation processes and reduced errors attributable to an over-riding effect of pattern completion while normalizing fMRI activation in the DG/CA3 and entorhinal cortex. Similar to findings in animal studies, higher dosing at 250 mg BID had no significant benefit on either task performance or fMRI activation. Consistent with predictions based on the computational functions of the DG/CA3 elucidated in basic animal research, these data support a dysfunctional encoding mechanism detected by fMRI in individuals with aMCI and therapeutic intervention using fMRI to detect target engagement in response to treatment.

Unfolded protein response in fuchs endothelial corneal dystrophy: a unifying pathogenic pathway?

PURPOSE To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients. DESIGN Retrospective, comparative case series of laboratory specimens. METHODS Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologic features other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as a sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy corneas, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for 3 unfolded protein response markers (GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein) and 2 apoptosis markers (caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression. RESULTS TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein, and caspase 9 compared with non-Fuchs dystrophy corneas (P < .05). CONCLUSIONS Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

A comprehensive analysis of eye bank-prepared posterior lamellar corneal tissue for use in endothelial keratoplasty.

Purpose: The purpose of this study was to assess eye bank-prepared corneal tissue with regards to the accuracy of postcut tissue thickness, endothelial cell loss, and rate of successful processing. Methods: Details of all 913 corneal tissues processed with an automated microkeratome for use in posterior lamellar transplantation, over a 1-year period, were obtained from a large eye bank. The number and success rate of all attempted cutting procedures were analyzed. The thickness of the corneal button obtained after cutting was compared with the graft thickness requested by the operating surgeon. Changes in endothelial cell density (ECD) during tissue processing were evaluated. Results: The rate of successful tissue preparation increased over the time period examined, from 95% in the first quarter to 99.5% in the fourth quarter. Graft material was frequently slightly thicker than requested by the operating surgeon with 28.3% of tissues cut thicker than requested. Postcut ECD over the entire period increased by an average of 4.7% and was closely related to the starting ECD. Conclusions: There was a very high rate of successful tissue preparation (98.5%), and early failed attempts at tissue cutting were likely the result of the initial learning curve of the involved technicians. Practical considerations resulted in tissue being cut marginally thicker than requested; this is an issue about which the operating surgeon should be aware, because it may possibly influence tissue handling. The quality of the obtained material, as measured by ECD, was excellent, although the calculated ECD may be prone to measurement artifact.

L450W and Q455K Col8a2 knock-in mouse models of fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy

PURPOSE We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues. METHODS In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using total RNA, and protein extracted from mouse CECs and human CECs obtained from FECD and autopsy patients. RESULTS Both Col8a2 mouse models exhibited hallmarks of FECD; however, the Col8a2(L450W/L450W) mice exhibited a milder phenotype compared to the Col8a2(Q455K/Q455K) mice. Both models exhibited upregulation of the unfolded protein response (UPR) as evidenced by dilated rough endoplasmic reticulum (RER), and upregulation of UPR-associated genes and proteins. Real-time PCR of Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) CECs at 40 weeks revealed a 2.1-fold (P < 0.05) and a 5.2-fold (P < 0.01) upregulation of the autophagy marker Dram1, respectively. Real-time PCR of human FECD endothelium revealed a 10.4-fold upregulation of DRAM1 (P < 0.0001) compared to autopsy controls. CONCLUSIONS The Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) mouse models of FECD showed distinct endothelial cell phenotypes. Dram1 was associated with activation of the UPR and increased autophagy. Overexpression of this gene in mouse and human FECD endothelial cells suggested a role for altered autophagy in this disease.

Assessment of attachment factors for primary cultured human corneal endothelial cells.

Purpose: To assess the ability of various attachment factors to promote attachment of primary cultured human corneal endothelial cells. Materials and Methods: Primary cultured human corneal endothelial cells (HCEC) were incubated for 2 hours in 24-well plates. Wells had been precoated with commercially available cell attachment improvement media (FNC coating mix), human collagen I, human fibronectin, fibronectin/collagen I, or poly-d-lysine. Ratios of cell count before and after rinsing with culture medium and ratios of cells showing morphological signs of spreading to total cells were calculated to measure effectiveness of attachment factors. Results: Incubation of HCEC for 2 hours in wells without precoating of attachment factors led to a rate of 41 ± 16% (mean ± SD) of cells showing signs of spreading. FNC coating mix, collagen I, and fibronectin/collagen I increased significantly the percentage of cells showing morphological features of attachment at 2 hours. Total cell loss was highest with poly-d-lysine and no pretreatment with attachment factor. Without the use of any attachment factor, 67 ± 19% of cells remained after rinsing. The lowest cell loss was observed with FNC coating mix where 108 ± 5% of cells remained after rinsing. Conclusion: Collagen I, collagen I/fibronectin, and FNC coating mix significantly enhance the spreading of human corneal endothelial cells to tissue culture plates after 2 hours. FNC coating mix significantly reduces cell loss due to rinsing. Without the use of any attachment factor, 67% of the cells remained in situ after rinsing.

Increased hippocampal activation in ApoE-4 carriers and non-carriers with amnestic mild cognitive impairment

Increased fMRI activation in the hippocampus is recognized as a signature characteristic of the amnestic mild cognitive impairment (aMCI) stage of Alzheimers disease (AD). Previous work has localized this increased activation to the dentate gyrus/CA3 subregion of the hippocampus and showed a correlation with memory impairments in those patients. Increased hippocampal activation has also been reported in carriers of the ApoE-4 allelic variation independently of mild cognitive impairment although these findings were not localized to a hippocampal subregion. To assess the ApoE-4 contribution to increased hippocampal fMRI activation, patients with aMCI genotyped for ApoE-4 status and healthy age-matched control participants completed a high-resolution fMRI scan while performing a memory task designed to tax hippocampal subregion specific functions. Consistent with previous reports, patients with aMCI showed increased hippocampal activation in the left dentate gyrus/CA3 region of the hippocampus as well as memory task errors attributable to this subregion. However, this increased fMRI activation in the hippocampus did not differ between ApoE-4 carriers and ApoE-4 non-carriers and the proportion of memory errors attributable to dentate gyrus/CA3 function did not differ between ApoE-4 carriers and ApoE-4 non-carriers. These results indicate that increased fMRI activation of the hippocampus observed in patients with aMCI is independent of ApoE-4 status and that ApoE-4 does not contribute to the dysfunctional hippocampal activation or the memory errors attributable to this subregion in these patients.

The distribution of the alpha7 nicotinic acetylcholine receptor in healthy aging: An in vivo positron emission tomography study with [18F]ASEM

ABSTRACT Altered function of the alpha7 nicotinic acetylcholine receptor (&agr;7‐nAChR) is implicated in several neuropsychiatric diseases. Nevertheless, studies of the human cerebral &agr;7‐nAChR even in healthy aging are limited in number and to postmortem tissue. Methods: The distribution of the cerebral &agr;7‐nAChR was estimated in nine brain regions in 25 healthy volunteers (ages 21–86 years; median 57 years, interquartile range 52 years) using [18F]ASEM with positron emission tomography (PET) imaging. Regional total distribution volume (VT) measurements were calculated using the Logan method from each subjects 90 min dynamic PET data and their metabolite‐corrected plasma input function. Spearmans rank or Pearsons correlation analysis was used depending on the normality of the data. Correlation between age and regional 1) volume relative to intracranial volume (volume ratio) and 2) [18F]ASEM VT was tested. Correlation between regional volume ratio and [18F]ASEM VT was also evaluated. Finally, the relationship between [18F]ASEM VT and neuropsychological measures was investigated in a subpopulation of 15 elderly healthy participants (those 50 years of age and older). Bonferroni correction for multiple comparisons was applied to statistical analyses. Results: A negative correlation between tissue volume ratio and age was observed in six of the nine brain regions including striatum and five cortical (temporal, occipital, cingulate, frontal, or parietal) regions. A positive correlation between [18F]ASEM VT and age was observed in all nine brain regions of interest (ROIs). There was no correlation between [18F]ASEM VT and volume ratio in any ROI after controlling for age. Regional [18F]ASEM VT and neuropsychological performance on each of eight representative subtests were not correlated among the well‐performing subpopulation of elderly healthy participants. Conclusions: Our results suggest an increase in cerebral &agr;7‐nAChR distribution over the course of healthy aging that should be tested in future longitudinal studies. The preservation of the &agr;7‐nAChR in the aging human brain supports the development of therapeutic agents that target this receptor for use in the elderly. Further study of the relationship between &agr;7‐nAChR availability and cognitive impairment over aging is needed.

LATERAL ENTORHINAL CORTEX HYPOACTIVATION IN AMNESTIC MILD COGNITIVE IMPAIRMENT

Background: Increased hippocampal activation in the context of impaired memory function is considered a characteristic feature of the amnestic mild cognitive impairment (aMCI) phase of Alzheimer’s disease. However, the entorhinal cortex, which serves as the primary relay for both input and output to and from the hippocampus, is the site of the earliest pathological changes including neuronal, synaptic and volume loss. Specifically, the lateral entorhinal cortex is also the site of significant accumulation of tau neurofibrillary tangles early in the disease progression. Methods:To assess whether functional changes can be observed in the lateral and medial entorhinal cortex in patients with aMCI, 37 healthy older adults and 42 patients with aMCI completed a forced choice memory task designed to tax medial temporal lobe memory function while completing a high-resolution functionalmagnetic resonance imaging (fMRI) scan. Results: Consistent with their diagnosis and previous studies, patients with aMCI showed a significant memory impairment compared toage-matchedhealthy control subjects aswell as increased hippocampal activation. In this context, patients with aMCI also showed significantly decreased activation in both the left and right entorhinal cortex. The observed hypoactivation was localized to the lateral entorhinal cortex and was not observed in the medial entorhinal cortex. Decreased activation in the left lateral entorhinal cortex was correlated with poorer performance on the memory task in patients with aMCI. Finally, the volume of both the left and right lateral entorhinal cortex was significantly reduced in patients with aMCI compared to healthy control subjects while no volume differences between the groups were observed in the medial entorhinal cortex. Conclusions: These results show that, consistent with the locus of early disease related pathology, decreased activation and reduced volume in specifically the lateral entorhinal cortex is observed in patients with aMCI and is associated with impaired memory function in these patients.

The role of ApoE-4 in hippocampal hyperactivity in amnestic mild cognitive impairment

Controlling for age and sex, ratios of YKL-40/Ab42 and MCP-1/ Ab42 were associated with increased isotropic diffusivity in the right parahippocampal gyrus. In a subsequent analysis, the increased diffusivity predicted by MCP-1/Ab42 was associated with significantly lower RAVLT scores of immediate memory, learning ability, and delayed memory. Neither Ab42/Ab40, nor the markers of inflammation alone predicted altered neural microstructure. Conclusions:Medial temporal lobe structures may be especially susceptible to inflammation in incipient AD. Interestingly, amyloid alone was not associated with neural injury, suggesting that, while necessary, amyloid is not sufficient for injury to occur. Higher diffusivity in the parahippocampal gyrus may indicate cellular pathology or neuronal loss, and given the association with memory function, may be an early marker of cognitive decline. The results of this study further inform the preclinical picture of AD and shed light on the early mechanisms of neural injury.