Potential radiation dose reduction in clinical photon-counting CT by the small pixel effect: ultra-high resolution (UHR) acquisitions reconstructed to standard resolution.

OBJECTIVE: To assess the potential dose reduction achievable with clinical photon-counting CT (PCCT) in ultra-high resolution (UHR) mode compared to acquisitions using the standard resolution detector mode (Std). MATERIALS AND METHODS: With smaller detector pixels, PCCT achieves far higher spatial resolution than energy-integrating (EI) CT systems. The reconstruction of UHR acquisitions to the lower spatial resolution of conventional systems results in an image noise and radiation dose reduction. We quantify this small pixel effect in measurements of semi-anthropomorphic abdominal phantoms of different sizes as well as in a porcine knuckle in the first clinical PCCT system by using the UHR mode (0.2 mm pixel size at isocenter) in comparison to the standard resolution mode (0.4 mm). At different slice thicknesses (0.4 up to 4 mm) and dose levels between 4 and 12 mGy, reconstructions using filtered backprojection were performed to the same target spatial resolution, i.e., same modulation transfer function, using both detector modes. Image noise and the resulting potential dose reduction was quantified as a figure of merit. RESULTS: Images acquired using the UHR mode yield lower noise in comparison to acquisitions using standard pixels at the same resolution and noise level. This holds for sharper convolution kernels at the spatial resolution limit of the standard mode, e.g., up to a factor 3.2 in noise reduction and a resulting potential dose reduction of up to almost 90%. CONCLUSION: Using sharper convolution kernels, UHR acquisitions allow for a significant dose reduction compared to acquisitions using the standard detector mode. CLINICAL RELEVANCE: Acquisitions should always be performed using the ultra-high resolution detector mode, if possible, to benefit from the intrinsic noise and dose reduction. KEY POINTS: • Ionizing radiation used in computed tomography examinations is a concern to public health. • The ultra-high resolution of novel photon-counting systems can be invested towards a noise and dose reduction if only a spatial resolution below the resolution limit of the detector is desired. • Acquisitions should always be performed in ultra-high resolution mode, if possible, to benefit from an intrinsic dose reduction.

Are T2WI PI-RADS sub-scores of transition zone prostate lesions biased by DWI information? A multi-reader, single-center study.

PURPOSE: According to PI-RADS v2.1, T2-weighted imaging (T2WI) is the dominant sequence for transition zone (TZ) lesions. This study aimed to assess, whether diffusion-weighted imaging (DWI) information influences the assignment of T2WI scores. METHOD: Out of 283 prostate MRI examinations with correlated biopsy results, fourty-four patients were selected retrospectively: first, 22 patients with a TZ lesion with T2WI and DWI scores ≥ 4, to represent lesions with unequivocal suspicion on T2WI and DWI. Second, 22 additional patients with TZ lesions of similar T2WI appearance but with corresponding DWI score ≤ 3 were added as control. Four residents and one board-certified radiologist each performed two assessments of the included patients: First, only T2WI was available (T2-only read); second, both T2WI and DWI sequences were available (biparametric read). Lesion scores were assessed using Wilcoxon signed-rank test, inter-reader agreement using weighted kappa and Kendall's W statistics, and sensitivity/specificity using McNemar test. RESULTS: The T2WI scores were significantly different between the T2-only and biparametric read for 3 out of 4 residents (p ≤ 0.049) but not for the radiologist. The overall PI-RADS scores derived from the two reading sessions differed considerably for 35/220 cases (all readers pooled). Inter-reader agreement was fair for the T2WI and overall PI-RADS scores (mean kappa 0.27-0.30) and moderate for the DWI scores (mean kappa 0.43). CONCLUSIONS: For inexperienced readers, assessment of T2WI is variable and potentially biased by availability of DWI information, which can lead to changes of overall PI-RADS score and consequently clinical management. Assessment of T2WI should be performed before reviewing DWI to ensure non-biased interpretation of TZ lesions in the dominant sequence.

Test-retest, inter- and intra-rater reproducibility of size measurements of focal bone marrow lesions in MRI in patients with multiple myeloma.

OBJECTIVE: To investigate the reproducibility of size measurements of focal bone marrow lesions (FL) in MRI in patients with monoclonal plasma cell disorders under variation of patient positioning and observer. METHODS: A data set from a prospective test-retest study was used, in which 37 patients with a total of 140 FL had undergone 2 MRI scans with identical parameters after patient repositioning. Two readers measured long and short axis diameter on the initial scan in T1 weighted, T2 weighted short tau inversion recovery and diffusion-weighted imaging sequences. The first reader additionally measured FL on the retest-scan. The Bland-Altman method was used to assess limits of agreement (LoA), and the frequencies of absolute size changes were calculated. RESULTS: In the simple test-retest experiment with one identical reader, a deviation of ≥1 mm / ≥2 mm / ≥3 mm for the long axis diameter in T1 weighted images was observed in 66% / 25% / 8% of cases. When comparing measurements of one reader on the first scan to the measurement of the other reader on the retest scan, a change of ≥1 mm / ≥3 mm / ≥5 mm for the long axis diameter in T1 weighted images was observed in 78% / 21% / 5% of cases. CONCLUSION: Small deviations in FL size are common and probably due to variation in patient positioning or inter-rater variability alone, without any actual biological change of the FL. Knowledge of the uncertainty associated with size measurements of FLs is critical for radiologists and oncologists when interpreting changes in FL size in clinical practice and in clinical trials. ADVANCES IN KNOWLEDGE: According to the MY-RADs criteria, size measurements of focal lesions in MRI are now of relevance for response assessment in patients with monoclonal plasma cell disorders.Size changes of 1 or 2 mm are frequently observed due to uncertainty of the measurement only, while the actual focal lesion has not undergone any biological change.Size changes of at least 6 mm or more in T1 weighted or T2 weighted short tau inversion recovery sequences occur in only 5% or less of cases when the focal lesion has not undergone any biological change.

Deep Learning for Automatic Bone Marrow Apparent Diffusion Coefficient Measurements From Whole-Body Magnetic Resonance Imaging in Patients With Multiple Myeloma: A Retrospective Multicenter Study.

OBJECTIVES: Diffusion-weighted magnetic resonance imaging (MRI) is increasingly important in patients with multiple myeloma (MM). The objective of this study was to train and test an algorithm for automatic pelvic bone marrow analysis from whole-body apparent diffusion coefficient (ADC) maps in patients with MM, which automatically segments pelvic bones and subsequently extracts objective, representative ADC measurements from each bone. MATERIALS AND METHODS: In this retrospective multicentric study, 180 MRIs from 54 patients were annotated (semi)manually and used to train an nnU-Net for automatic, individual segmentation of the right hip bone, the left hip bone, and the sacral bone. The quality of the automatic segmentation was evaluated on 15 manually segmented whole-body MRIs from 3 centers using the dice score. In 3 independent test sets from 3 centers, which comprised a total of 312 whole-body MRIs, agreement between automatically extracted mean ADC values from the nnU-Net segmentation and manual ADC measurements from 2 independent radiologists was evaluated. Bland-Altman plots were constructed, and absolute bias, relative bias to mean, limits of agreement, and coefficients of variation were calculated. In 56 patients with newly diagnosed MM who had undergone bone marrow biopsy, ADC measurements were correlated with biopsy results using Spearman correlation. RESULTS: The ADC-nnU-Net achieved automatic segmentations with mean dice scores of 0.92, 0.93, and 0.85 for the right pelvis, the left pelvis, and the sacral bone, whereas the interrater experiment gave mean dice scores of 0.86, 0.86, and 0.77, respectively. The agreement between radiologists' manual ADC measurements and automatic ADC measurements was as follows: the bias between the first reader and the automatic approach was 49 × 10 -6 mm 2 /s, 7 × 10 -6 mm 2 /s, and -58 × 10 -6 mm 2 /s, and the bias between the second reader and the automatic approach was 12 × 10 -6 mm 2 /s, 2 × 10 -6 mm 2 /s, and -66 × 10 -6 mm 2 /s for the right pelvis, the left pelvis, and the sacral bone, respectively. The bias between reader 1 and reader 2 was 40 × 10 -6 mm 2 /s, 8 × 10 -6 mm 2 /s, and 7 × 10 -6 mm 2 /s, and the mean absolute difference between manual readers was 84 × 10 -6 mm 2 /s, 65 × 10 -6 mm 2 /s, and 75 × 10 -6 mm 2 /s. Automatically extracted ADC values significantly correlated with bone marrow plasma cell infiltration ( R = 0.36, P = 0.007). CONCLUSIONS: In this study, a nnU-Net was trained that can automatically segment pelvic bone marrow from whole-body ADC maps in multicentric data sets with a quality comparable to manual segmentations. This approach allows automatic, objective bone marrow ADC measurements, which agree well with manual ADC measurements and can help to overcome interrater variability or nonrepresentative measurements. Automatically extracted ADC values significantly correlate with bone marrow plasma cell infiltration and might be of value for automatic staging, risk stratification, or therapy response assessment.

Ultrahigh resolution whole body photon counting computed tomography as a novel versatile tool for translational research from mouse to man.

X-ray computed tomography (CT) is a cardinal tool in clinical practice. It provides cross-sectional images within seconds. The recent introduction of clinical photon-counting CT allowed for an increase in spatial resolution by more than a factor of two resulting in a pixel size in the center of rotation of about 150 µm. This level of spatial resolution is in the order of dedicated preclinical micro-CT systems. However so far, the need for different dedicated clinical and preclinical systems often hinders the rapid translation of early research results to applications in men. This drawback might be overcome by ultra-high resolution (UHR) clinical photon-counting CT unifying preclinical and clinical research capabilities in a single machine. Herein, the prototype of a clinical UHR PCD CT (SOMATOM CounT, Siemens Healthineers, Forchheim, Germany) was used. The system comprises a conventional energy-integrating detector (EID) and a novel photon-counting detector (PCD). While the EID provides a pixel size of 0.6 mm in the centre of rotation, the PCD provides a pixel size of 0.25 mm. Additionally, it provides a quantification of photon energies by sorting them into up to four distinct energy bins. This acquisition of multi-energy data allows for a multitude of applications, e.g. pseudo-monochromatic imaging. In particular, we examine the relation between spatial resolution, image noise and administered radiation dose for a multitude of use-cases. These cases include ultra-high resolution and multi-energy acquisitions of mice administered with a prototype bismuth-based contrast agent (nanoPET Pharma, Berlin, Germany) as well as larger animals and actual patients. The clinical EID provides a spatial resolution of about 9 lp/cm (modulation transfer function at 10%, MTF10%) while UHR allows for the acquisition of images with up to 16 lp/cm allowing for the visualization of all relevant anatomical structures in preclinical and clinical specimen. The spectral capabilities of the system enable a variety of applications previously not available in preclinical research such as pseudo-monochromatic images. Clinical ultra-high resolution photon-counting CT has the potential to unify preclinical and clinical research on a single system enabling versatile imaging of specimens and individuals ranging from mice to man.

Combining Deep Learning and Radiomics for Automated, Objective, Comprehensive Bone Marrow Characterization From Whole-Body MRI: A Multicentric Feasibility Study.

OBJECTIVES: Disseminated bone marrow (BM) involvement is frequent in multiple myeloma (MM). Whole-body magnetic resonance imaging (wb-MRI) enables to evaluate the whole BM. Reading of such whole-body scans is time-consuming, and yet radiologists can transfer only a small fraction of the information of the imaging data set to the report. This limits the influence that imaging can have on clinical decision-making and in research toward precision oncology. The objective of this feasibility study was to implement a concept for automatic, comprehensive characterization of the BM from wb-MRI, by automatic BM segmentation and subsequent radiomics analysis of 30 different BM spaces (BMS). MATERIALS AND METHODS: This retrospective multicentric pilot study used a total of 106 wb-MRI from 102 patients with (smoldering) MM from 8 centers. Fifty wb-MRI from center 1 were used for training of segmentation algorithms (nnU-Nets) and radiomics algorithms. Fifty-six wb-MRI from 8 centers, acquired with a variety of different MRI scanners and protocols, were used for independent testing. Manual segmentations of 2700 BMS from 90 wb-MRI were performed for training and testing of the segmentation algorithms. For each BMS, 296 radiomics features were calculated individually. Dice score was used to assess similarity between automatic segmentations and manual reference segmentations. RESULTS: The "multilabel nnU-Net" segmentation algorithm, which performs segmentation of 30 BMS and labels them individually, reached mean dice scores of 0.88 ± 0.06/0.87 ± 0.06/0.83 ± 0.11 in independent test sets from center 1/center 2/center 3-8 (interrater variability between radiologists, 0.88 ± 0.01). The subset from the multicenter, multivendor test set (center 3-8) that was of high imaging quality was segmented with high precision (mean dice score, 0.87), comparable to the internal test data from center 1. The radiomic BM phenotype consisting of 8880 descriptive parameters per patient, which result from calculation of 296 radiomics features for each of the 30 BMS, was calculated for all patients. Exemplary cases demonstrated connections between typical BM patterns in MM and radiomic signatures of the respective BMS. In plausibility tests, predicted size and weight based on radiomics models of the radiomic BM phenotype significantly correlated with patients' actual size and weight ( P = 0.002 and P = 0.003, respectively). CONCLUSIONS: This pilot study demonstrates the feasibility of automatic, objective, comprehensive BM characterization from wb-MRI in multicentric data sets. This concept allows the extraction of high-dimensional phenotypes to capture the complexity of disseminated BM disorders from imaging. Further studies need to assess the clinical potential of this method for automatic staging, therapy response assessment, or prediction of biopsy results.

Pseudoprospective Paraclinical Interaction of Radiology Residents With a Deep Learning System for Prostate Cancer Detection: Experience, Performance, and Identification of the Need for Intermittent Recalibration.

OBJECTIVES: The aim of this study was to estimate the prospective utility of a previously retrospectively validated convolutional neural network (CNN) for prostate cancer (PC) detection on prostate magnetic resonance imaging (MRI). MATERIALS AND METHODS: The biparametric (T2-weighted and diffusion-weighted) portion of clinical multiparametric prostate MRI from consecutive men included between November 2019 and September 2020 was fully automatically and individually analyzed by a CNN briefly after image acquisition (pseudoprospective design). Radiology residents performed 2 research Prostate Imaging Reporting and Data System (PI-RADS) assessments of the multiparametric dataset independent from clinical reporting (paraclinical design) before and after review of the CNN results and completed a survey. Presence of clinically significant PC was determined by the presence of an International Society of Urological Pathology grade 2 or higher PC on combined targeted and extended systematic transperineal MRI/transrectal ultrasound fusion biopsy. Sensitivities and specificities on a patient and prostate sextant basis were compared using the McNemar test and compared with the receiver operating characteristic (ROC) curve of CNN. Survey results were summarized as absolute counts and percentages. RESULTS: A total of 201 men were included. The CNN achieved an ROC area under the curve of 0.77 on a patient basis. Using PI-RADS ≥3-emulating probability threshold (c3), CNN had a patient-based sensitivity of 81.8% and specificity of 54.8%, not statistically different from the current clinical routine PI-RADS ≥4 assessment at 90.9% and 54.8%, respectively ( P = 0.30/ P = 1.0). In general, residents achieved similar sensitivity and specificity before and after CNN review. On a prostate sextant basis, clinical assessment possessed the highest ROC area under the curve of 0.82, higher than CNN (AUC = 0.76, P = 0.21) and significantly higher than resident performance before and after CNN review (AUC = 0.76 / 0.76, P ≤ 0.03). The resident survey indicated CNN to be helpful and clinically useful. CONCLUSIONS: Pseudoprospective paraclinical integration of fully automated CNN-based detection of suspicious lesions on prostate multiparametric MRI was demonstrated and showed good acceptance among residents, whereas no significant improvement in resident performance was found. General CNN performance was preserved despite an observed shift in CNN calibration, identifying the requirement for continuous quality control and recalibration.

Repeatability and Reproducibility of ADC Measurements and MRI Signal Intensity Measurements of Bone Marrow in Monoclonal Plasma Cell Disorders: A Prospective Bi-institutional Multiscanner, Multiprotocol Study.

BACKGROUND/OBJECTIVES: Apparent diffusion coefficient (ADC) and signal intensity (SI) measurements play an increasing role in magnetic resonance imaging (MRI) of monoclonal plasma cell disorders. The purpose of this study was to assess interrater variability, repeatability, and reproducibility of ADC and SI measurements from bone marrow (BM) under variation of MRI protocols and scanners. PATIENTS AND METHODS: Fifty-five patients with suspected or confirmed monoclonal plasma cell disorder were prospectively included in this institutional review board-approved study and underwent several measurements after the standard clinical whole-body MR scan, including repeated scan after repositioning, scan with a second MRI protocol, scan at a second 1.5 T scanner with a harmonized MRI protocol, and scan at a 3 T scanner. For T1-weighted, T2-weighted STIR, B800 images, and ADC maps, regions of interest were placed in the BM of the iliac crest and sacral bone, and in muscle tissue for image normalization. Bland-Altman plots were constructed, and absolute bias, relative bias to mean, limits of agreement, and coefficients of variation were calculated. RESULTS: Interrater variability and repeatability experiments showed a maximal relative bias of -0.077 and a maximal coefficient of variation of 16.2% for all sequences. Although the deviations at the second 1.5 T scanner with harmonized MRI protocol to the first 1.5 T scanner showed a maximal relative bias of 0.124 for all sequences, the variation of the MRI protocol and scan at the 3 T scanner led to large relative biases of up to -0.357 and -0.526, respectively. When comparing the 3 T scanner to the 1.5 T scanner, normalization to muscle reduced the bias of T1-weighted and T2-weighted sequences, but not of ADC maps. CONCLUSIONS: The MRI scanners with identical field strength and harmonized MRI protocols can provide relatively stable quantitative measurements of BM ADC and SI. Deviations in MRI field strength and MRI protocol should be avoided when applying ADC cutoff values, which were established at other scanners or when performing multicentric imaging trials.

Histamine causes endothelial barrier disruption via Ca2+-mediated RhoA activation and tension at adherens junctions.

During inflammation, the disruption of the endothelial barrier leads to increased microvascular permeability. Whether tension along cell junctions contributes to histamine-induced endothelial barrier disruption remains unknown. Rapid Ca2+ influx induced by both histamine and thrombin was accompanied by endothelial barrier breakdown revealed as drop of transendothelial electric resistance in primary human microvascular endothelial cells. Interestingly, GLISA measurements revealed activation of RhoA but not inactivation of Rac1 at the time-point of barrier breakdown. FRET measurements showed activation of RhoA at intercellular junctions after both thrombin and histamine exposure. Breakdown coincided with increased stress fiber formation but not with translocation of vinculin, which was located along junctions in the resting state similar to postcapillary venules ex vivo. Moreover, increased tension at AJs was indicated by immunostaining with a conformation-sensitive antibody targeting the α18-subunit of α-catenin. Ca2+ chelation by BAPTA-AM and ROCK1 inhibition by Y27632 abolished both increase of tension along AJs as well as barrier dysfunction. Moreover, BAPTA-AM decreased RhoA activation following histamine stimulation, indicating a key role of Ca2+ signaling in barrier breakdown. Taken together, in response to histamine, Ca2+ via RhoA/ROCK activation along endothelial adherens junctions (AJs) appears to be critical for barrier disruption and presumably correlated with enhanced tension. However, vinculin appears not to be critical in this process.

Compound heterozygote mutations in SPG7 in a family with adult-onset primary lateral sclerosis.

OBJECTIVE: To identify the genetic defect for adult-onset primary lateral sclerosis (PLS) in a family with 5 patients. METHODS: Whole-exome sequencing was performed to identify the shared genetic variants in 3 affected members in a PLS family with 5 affected individuals. Sanger sequencing was used for validation of the variants and for cosegregation analysis. Mitochondrial activity for both patients and unaffected siblings was measured using a SeaHorse metabolic analyzer. RESULTS: Whole-exome sequencing and subsequent cosegregation analysis demonstrated that compound heterozygous missense variants L695P and I743T in SPG7 were the only mutations cosegregating with the disease in an autosomal recessive fashion in this family. The parents and siblings are genetically heterozygous and clinically unaffected. Functional studies suggested that the PLS-associated SPG7 mutants affect mitochondrial function when glucose is reduced. CONCLUSIONS: Compound heterozygote mutations in SPG7 are associated with adult-onset PLS, extending the spectrum of SPG7-linked neurologic diseases. Patients with the PLS phenotype should have genetic testing for paraplegin, especially when the condition is familial.

ACTH (Acthar Gel) Reduces Toxic SOD1 Protein Linked to Amyotrophic Lateral Sclerosis in Transgenic Mice: A Novel Observation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a complex etiology and pathology that makes the development of new therapies difficult. ACTH has neurotrophic and myotrophic effects, but has not been tested in an ALS mouse model. The G93A-SOD1 mouse model of ALS was used to test the ability of this drug to delay ALS-like symptoms. We showed that within a specific dose range, ACTH significantly postponed the disease onset and paralysis in the mouse model. To our surprise and of greater significance is that ACTH significantly reduced the levels of soluble SOD1 in the spinal cord and CNS tissues of G93A-SOD1 treated mice as well as cultured fibroblasts.

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation. GLIA 2015;63:736-753.

Pharmacodynamics of potassium channel openers in cultured neuronal networks.

A novel class of drugs - potassium (K(+)) channel openers or activators - has recently been shown to cause anticonvulsive and neuroprotective effects by activating hyperpolarizing K(+) currents, and therefore, may show efficacy for treating tinnitus. This study presents measurements of the modulatory effects of four K(+) channel openers on the spontaneous activity and action potential waveforms of neuronal networks. The networks were derived from mouse embryonic auditory cortices and grown on microelectrode arrays. Pentylenetetrazol was used to create hyperactivity states in the neuronal networks as a first approximation for mimicking tinnitus or tinnitus-like activity. We then compared the pharmacodynamics of the four channel activators, retigabine and flupirtine (voltage-gated K(+) channel KV7 activators), NS1619 and isopimaric acid ("big potassium" BK channel activators). The EC50 of retigabine, flupirtine, NS1619, and isopimaric acid were 8.0, 4.0, 5.8, and 7.8µM, respectively. The reduction of hyperactivity compared to the reference activity was significant. The present results highlight the notion of re-purposing the K(+) channel activators for reducing hyperactivity of spontaneously active auditory networks, serving as a platform for these drugs to show efficacy toward target identification, prevention, as well as treatment of tinnitus.

Discovery of 1,3,4-oxidiazole scaffold compounds as inhibitors of superoxide dismutase expression.

The treatment of neurodegenerative diseases is difficult because of multiple etiologies and the interplay of genetics and environment as precipitating factors. In the case of amyotrophic lateral sclerosis (ALS), we have knowledge of a handful of genes that cause disease when mutated. However, drugs to counteract the effect of genetic mutations have not yet been found. One of the causative genes, Cu, Zn-superoxide dismutase (SOD1) is responsible for about 10-15% of the genetically linked autosomal dominant disease. Our rationale was that compounds that reduce expression of the mutant protein would be beneficial to slow onset and/or disease progression. We screened candidate compounds using a cell-based in vitro assay for those that reduce mutant SOD1 (G93A) protein expression. This led to the discovery of 2-[3-iodophenyl)methylsulfanyl]-5pyridin-4-yl-1,3,4-oxadiazole, a known protein kinase inhibitor that decreases G93A-SOD1 expression in vitro and in the brain and spinal cord in vivo. However, this compound has a biphasic dose response curve and a likely toxophore which limit its therapeutic window for chronic disease such as ALS. Therefore, we designed and tested a focused library of analogs for their ability to decrease SOD1 expression in vitro. This exercise resulted in the identification of a lead compound with improved drug-like characteristics and activity. Development of small molecules that reduce the expression of etiologically relevant toxic proteins is a strategy that may also be extended to familial ALS linked to gain of function mutations in other genes.

Kcnq1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish.

BACKGROUND: KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ1), epilepsy in adults (KCNQ2/3), benign familial neonatal convulsions in children (KCNQ3), and hearing loss or tinnitus in humans (KCNQ4, but not KCNQ5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues. RESULTS: Overall expression of the kcnqx channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues. CONCLUSIONS: We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.

Tumor-suppressive maspin functions as a reactive oxygen species scavenger: importance of cysteine residues.

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth.

Isolation and culture of astrocytes from the retina and optic nerve.

Detailed methods for the preparation of optic nerve head (ONH) astrocytes from human donor eyes and retinal astrocytes from rat eyes are described. Included is the immunopanning method used for ONH astrocyte isolation as well as cell characterization. The isolation of purified retinal astrocytes is outlined as a method applicable to rodent and other mammalian retinas.

An in vitro model for testing drugs to treat tinnitus.

Tinnitus affects approximately 50 million people in the USA alone, with 10 million being highly debilitated. Pharmacotherapy for tinnitus is still in emerging stages due to time consuming clinical trials and/or animal experiments. We tested a new cellular model where induced rapid neuronal firing or spiking was used as a mimic for the type of aberrant activity that may occur in tinnitus. Spontaneously active auditory cortical networks growing on microelectrode arrays were exposed to pentylenetetrazol (PTZ), a proconvulsant and an antagonist of GABA(A) receptor, which is implicated in tinnitus. Auditory cortical networks were then exposed to experimental tinnitus drugs linopirdine (Dup966, a potassium channel blocker), L-carnitine (an antioxidant), or selective Ca(2+) channel antagonists pregabalin (Lyrica), or gabapentin (Neurontin) at various concentrations. PTZ increased spike rate by 139.6±27% and burst rate by 129.7±28% in auditory cortical networks with a phenotypic high firing of excitable neurons. Reductions of increased activity were observed to varying degrees using the experimental tinnitus drugs. The potency of the drugs was linopirdine (EC(50): 176±7.0 µM)>L-carnitine (EC(50): 1569±41 µM)>pregabalin (EC(50): 8360±340 µM), >gabapentin, with 34.2±7.5% efficacy (EC(50): 2092±980 µM). These studies provide proof of principle for the use of auditory cortical networks on microelectrode array as a feasible platform for semi-high throughput application for screening of drugs that might be used for the treatment of tinnitus.

Modulation of factors affecting optic nerve head astrocyte migration.

PURPOSE: The authors investigated the role of myosin light chain kinase (MYLK) and transforming growth factor beta (TGFbeta) receptor pathways in optic nerve head (ONH) astrocyte migration. They further investigated how the expression of these genes is altered by elevated hydrostatic pressure (HP). METHODS: PCR was used to determine the isoforms of MYLK expressed in ONH astrocytes. siRNAs against MYLK (all isoforms) and TGFbeta receptor 2 (TGFBR2) were prepared and tested for effects on the migration of cultured ONH astrocytes. Finally, the effects of elevated HP (24-96 hours) on the expression of MYLK isoforms and selected TGFbeta pathway components were measured. RESULTS: Multiple isoforms of MYLK are present in ONH astrocytes from Caucasian (CA) and African American (AA) donors. Both populations express the short form (MYLK-130) and the long form (MYLK-210) of MYLK and a splicing variant within MYLK-210. MYLK-directed siRNA decreased MYLK expression and cell migration compared with control siRNA. siRNA directed against TGFbeta receptor 2 also decreased cell migration compared with control and decreased extracellular matrix genes regulated by TGFbeta signaling. Elevated HP increased the expression of MYLK-130 and MYLK-210 in both populations of astrocytes. However, TGFbeta2 was uniquely upregulated by exposure to elevated HP in CA compared with AA astrocytes. CONCLUSIONS: Differential expression of TGFbeta pathway genes and MYLK isoforms observed in populations of glaucomatous astrocytes applies to the elevated HP model system. MYLK may be a new target for intervention in glaucoma to alter reactive astrocyte migration in the ONH.

Age-related increase in mitochondrial DNA damage and loss of DNA repair capacity in the neural retina.

With age, there is increased mitochondrial DNA (mtDNA) damage in the central nervous system (CNS) that may underlie, contribute or increase the susceptibility to certain neurodegenerative diseases. We examined retinas from the eyes of young and old rodents for mtDNA damage and for changes in selected DNA repair enzymes. We found increased levels of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) by immunohistochemical labeling for the oxidative DNA damage marker in aged rodent retinas, which was confirmed by quantitative ELISA. 8-OHdG co-localized with the mitochondrial enzyme superoxide dismutase (MnSOD), suggesting damage to mtDNA. Most of the damaged mtDNA was in the photoreceptors and retinal ganglion cells. Measurements of nuclear DNA (nDNA) and mtDNA lesions indicated that DNA damage was primarily in mtDNA in aged retinas. The increased damage to mtDNA may be due to decreased levels of DNA repair enzymes in the aged retina. Using qPCR, Western blots and immunohistochemistry, we determined the levels of DNA repair enzymes for oxidative damage. In retinas from old eyes compared to retinas from young eyes, we found decreased levels of poly (ADP-ribose) polymerase 1 (PARP1), mutY homolog (MYH) and endonuclease III homologue 1 (NTH1). Our results suggest that normal, age-related, increased mtDNA damage, likely due to decreased repair capacity in aged retinas, may be a susceptibility factor that underlies age-related retinal diseases.