Blood‐based markers (BBMs) have recently shown promise to revolutionize the diagnostic and prognostic work‐up of Alzheimer's disease (AD), as well as to improve the design of interventional trials. ...Here we discuss in detail further research needed to be performed before widespread use of BBMs. We already now recommend use of BBMs as (pre‐)screeners to identify individuals likely to have AD pathological changes for inclusion in trials evaluating disease‐modifying therapies, provided the AD status is confirmed with positron emission tomography (PET) or cerebrospinal fluid (CSF) testing. We also encourage studying longitudinal BBM changes in ongoing as well as future interventional trials. However, BBMs should not yet be used as primary endpoints in pivotal trials. Further, we recommend to cautiously start using BBMs in specialized memory clinics as part of the diagnostic work‐up of patients with cognitive symptoms and the results should be confirmed whenever possible with CSF or PET. Additional data are needed before use of BBMs as stand‐alone diagnostic AD markers, or before considering use in primary care.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Introduction
Pre‐analytical sample handling might affect the results of Alzheimer's disease blood‐based biomarkers. We empirically tested variations of common blood collection and handling ...procedures.
Methods
We created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze–thawing. We measured amyloid beta (Aβ)42 and 40 peptides with six assays, and Aβ oligomerization‐tendency (OAβ), amyloid precursor protein (APP)699‐711, glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t‐tau), and phosphorylated tau181.
Results
Collection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aβ and t‐tau; t‐tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations.
Discussion
We constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood‐based biomarkers into the research and clinical settings.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Injury to the peripheral or central nervous system can induce changes within the nervous tissues that promote a state of sensitization that may underlie conditions of pathological chronic pain. A key ...biochemical event in the initiation and maintenance of peripheral and central neuronal sensitization associated with chronic pain is the phosphorylation and subsequent activation of mitogen-activated protein kinases (MAPKs) and immediate early gene transcription factors, in particular cAMP-response element binding protein (CREB). In this commentary we review the preclinical data that describe anatomical and mechanistic aspects of nociceptive-induced signaling along nociceptive pathways including peripheral cutaneous axons, the dorsal root ganglia, spinal cord dorsal horn and cerebral cortex. In addition to the regional manifestation of nociceptive signaling, investigations have attempted to elucidate the cellular origin of biochemical nociceptive processing in which communication, i.e. cross-talk between neurons and glia is viewed as an essential component of pathogenic pain development. Here, we outline a research strategy by which nociceptive-induced cellular signaling in experimental pain models, specifically MAPK and CREB phosphorylation can be utilized to provide mechanistic insight into drug-target interaction along the nociceptive pathways. We describe a series of studies using nociceptive inflammatory and neuropathic pain models to investigate the effects of known pain therapeutics on nociceptive-induced biochemical signaling and present this as a complementary research strategy for assessing antinociceptive activity useful in the preclinical development of novel pain therapeutics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background
Prior studies have shown that decreased meningeal pH activates dural afferents via opening of acid‐sensing ion channels (ASICs), suggesting one pathophysiological mechanism for the ...generation of headaches. The studies described here further examined the ASIC subtype mediating pH‐induced dural‐afferent activation and examined whether sensitization influences pH responses.
Objective
Given the potential importance of meningeal mast cells to headache, the goal of this study was to evaluate dural afferent responses to pH following sensitization with mast cell mediators.
Methods
Cutaneous allodynia was measured in rats following stimulation of the dura with decreased pH alone or in combination with mast cell mediators. Trigeminal ganglion neurons retrogradely labeled from the dura were stained with an ASIC3 antibody using immunohistochemistry. Current and action potentials evoked by changes in pH alone or in combination with mast cell mediators were measured in retrogradely labeled dural afferents using patch‐clamp electrophysiology.
Results
pH‐sensitive dural afferents generated currents in response to the ASIC3 activator 2‐guanidine‐4‐methylquinazoline (GMQ), approximately 80% of these neurons express ASIC3 protein, and pH‐evoked behavioral responses were inhibited by the ASIC3 blocker APETx2. Following exposure to mast cell mediators, dural afferents exhibited increased pH‐evoked excitability, and cutaneous allodynia was observed at higher pH than with pH stimuli alone.
Conclusions
These data indicate that the predominant ASIC subtype responding to decreased meningeal pH is ASIC3. Additionally, they demonstrate that in the presence of inflammation, dural afferents respond to even smaller decreases in pH providing further support for the ability of small pH changes within the meninges to initiate afferent input leading to headache.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Objective
To develop and validate a model of cutaneous allodynia triggered by dural inflammation for pain associated with headaches. To explore neural mechanisms underlying cephalic and extracephalic ...allodynia.
Methods
Inflammatory mediators (IM) were applied to the dura of unanesthetized rats via previously implanted cannulas, and sensory thresholds of the face and hind‐paws were characterized.
Results
IM elicited robust facial and hind‐paw allodynia, which peaked within 3 hours. These effects were reminiscent of cutaneous allodynia seen in patients with migraine or other primary headache conditions, and were reversed by agents used clinically in the treatment of migraine, including sumatriptan, naproxen, and a calcitonin gene–related peptide antagonist. Consistent with clinical observations, the allodynia was unaffected by a neurokinin‐1 antagonist. Having established facial and hind‐paw allodynia as a useful animal surrogate of headache‐associated allodynia, we next showed that blocking pain‐facilitating processes in the rostral ventromedial medulla (RVM) interfered with its expression. Bupivacaine, destruction of putative pain‐facilitating neurons, or block of cholecystokinin receptors prevented or significantly attenuated IM‐induced allodynia. Electrophysiological studies confirmed activation of pain‐facilitating RVM “on” cells and transient suppression of RVM “off” cells after IM.
Interpretation
Facial and hind‐paw allodynia associated with dural stimulation is a useful surrogate of pain associated with primary headache including migraine and may be exploited mechanistically for development of novel therapeutic strategies for headache pain. The data also demonstrate the requirement for activation of descending facilitation from the RVM for the expression of cranial and extracranial cutaneous allodynia, and are consistent with a brainstem generator of allodynia associated with headache disorders. Ann Neurol 2009;65:184–193
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
TRPA1 is expressed on dural afferent neurons and activation of dural TRPA1 produces behavioral responses consistent with headache.
Activation of transient receptor potential ankyrin-1 (TRPA1) on ...meningeal nerve endings has been suggested to contribute to environmental irritant–induced headache, but this channel may also contribute to other forms of headache, such as migraine. The preclinical studies described here examined functional expression of TRPA1 on dural afferents and investigated whether activation of TRPA1 contributes to headache-like behaviors. Whole-cell patch-clamp recordings were performed in vitro with 2 TRPA1 agonists, mustard oil (MO), and the environmental irritant umbellulone (UMB) on dural-projecting trigeminal ganglion neurons. Application of MO and UMB to dural afferents produced TRPA1-like currents in approximately 42% and 38% of cells, respectively. By means of an established in vivo behavioral model of migraine-related allodynia, dural application of MO and UMB produced robust time-related tactile facial and hind paw allodynia that was attenuated by pretreatment with the TRPA1 antagonist HC-030031. Additionally, MO or UMB were applied to the dura, and exploratory activity was monitored for 30min with an automated open-field activity chamber. Dural MO and UMB decreased the number of vertical rearing episodes and the time spent rearing in comparison to vehicle-treated animals. This change in activity was prevented in rats pretreated with HC-030031 as well as sumatriptan, a clinically effective antimigraine agent. These data indicate that TRPA1 is expressed on a substantial fraction of dural afferents, and activation of meningeal TRPA1 produces behaviors consistent with those observed in patients during migraine attacks. Further, they suggest that activation of meningeal TRPA1 via endogenous or exogenous mechanisms can lead to afferent signaling and headache.
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GEOZS, IJS, IMTLJ, KILJ, OILJ, SBJE, UL, UPUK
Background: The mechanisms contributing to the pain of migraine are poorly understood although activation of afferent nociceptors in the trigeminovascular system has been proposed as a key event. ...Prior studies have shown that dural-afferent nociceptors are sensitive to both osmotic and mechanical stimuli. Based on the sensitivity to these stimuli we hypothesized that dural afferents express the osmo/mechano-sensitive channel transient receptor-potential vanilloid 4 (TRPV4).
Methods: These studies used in vitro patch-clamp electrophysiology of trigeminal neurons retrogradely labeled from the dura to examine the functional expression of TRPV4. Additionally, we used a rat headache model in which facial/hind paw allodynia following dural stimulation is measured to determine whether activation of meningeal TRPV4 produces responses consistent with migraine.
Results: These studies found that 56% and 49% of identified dural afferents generate currents in response to hypotonic solutions and 4α-PDD, respectively. The response to these stimuli indicates that dural afferents express TRPV4. Activation of meningeal TPRV4 using hypotonic solution or 4α-PDD in vivo resulted in both facial and hind paw allodynia that was blocked by the TRPV4 antagonist RN1734.
Conclusion: These data indicate that activation of TRPV4 within the meninges produces afferent nociceptive signaling from the head that may contribute to migraine headache.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aβ42 and Aβ40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high ...diagnostic performance for AD, including the early phases of the disease. However, between‐center differences in pre‐analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre‐analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut‐off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Over 5 million Americans and 50 million individuals worldwide are living with Alzheimer's disease (AD). The progressive dementia associated with AD currently has no cure. Although clinical trials in ...patients are ultimately required to find safe and effective drugs, animal models of AD permit the integration of brain pathologies with learning and memory deficits that are the first step in developing these new drugs. The purpose of the Alzheimer's Association Business Consortium Think Tank meeting was to address the unmet need to improve the discovery and successful development of Alzheimer's therapies. We hypothesize that positive responses to new therapies observed in validated models of AD will provide predictive evidence for positive responses to these same therapies in AD patients. To achieve this goal, we convened a meeting of experts to explore the current state of AD animal models, identify knowledge gaps, and recommend actions for development of next‐generation models with better predictability. Among our findings, we all recognize that models reflecting only single aspects of AD pathogenesis do not mimic AD. Models or combinations of new models are needed that incorporate genetics with environmental interactions, timing of disease development, heterogeneous mechanisms and pathways, comorbidities, and other pathologies that lead to AD and related dementias. Selection of the best models requires us to address the following: (1) which animal species, strains, and genetic backgrounds are most appropriate; (2) which models permit efficient use throughout the drug development pipeline; (3) the translatability of behavioral‐cognitive assays from animals to patients; and (4) how to match potential AD therapeutics with particular models. Best practice guidelines to improve reproducibility also need to be developed for consistent use of these models in different research settings. To enhance translational predictability, we discuss a multi‐model evaluation strategy to de‐risk the successful transition of pre‐clinical drug assets to the clinic.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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