Afraxis ESP Methods

The increased optical resolution obtained over conventional fluorescence confocal microscopy is critical for evaluation.


fluorescence confocal microscopy

Dendritic spines are protrusions that emerge from dendrites and house the postsynaptic signaling machinery at the vast majority of excitatory synapses. With a quick glance at a segment of dendrite, one can easily note a diversity of spine morphological phenotypes – short, fat, long, thin. The dimensions of a spine’s key morphological features, it turns out, strongly predict the (a) size of the postsynaptic density, (b) the number of inserted glutamate receptors at the synapses, and (c) excitatory neurotransmission of that spine.

We can therefore measure individual spine structural features to estimate its individual contribution to its local synaptic network.

Furthermore, dendritic spine plasticity (i.e. long-term potentiation) requires dynamic dendritic spine cytoskeletal processes for its expression and fine-tuning – far from a simple relationship to synaptic function, dendritic spine morphology is a fundamental biomarker of synaptic efficacy.

Measuring Synapses: Beyond The HCS Analysis

laser scanning confocal microscopy

Two fundamental tenets of any real-world memory system are that it maintains large capacity for information encoding, and that signal strength is retained for very long periods at the positon of encoding (input-specificity). The numbers of synapses required to produce a disease condition (or for a drug to produce beneficial effects) therefore are necessarily (a) sparse and (b) expressed in a distributed manner.

Why is this important? It means that to decipher how the brain processes information, we need to measure large numbers of individual synapses and measure them in distributed locations in the brain.

The Afraxis ESP platform is designed to do exactly that.

Why is this such a daunting task? Dendritic spines are really small (< 1 µm in diameter) – so precise and reliable measurement of spine features requires us to push our imaging and analytical capabilities to the limit. Then, we need to repeat this thousands of times and with pinpoint accuracy within diverse areas of brain anatomy. This kind of sensitive readout on large numbers of spines is not currently available as a high-throughput screening (HTS) or high-content screening (HCS) – but we provide the next best thing – workflow-optimized in vivo platform (WIP) technology.

Workflow-optimized In Vivo Platform (WIP) Technology

image analysis software

HTS and HCS brought about a boon for drug discovery – they injected real cell biology into mass drug screening. Despite these advances, CNS drug researchers have continued to struggle to identify efficacious new molecular entities. One reason for this is the lack of cost-efficient in vivo evaluation assays, rendering the available assays as expensive and a rarely implemented spot check.

To tackle this issue, Afraxis ESP is built on patented digital technology that optimizes the training and activities of study team members. This approach reduces a single compound evaluation (typically a 4-9 month process) to less than 2 weeks. Importantly, no elements of the study are compromised.

Our rapid turn-around times combined with parallel processing permit:

  • Large-scale in vivo drug screening
  • Adaptive structure-activity relationship (SAR) testing
  • Broad pathology screening in novel animal disease models

Contact us to learn more information.

In Vivo Analysis in CNS

Why is in vivo analysis so important in CNS drug discovery? Neurons within the cerebral cortex and lower brain regions are densely interconnected – therefore the behavior and vulnerability of individual neurons is intimately aligned with the activity of distant synaptic networks in coordination with its local networks.

The Afraxis ESP platform addresses this by measuring disease and therapeutic impact in the fully intact nervous system.

Afraxis ESP:
In-Life Administration

laser confocal microscopy

Compound formulation and administration can be performed:

  • By the client or a third-party CRO hired by the client
  • A trusted Afraxis partner CRO

Afraxis has completed a large number of studies working closely with R&D teams at large pharmaceutical and biotech companies, as well as small start-ups and academic labs. In these cases, the client performs the study and delivers formaldehyde-fixed tissue to Afraxis upon completion.

We provide free-of-charge tissue quality evaluation services to ensure our client’s processing is compatible with the ESP platform.

Compound formulation and administration can be performed by a third-party CRO selected by the client. We will work closely with the CRO to establish optimal tissue processing and delivery procedures.

Alternatively, Afraxis maintains close collaborations with CROs that routinely perform in-life studies for the ESP platform. Afraxis ESP efficacy or safety evaluations can be combined with toxicology, safety, or other types of analyses.

Contact us to learn more information.

Labeling: Fluorescence Confocal Microscopy

Afraxis ESP utilizes ballistic dye labeling for fluorescent staining of individual neurons. The fluorescence confocal microscopy permits detailed investigation of extensive dendritic arbors on each neuron. Our trained microscopists accurately identify individual neurons within precise neuroanatomical regions and subregions. Narrow criteria (e.g. branch type, branch order) are applied based on microscopic measurements collected on each individual neuron. These steps are required to produce the most accurate, reliable, and reproducible data for our evaluations. Our methods are also compatible with GFP and other fluorescent protein labels.

Dendritic Spine Analysis:
A human-computer hybrid approach

Afraxis’ patented analytical schema unifies the careful and discerning eye of human observers with the strict organization of digital automation. Multiply-redundant analyses are employed to ensure optimal accuracy. This approach is critical in establishing highly reproducible baseline data for large-scale serial studies and corroborating data across multiple studies.

Confocal Microscopy:
Send us your Z-stacks!

Afraxis ImageDirect image analysis software allows to take fluorescence confocal microscopy to the next level. ImageDirect is a new generation 3D image upload service, optimized for ‘Z-stack’ images of neural tissue acquired by confocal scanning laser microscopy. Academic research labs and drug development teams can now submit images directly to a persistent image container object on Afraxis’ secure cloud, define the target features, type of analysis, and the desired report format. We provide timely responses on cost and turn-around time for a full quantitation of any submitted image deck. Our expert crowd-sourced analyst team then provides unbiased measurement and full analytics with a one-week or shorter turn-around time for most studies.

Contact Us

Afraxis, Inc.
6605 Nancy Ridge Rd. Suite 224
San Diego, CA 92121
Phone: 858.436.1539