A rapid microfluidic in VIVO bioassay for evaluating the cognitive health benefits of compounds

Abstract

Provided herein are systems and methods for a rapid microfluidic in vivo bioassay for evaluating the cognitive health benefits of compounds.

Description

Docket No. NEM-102-PCT TITLE A RAPID MICROFLUIDIC IN VIVO BIOASSAY FOR EVALUATING THE COGNITIVE HEALTH BENEFITS OF COMPOUNDS BACKGROUND

[0001] The invention relates to high-throughput microfluidic screening of compounds for their biological effect on cognition in an intact living microorganism. Specifically, an in vivo assay with nematodes where a flow stimulus is applied and the behavioral response is characterized by a number of phenotypic metrics.

[0002] There is a rise in cognitive disorders due to the growing aging population. It is estimated that more than 55 million people were affected by dementia in 2019, and that a major cause of dementia are neurological disorders like Alzheimer’s disease (AD). Thus, there is a significant need to discover interventions that may act as preventive or therapeutic measures is critical. Bioactive substances including phytochemicals, botanical extracts, prebiotics, probiotics, postbiotics, pharmaceutical compounds, and micronutrients could be a potential source of novel interventions to improve cognitive health. However, the challenge is availability of high throughput screening tools that can evaluate and identify promising candidates that can deliver cognitive health benefits.

[0003] Cognition is an organismal-level response where in humans it can span diverse neural activities including attention, stimuli-evoked alertness, memory and pattern recognition. Clearly, in vitro cell-based models have difficulty in capturing such organismal-level response. Mammalian (e.g., mice) models are an established approach to evaluate cognition, and assays have been developed to mimic aspects of human cognitive abilities. However, the challenge is that mice models are not suitable for screening a large number of compound conditions, due to high cost and labor requirements. Moreover, regulatory policies supported by public sentiment are being instituted to reduce the reliance on use of mammals for testing.

[0004] Addressing the gap of high throughput in vivo screening, the invertebrate C. elegans is a popular choice owing to its fast reproductive cycle, small size, and a high degree of genetic conservation with mammals. The adult hermaphrodite has 302 neurons, including 52 glial cells and 118 different neuronal classes. Its neurobiology is highly conserved with similar ion channels, receptors, vesicular transporters and synaptic components. Similar to mammals, it uses neurotransmitters such as glutamate, γ-aminobutyric acid, dopamine, serotonin and acetylcholine.Docket No. NEM-102-PCT This nervous system allows the nematode to sense and react to environmental cues and exhibit diverse brain-states that lend to learning, memory and sleep.

[0005] The present invention attempts to solve these problems as well as others. SUMMARY OF THE INVENTION

[0006] Provided herein are systems and methods for a rapid microfluidic in vivo bioassay for screening compounds for improving cognition.

[0007] The systems and methods are set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the systems and methods. The advantages of the systems and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the systems and methods, as claimed.

[0008] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the accompanying figures, like elements are identified by like reference numerals among the several preferred embodiments of the present invention.

[0010] FIG.1A is a schematic of the microfluidic chamber that enables flow-induced stimulation of nematodes. The fluid exchange ports allow liquid to be injected. Young adult nematodes are introduced from the loading port. The sieve channels allow the progeny to be washed-off, whileDocket No. NEM-102-PCT retaining the adult nematodes. The micropillar arena is conducive for nematodes to crawl and prevent wash-off. (FIG.1B) Mean speed of the nematode population versus time in the micropillar arena from a typical flow-stimulation experiment. The inset shows the nematode trajectories during pre- and post-stimulus. The track density is much higher after stimulus indicating enhanced locomotory activity. The grey shaded area indicates the duration of flow stimulus. (FIG. 1C) Speed response curve shows a diminished response in mec-4 mutant in comparison to wildtype (WT) demonstrating that mechanosensory neural circuitry is involved in the stimulated response.

[0011] FIGS. 1A-2B are graphs showing the diminished response in neurodegenerative models and in aged nematodes. FIG. 2A is a graph showing both neurodegenerative models-A^^ (GRU102) and tau (BR5270)- show diminished response in comparison to wild-type in their speed-response curve confirming that the microfluidic bioassay captures cognitive deficits. FIG. 2B is a graph comparing the distribution of post-stimulus speed values in the models with neuronal impairment in comparison to those in wild-type (Mann-Whitney test, all p-values<0.001). In addition to disease-dependent cognitive decline, this bioassay also captures age-dependent decline.

[0012] FIG. 3 is a graph showing the microfluidic bioassay confirming that Ashwagandha provides improvement in cognitive function with age. The post-stimulus speed of Ashwagandha- treated worms is statistically significant (p-values<0.0001, Mann-Whitney test) from that of no- treatment control across all four ages of adult nematodes. The exposure concentration of Ashwagandha is 1.28 mg / mL. The violin plots were generated by using > 150 post-stimulus speed values from the worm-speed response curve.

[0013] FIG. 4 is a graph showing the microfluidic bioassay confirming that caffeine provides sustained alertness. The post-stimulus speed of caffeine-treated worms of age day 8 is statistically significant (p-values<0.0001, Mann-Whitney test) from that of no-treatment control across all three durations of exposure. The exposure concentration of caffeine is 5 mM. The violin plots were generated by using > 450 post-stimulus speed values from the worm-speed response curve. DETAILED DESCRIPTION OF THE INVENTION

[0014] The foregoing and other features and advantages of the invention are apparent from the following detailed description of exemplary embodiments, read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.Docket No. NEM-102-PCT

[0015] Embodiments of the invention will now be described with reference to the Figures, wherein like numerals reflect like elements throughout. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive way, simply because it is being utilized in conjunction with detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, no single one of which is solely responsible for its desirable attributes or which is essential to practicing the invention described herein.

[0016] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms “comprises,” “comprising,” “includes,” and / or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0017] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The word “about,” when accompanying a numerical value, is to be construed as indicating a deviation of up to and inclusive of 10% from the stated numerical value. The use of any and all examples, or exemplary language (“e.g.” or “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0018] References to “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.

[0019] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques andDocket No. NEM-102-PCT procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0020] Description of the Embodiments

[0021] Generally speaking, a method for rapid microfluidic in vivo bioassay for screening functional ingredients for measuring cognitive improvement comprises applying a flow-induced stimulation to C. elegans in a microfluidic device and tracking the C. elegans response to the flow- induced stimulation as a measure of cognitive improvement; and screening bioactive compounds for cognitive benefits including phytochemicals, pre / pro / postbiotics, dietary proteins, lipids, carbohydrates, or pharmaceutical compounds. The assay is compound-agnostic, as long as the worms can “eat” the substance, which is based on particle size being less than 3 microns.

[0022] The biological optimality of C. elegans is operably coupled with the technical advances in microfluidics, automated imaging and AI-based behavioral tracking to develop a high throughput in vivo assay with cognition-relevant readouts that can be used for pre-clinical testing of compounds and prioritizing safe compounds for human studies. C. elegans and other nematodes rely on mechanoreceptor neurons to detect collisions with soil particles and other animals, as well as forces generated by their own body movement. The microfluidic assay uses the conversion of mechanical energy into electrical signals that drives neural circuitry. The microfluidic bioassay comprises stimulating the mechanoreceptor neurons of C. elegans, and testing with compounds known to positively impact cognitive health in humans, and the tested compounds show a marked difference in the cognition response of C. elegans.

[0023] Description of the microfluidic bioassay for evaluating cognitive health

[0024] As shown in Fig.1A, the microfluidic assay 100 for evaluating cognitive health comprises a microfluidic chamber 102, a first fluid exchange port 110 and a second fluid exchange port 112, an animal loading port 116, a first sieve channel 120, a second sieve channel 122, and a micropillarDocket No. NEM-102-PCT arena 130. Nematodes are housed in the microfluidic chamber 102 with one or more inlet or outlet ports that allows worms of a defined age to be cultured (Fig.1A). The microfluidic chamber 102 has the capability to add or remove fluid so that worms can be cultured from young to old age, where Larval stages (e.g. day 2 from hatching of eggs) to old age (e.g. days 21 - 30). Worms can be crawling or swimming in the microfluidic chamber. Micropillars are introduced in the chamber to induce worm crawling.

[0025] In one embodiment, nematodes could be C. elegans, parasitic nematodes, mutant or transgenic nematode strains that report on specific molecular pathways or disease models.

[0026] In one embodiment, the mechanical stimulation is provided to the worms in the microfluidic chamber (Fig. 1B). The stimulation comprises of injection of fluid into the microfluidic chamber which agitates the worms and increases their activity. The fluid stress stimulates the mechanosensory neurons thereby eliciting a rapid increase in the nematode movement. The mechanosensory apparatus could also be stimulated via acoustic, light forces, chemical agent, and fluid forces or a combination of acoustic-light, light-fluid forces, acoustic- fluid forces. The type of stimulation (acoustic, optical, mechanical, or chemical) and dose of stimulation (magnitude and duration) is determined to activate specific ion channels or neuronal pathways. In other embodiments, the specific microfluidic output and screening of bioactive compounds or research question may be considered, including stimulation, adaptation / habituation, resilience, fatigue, or ablation.

[0027] The microfluidic assay comprises recording videos and imaging the nematodes in the microfluidic chamber prior to stimulus, during the stimulus, and after stimulus. The videos are analyzed using image processing and artificial intelligence based neural networks to obtain a variety of readouts including pre- and post-stimulus worm velocity, reflex latency, response time (rise) to stimulation and decay time, frequency of turns, reversal, and the like. In one embodiment, the video-recording protocol images the pre-stimulus state for about 60 seconds or more, while the stimulus is provided for about 1 second or more, and the post-stimulus state for about 60 seconds or more. This embodiment for the video-recording protocol could involve recording images at about 1 frame per second or more.

[0028] The microfluidic assay comprises comparing the response prior to stimulus and post stimulus that informs on the magnitude of excitation of neural circuity in C. elegans. Given that the locomotory activity is enhanced upon stimulation in nematodes, the response is similar toDocket No. NEM-102-PCT alertness in mammals, where audio / visual / olfactory stimuli elicit a reactionary response. The alertness response is a cognition state that can decline with age, or due to neurological disorders. In an alternative embodiment, the in vivo alertness response can be enhanced due to consumption of bioactive substances that target signaling pathways in the nervous system or between the gut and the nervous system.

[0029] Description of readouts from the microfluidic bioassay for evaluating cognitive health

[0030] The stimulation protocol provides a response curve as shown in Fig. 1B. The response curve can be generated either by determining the locomotory speed, or some other behavioral measure, including but not limiting to, turn frequency, pauses, angular velocity, reversals, and the like. From these response curves, a variety of readouts can be generated, including reflex latency, response time, response peak, decay time, pre-stimulus speed, and post-stimulus speed, as discussed below.

[0031] Reflex latency is the delay in the response to the stimulus and is determined as time which is the difference between the start of the stimulus and the start of the rise of the response curve, as shown in Fig 1B. Reflex time can vary depending on multiple factors, including the type of stimulus, the sensory pathway involved, individual differences, and the complexity of the response. Generally, reflex times are relatively quick because reflexes are rapid, involuntary responses that help protect the body or allow for quick adjustments in response to external stimuli. The reflex latency is a combined metric for detection of the stimuli type and magnitude, transmission of the stimuli, processing, and motor response. Factors that influence reflex latency are age, physical condition, nutrition, alertness, and genetic defects. Additionally, practice or training can also greatly influence the reflex latency.

[0032] Response time (Rise) represents the time required to reach the peak response which is determined from the start of the rise of response curve to the peak of the response shown in Fig 1B. Rise provides critical information on the motor function, physical / nutritional condition, and age of the animal.

[0033] Response peak is the strength of the response which is determined by the peak speed of the animal population. Peak response is the reflection of the maximum activity or firing rate of the sensory neurons or mechanoreceptors in response to the given stimulus intensity. Peak response is the function of the intensity, duration and nature of the mechanical stimulus which helpsDocket No. NEM-102-PCT researcher / scientists the sensitivity, dynamic range, and functional properties of mechanosensitive cells.

[0034] Decay time is the duration between the peak response and the post stimulus speed plateau. Decay time represents neuronal plasticity, adaptation, peripheral adaptation and fatigue. Neuronal plasticity, adaptation or fatigue is interrogated by changing the duration or frequency of the stimuli.

[0035] Pre-stimulus speed is the baseline / resting speed distribution is defined as the pre-stimulus speed shown in Fig.1B.

[0036] Post-stimulus speed is the post-stimulus speed is determined by the speed distribution after the decay in of the peak response shown in Fig.1B. Both pre- and post-stimulus speed is used to understand the short-term and long-term effect of the stimuli.

[0037] Results

[0038] A. Microfluidic bioassay detects cognitive response

[0039] To implement the microfluidic bioassay, age synchronized L4 worms are loaded into the microfluidic chamber and kept overnight at about 20°C in liquid NGM buffer containing about 20 mg / ml of bacterial diet (e.g., E. coli strain OP50 or another microbial strain) as a food source for the worms. Flow-induced stimulation is provided on the desired day of assessment by introducing a fluid pulse for about 5 seconds at a flow rate of about 7.5 mL / min, while the microfluidic chamber was being video recorded. Fig.1A shows a representative microfluidic chamber with one inlet and outlet port. The inlet port is used to introduce a fluid stimulus. The stimulus period could be higher than about 1 second, and the flow rate could be lower or higher than about 1 mL / min.

[0040] Fig. 1B shows the analyzed mean velocity of the nematode population obtained from a typical stimulation experiment. As shown, the mean velocity of the population is nearly constant during the pre-stimulus, but increases dramatically, when the flow stimulus is perceived by the nematodes. Importantly, the mean velocity is sustained to be higher than the pre-stimulus value for tens of seconds, even after the cessation of the flow stimulus.

[0041] The enhanced locomotory response could be due to stimulation of the mechanoreceptor neurons. To support this, the stimulation response between wild-type and mec-4 mutant were compared, where mec-4 mutant is defective in a gene necessary for mechanosensation. The speed response is diminished (Fig.1C), confirming that the mechanoreceptor neurons are stimulated in microfluidic bioassay.Docket No. NEM-102-PCT

[0042] To provide additional evidence that the stimulated behavior is linked to cognitive response, two transgenic strains of C. elegans that have impaired neuronal health were tested. The nematode strains A^^ (GRU102) and Tau (BR5270) capture the neuropathology of Alzheimer’s Disease (AD). The two mechanisms separately contribute to accumulation of A^^ plaques and neurofibrillary tangles composed of tau-protein respectively, both of which are implicated in dementia. Further, young to old nematodes were investigated to demonstrate that cognitive deficits occur in older worms.

[0043] Fig. 2 shows the worm-speed response in wildtype, A^^ (GRU102) and Tau (BR5270) strains of ages day 6, day 8 and day 10 (Note: day 0 is hatching, and day 4 animals are young adults). Even though the pre-stimulus speed is nearly the same for all three strains, the neuronally impaired strains have diminished response upon stimulation indicating cognitive deficits. Moreover, the response declines with age supporting that the microfluidic bioassay informs on age-induced cognitive decline. The post-stimulus speed values were obtained and plotted them as distributions in Fig. 2B to clearly highlight the differences between individual strains, and the differences with age.

[0044] B. Microfluidic bioassay detects cognitive improvements due to consumption of bioactive substances

[0045] To determine the effectiveness of the microfluidic bioassay for testing bioactive substances, the nematodes of given ages in the microfluidic chamber were fed for specified durations and compared the post-stimulus response against control (no-treatment wildtype worms).

[0046] In human studies, the botanical extract Ashwagandha has been shown to improve cognition, alertness and short-term memory. Therefore, Ashwagandha was tested using the microfluidic bioassay protocol described above. Fig.3 shows the post-stimulus speed distributions when the subjects were treated with Ashwagandha. Ashwagandha was found to enhance the cognitive response compared to control, and also in older worms.

[0047] Caffeine was tested, which has been known to enhance human cognitive function in terms of increased wakefulness, and reduced reaction time. The microfluidic bioassay is able to capture this enhanced cognitive effect of caffeine, where nematodes treated with caffeine for about 30 min, about 2 hours, and about 5 hours show elevated mechanosensory response indicative of sustained alertness, as shown in Fig.4.Docket No. NEM-102-PCT

[0048] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0049] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.

Claims

Docket No. NEM-102-PCT CLAIMS What is claimed is:

1. A method for rapid microfluidic in vivo bioassay for screening functional ingredients for measuring cognitive improvement comprising: a. applying a stimulation to nematodes in a microfluidic device and tracking the nematodes response to the flow-induced stimulation as a measure of cognitive improvement; and b. treating the nematodes with a plurality of bioactive compounds; and c. screening bioactive compounds for cognitive benefits.

2. The method of Claim 1, wherein the bioactive compounds are selected from the group consisting of phytochemicals, pre / pro / postbiotics, dietary proteins, lipids, carbohydrates, and pharmaceutical compounds.

3. The method of Claim 1, wherein the nematodes are selected from the group consisting of C. elegans, parasitic nematodes, mutant nematode strains, and transgenic nematode strains that report on specific molecular pathways or disease models.

4. The method of Claim 1, wherein applying the stimulation comprises of injection of fluid into the microfluidic chamber which agitates the nematodes and increases their activity.

5. The method of Claim 1, wherein applying the stimulation is acoustic, light forces, fluid forces, chemical agent or a combination of acoustic-light forces, light-fluid forces, acoustic-fluid forces, acoustic-chemical forces, light-chemical forces, and fluid-chemical forces.

6. The method of Claim 5, wherein the type of stimulation and dose of stimulation is determined to activate specific ion channels or neuronal pathways.Docket No. NEM-102-PCT 7. The method of Claim 1, wherein screening bioactive compounds comprises recording videos and imaging the nematodes in the microfluidic chamber prior to stimulation, during the stimulation, and after stimulation, analyzing the videos using image processing and artificial intelligence based neural networks to obtain a plurality of readouts including pre- and post-stimulus worm velocity, reflex latency, response time (rise) to stimulation and decay time, frequency of turns, reversal, and the like.

8. The method of Claim 7, wherein the recording videos and imaging the nematodes comprises a video-recording protocol imaging a pre-stimulation state for about 60 seconds or more, while the simulation is provided for about 1 second or more, and the post- stimulation state for about 60 seconds or more.

9. The method of Claim 8, wherein the video-recording protocol comprises recording images at about 1 frame per second or more.

10. The method of Claim 7, wherein comparing the response prior to stimulus and post stimulus that informs on the magnitude of excitation of neural circuity in the nematodes, and the in vivo alertness response is enhanced due to consumption of bioactive substances that target signaling pathways in the nervous system or between the gut and the nervous system.

11. The method of Claim 7, wherein the response curve is generated by determining the locomotory speed, or some other behavioral measure, including but not limiting to, turn frequency, pauses, angular velocity, or reversals.

12. The method of Claim 11, further comprising generating a plurality of readouts from the response curve, wherein the plurality of readouts include reflex latency, response time, response peak, decay time, pre-stimulus speed, and post-stimulus speed.Docket No. NEM-102-PCT 13. The method of Claim 7 or 12, wherein reflex latency is the delay in the response to the stimulus and is determined as time which is the difference between the start of the stimulus and the start of the rise of the response curve, the reflex latency is a combined metric for detection of the stimuli type and magnitude, transmission of the stimuli, processing, and motor response.

14. The method of Claim 7 or 12, wherein the response time represents the time required to reach the peak response which is determined from the start of the rise of response curve to the peak of the response.

15. The method of Claim 12, wherein the response peak is the strength of the response which is determined by the peak speed of the animal population, and the peak response is the function of the intensity, duration and nature of the mechanical stimulus.

16. The method of Claim 12, wherein the decay time is the duration between the peak response and the post stimulus speed plateau, and the decay time represents neuronal plasticity, adaptation peripheral adaptation and fatigue.

17. The method of Claim 12, wherein the pre-stimulus speed is the baseline / resting speed distribution is defined as the pre-stimulus speed.

18. The method of Claim 12, wherein the post-stimulus speed is the post-stimulus speed is determined by the speed distribution after the decay in of the peak response.

19. The method of Claim 12, wherein the microfluidic chamber comprises a first fluid exchange port and a second fluid exchange port, an animal loading port, a first sieve channel, a second sieve channel, and a micropillar arena.

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