This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo and the Ministry of Science and ICT (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021).

1. Hornworm preparation and cannabidiol treatment
<ol>
	<li>Obtain 150-200 viable M. sexta eggs and wheat germ-based artificial diets (see Table of Materials).</li>
	<li>Place the hornworm eggs in a polystyrene Petri dish with a wheat germ-based artificial diet (AD) layer and transfer the eggs to an insect rearing chamber (see Table of Materials) maintained at 25 &#176;C with 40%-60% relative humidity.</li>
	<li>Allow tobacco hornworm eggs for 1-3 days to hatch inside the insect rearing chamber maintained at 25 &#176;C with 40%-60% relative humidity.</li>
	<li>Prepare cannabidiol (CBD) stock solution (200 mM) by adding 1.26 g of &#62;98% purity CBD isolate in 20 mL of EtOH (200 proof) or 100% medium chain triglycerides (MCT) oil (see Table of Materials). 
	NOTE: CBD isolate is light-sensitive, so handle at dark.</li>
	<li>Add 5 mL and 10 mL of the 200 mM CBD stock solution to the 1,000 g of AD to bring the final concentrations of the diets 1 mM and 2 mM of CBD, respectively. 
	NOTE: Ensure the diet and CBD stock solution are well-blended until a completely homogeneous mixture is formed. Blend the AD containing stock of CBD in a plastic bag for at least 45 min by hand. 
	CAUTION: Coffee mixer or any other metal grinder appeared to be ineffective.</li>
	<li>Dispense 20 g of the three media, control (AD), vehicle (AD + 0.1% of EtOH or MCT oil), and CBD containing media (AD + 0.1% of EtOH or MCT oil + 1 mM/2 mM of CBD) to the bottom of the 50 mL tube.</li>
	<li>Randomly distribute 1st instar larvae (~2 mm long) individually in a 50 mL test tube and cover with a perforated lid or cheesecloth (see Table of Materials). 
	NOTE: Place the tube upside down and grow insects at an insect rearing chamber maintained 25 &#176;C with 40%-60% relative humidity.</li>
	<li>Grow them inside an insect rearing chamber (see Table of Materials) maintained at 25 &#176;C with a 12 h light/dark cycle.</li>
</ol>
2. M. sexta larval growth, diet consumption, and mortality measurements
<ol>
	<li>Measure the larval growth (i.e., size and weight) with an analytical balance and mortality at 2-day intervals after being transferred to individual containers until pupation is recognized as the dark brown coloration of a hardened exocuticle layer.
	<ol>
		<li>Record the initial mass (in grams) of each group of larvae before introducing the larvae to their respective diets and subtract the mass of the larvae at each measurement from the initial mass to determine mass gains between larvae developmental stages until the larvae complete the pupation stage.</li>
		<li>Record the number of days between the instar developmental stages to understand differences in the developmental timeframe between stages of larvae growth until pupation on each diet. 
		NOTE: Scrape off the fecal matter from the container to avoid any mold contamination. Collect the matter for future testing dependent on experiment purposes (e.g., CBD accumulation rate calculation, microbial profiling). It is important to carefully handle the insect during the fragile periods of apolysis or ecdysis. When taking out of the larvae from a container, gently grab the main body of the insect with a flat-tip and wide forceps and do not force to remove the outer layer of skin when an insect is in the process of shedding.</li>
	</ol>
	</li>
	<li>Measure the diet consumption31 by weighing the diet loss of the container between 1st instar larvae and pupation. Record the initial grams of diet at the beginning of the experiment and subtract the initial amount from the remaining amount of diet when the larvae entered the complete pupation stage. 
	NOTE: The fecal matter should be excluded from the diet measurement. The fecal matter and other debris (i.e., skin sheds) can be easily removed from the media by placing the container upside down.</li>
	<li>For mobility measurements, allow the subjected insect to acclimate the chamber environment for at least 5 min and track the distances31 that three groups of 5th instar insects (80-100 mm in length) traveled using an automated, computerized fear conditioning chamber (see Table of Materials).</li>
	<li>Analyze the mobility response31 through video recorded 60 frames/s for 5 min using a motion detection software (see Table of Materials) which generates a motion index.</li>
</ol>
3. Statistical analysis
<ol>
	<li>Analyze the differences in the larval growth (i.e., size and weight) and the motion index by one-way ANOVA with Tukey&#39;s post-test32.</li>
	<li>Use the log-rank (Mantel-Cox) test33 for survival curve comparisons. 
	NOTE: All the statistical analyses were performed using statistical analysis software (see Table of Materials).</li>
</ol>

<ol>
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The authors have no conflicts of interest.

The feeding study demonstrated that high doses of CBD (2 mM) inhibited the insect&#39;s growth and increased mortality31. The insect model also showed sensitivity to ethanol; however, CBD effectively detoxicated the ethanol toxicity, increasing their survival rate, diet consumption, and food searching behaviors to similar levels to the control group (Figure 3A,B)31. The described insect model system is composed of three critica...

Over the past years, public attention has been centered on cannabinoids due to their therapeutic potential, including the treatment of epilepsy1, Parkinson&#39;s disease2, multiple sclerosis3, and various forms of cancer4,5,6 with cannabidiol (CBD). Since Cannabis is legalized as an agricultural commodity in the Agricultural Improvement Act of 2018, Public Law 115-334 (the 2018 Farm Bill), Cannabis and its cannabinoid derivatives in the food, cosmetic, and pharmaceutical industries have exponentially increased. Additionally, clinical-grade isolates of single cannabinoids and cannabinoid mixtures have been successfully tested in human subjects7, cell lines5,8, and diverse animal model systems9,10.
A clinical trial would be ideal for validating the efficacy and adverse effects of cannabinoids on a specific disease. However, there are numerous challenges in clinical trials, including ethical/IRB approval, recruitment, and retention of the subjects11. To overcome these hurdles, various human cell lines were used because human-derived cell lines are cost-effective, easy to handle, can bypass the ethical issues, and provide consistent and reproducible results as the cell lines are a &#39;pure population of cells that have no cross-contamination of other cells and chemicals&#39;12.
Alves et al. (2021)13 tested CBD in a dose-dependent manner in the placental trophoblasts, which are specialized cells of the placenta that play an essential role in embryo implantation and interaction with the decidualized maternal uterus14. Their results showed that CBD caused cell viability loss, cell cycle progression disruption, and apoptosis induction. These observations demonstrate the potential negative impacts of Cannabis use by pregnant women13. Likewise, a series of cell lines were also used to examine the pharmacological effects of CBD in human diseases, in particular, various forms of cancer. The in vitro studies successfully demonstrated anti-cancer effects in pancreatic15, breast8, and colorectal cancer cells16. However, while being widely available and easy to handle, specific cell lines such as HeLa, HEK293 are prone to genetic and phenotypic changes due to alterations in their growth conditions or handling17.
In Cannabis research, various animal model systems, ranging from small animals such as mouse18, guinea-pig19, and rabbit19 to large animals such as canine20, piglet21, monkey22, horse23, have been used to explore unknown therapeutic effects. Mice have been the most preferred animal model system for cannabinoid research due to their anatomical, physiological, and genetic similarity to humans24. Most significantly, mice have CB1/2 receptors in their nervous system, which are present in humans. They also have a shorter life cycle than human subjects, with easier maintenance and abundant genetic resources, thus making it much easier to monitor the effects of cannabinoids throughout an entire life cycle. The mammalian system is widely used and has successfully demonstrated that CBD relieves seizure disorders1, post-traumatic stress disorder9, oral ulcers25, and dementia-like symptoms10. The mouse model has also enabled a social interaction study of individuals within a community which is extremely difficult in large animals and humans26.
Despite all the advantages of the animal model system, it is still costly and requires intensive care during drug administration and data collection. Additionally, there is scrutiny of using mice in research because of irreproducibility and poor recapitulation of human conditions due to limitations in experimental design and rigor27.
With the increasing demand for medical/preclinical studies of cannabinoids, a non-mammalian model system is needed. Invertebrate models traditionally conferred distinctive benefits over vertebrate models. The significant benefits include the ease and low cost of rearing many specimens and enabling researchers to monitor multiple generations of genetically homogeneous populations28. A recent study proved the fruit fly, Drosophila melanogaster, to be an effective insect model system to investigate pharmacological functions of cannabinoids in modulating feeding behaviors29. Among the insect model systems, the authors focused on the tobacco hornworm, Manduca sexta, also known as Carolina sphinx moth or hawk moth, as a novel insect model system for cannabinoid research.
Manduca sexta belongs to the family of Sphingidae. The insect is the most common plant pest in the southern United States, where they feed on solanaceous plants. The insect model has a long history in research in insect physiology, biochemistry, neurobiology, and drug interaction studies. Manduca sexta&#39;s&#160;research portfolio includes a draft genome sequence, allowing for a molecular-level understanding of essential cellular processes30. Another crucial benefit of this model system is its large size, reaching more than 100 mm in length and 10 g in weight in the 18-25 days of larval development. The large size enables researchers to easily monitor morphological and behavioral changes in real-time in response to the CBD treatment. Also, due to the size, electrophysiological responses were examined with the abdominal nervous system, including ganglia dissected from the larvae without high-resolution microscope settings. The unique feature allows researchers to readily investigate acute and long-term responses to the administered cannabinoid(s).
Despite such versatility, M. sexta has only recently been explored for its suitability as an experimental model for Cannabis and cannabinoid&#160;studies. In 2019, the authors used the insect model system for the first time to address the hypothesis that Cannabis has evolved to produce Cannabidiol to protect itself from insect herbivores30,31. The result clearly showed that the plants exploited CBD as a feeding deterrent and inhibited the growth of the pest insect M. sexta caterpillar, as well as causing increased mortality31. The study also demonstrated the rescuing effects of CBD to intoxicated ethanol larvae, identifying the potential vehicle effect of ethanol as a carrier of the CBD. As shown, the insect model system effectively investigated the therapeutic effects of cannabinoids within 3-4 weeks with less labor and costs than other animal systems. Although the insect model lacks cannabinoid receptors (i.e., no CB1/2 receptors), the model system provides a valuable tool to understand the pharmacological roles of cannabinoids through a cannabinoid receptor-independent manner.
The authors of this study have previously worked with the tobacco hornworm as a model system for cannabinoid research31. After careful consideration of the benefits and risks of using M. sexta, we have provided a method involving the proper care and preparation of diet for preclinical trials that allow for opportunities for future preclinical laboratory use.

<table><tbody><tr><td>Analytic balance</td><td>Mettler Instrument Corp.</td><td>AE100S</td><td></td></tr><tr><td>Cannabidiol isolate (&gt;99.4%)</td><td>Lilu&#039;s Garden</td><td></td><td></td></tr><tr><td>Cheesecloth</td><td>VWR INTERNATIONAL</td><td>470150-438</td><td></td></tr><tr><td>Corning 50mL clear polypropylene (PP) centrifuge tubes</td><td>VWR</td><td>89093-192</td><td></td></tr><tr><td>Ethyl Alcohol, 200 Proof</td><td>Sigma-Aldrich</td><td>EX0276-1</td><td></td></tr><tr><td>Fear conditioning chamber</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Insect rearing chamber</td><td>Darwin Chambers</td><td>INR034</td><td></td></tr><tr><td>Medium chain triglycerides (MCT) oil</td><td>Walmart</td><td></td><td></td></tr><tr><td>Motion detection software (Actimetrics)</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Polystyrene petri dish (120 mm x 120 mm x 17mm)</td><td>VWR INTERNATIONAL</td><td>688161</td><td></td></tr><tr><td>Tobacco hormworm artificial diet</td><td>Carolina Biological Supply Company</td><td>Item # 143908</td><td>Ready-To-Use-Hornworm-Diet</td></tr><tr><td>Tobacco hormworm eggs</td><td>Carolina Biological Supply Company</td><td>Item # 143880</td><td>Unit of 30-50</td></tr></tbody></table>

tobacco hornworm as an insect model system for cannabinoid pre-clinical studies

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 &#176;C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

Manduca sexta as a model system to examine cannabinoids toxicity 
Figure 1 depicts the key components of the CBD experiment using tobacco hornworm Manduca sexta. Large numbers of insects (&#62;20) were individually reared at 25 &#176;C on a 12 h:12 h = light: dark cycle. The insects&#39; size, weight, and mortality were measured at 2-day intervals to monitor for short-and long-term responses after high-dose CBD (2 mM) treatment.
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Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

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Research

JoVE Journal

Biology

2.5K Views.  Colorado State University-Pueblo. Het protocol stelt voor om tabakshoornworm te gebruiken als een insectenmodelsysteem voor preklinische onderzoeken naar cannabinoïden. Dit systeem stelt onderzoekers in staat om acute en langetermijnreacties op cannabinoïden gemakkelijk te onderzoeken. Het insectenmodelsysteem stelt onderzoekers in staat om cannabinoïde therapeutische effecten in een grote populatie binnen drie tot vier weken te onderzoeken met minder arbeid en kosten dan andere dierlijke systemen.Verkrijg 150 tot 2...

Video: Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Environment

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Immunology and Infection

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Bioengineering

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Medicine

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Preclinical Model of Prenatal Delta-9-Tetrahydrocannabinol Exposure to Assess Its Impact on Neurodevelopmental Outcomes

Prenatal cannabinoid exposure (PCE) is becoming increasingly frequent as more states across the United States legalize recreational marijuana (cannabinoids). The consumption of cannabinoid products during pregnancy has been associated with various abnormal outcomes, although historical studies were conducted during a time when the potency of these products was approximately 300% lower than that of current products. Given the rising use of cannabinoids, it is essential to understand the potential impacts PCE may have on fetal neurodevelopment and subsequent infant and child development. Previous studies have demonstrated that PCE negatively affects learning and memory, behavioral skills, sleep, and attention in offspring. The aim of this study is to recapitulate PCE through voluntary ingestion of delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabinoid products, during pregnancy in a preclinical model. This article outlines the procedure for achieving moderate PCE throughout gestation. In this model, the control group consumes plain mini-chocolate/peanut butter cookies, while the PCE group consumes THC mixed into peanut butter paired with mini-chocolate cookies. This approach enables further investigation into the impact of PCE on developmental outcomes.

As legislative restrictions change, resulting in increased accessibility of cannabinoid products, it is critically important to develop models to study the impact of these exposures, particularly during pregnancy. A preclinical model of moderate prenatal cannabinoid exposure through voluntary ingestion has been developed, enabling more in-depth investigations.

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

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