Name: a surgical procedure for the administration of drugs to the inner ear in a non-human primate common marmoset (callithrix jacchus)
Uploaded: 2018-02-27T12:30:00
Description: Watch this Scientific Journal Video about A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus at JoVE.com

The work is supported by grants to M.F. from the Japanese government MEXT KAKENHI (24592560, 15H04991, and 15K15624) and the Takeda Science Foundation to M.F. and the Jikei University Strategic Prioritizing Research Fund to H.J.O.

All experimental procedures were performed at the Jikei University, Tokyo Japan. Animal handling and experimental procedures were approved by The Institutional Animal Care and Use Committee of The Jikei University (Approval no: 26-060) and performed in accordance with the Institutional guidelines for use of laboratory animals at the Jikei University, which agree with the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan (2006). Animal care was conducted in accordance with the recommendations of the Guide for...

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The volume of cochlear blood flow is extremely small, estimated to be in the order of 1/1,000,000 of the total cardiac output in humans, and access is limited by the presence of the blood-cochlea barrier which separates the inner ear from the general circulation19,20. For these reasons, drug or viral vector administration into the inner ear is favorable, not via systemic administration but rather by direct administration. In mouse and guinea pig experiments, vari...

Sensorineural hearing loss (SNHL) arises predominantly from damage or deficiency in the cochlea. Common causes of SNHL include aging (e.g. presbycusis), genetic defects, exposure to loud noise, infection, and ototoxic drugs1. The World Health Organization (WHO) estimated that over 360 million people, representing 5.3% of the world&#39;s population, suffer from hearing loss2. It is also estimated that 1 in 900 to 1 in 2,500 newborns have moderate, severe, and profound congenital permanent hearing loss, and around one in three adults older than 65 years have some degree of hearing loss3. However, there is no effective clinical treatment of hearing loss for these patients.
Hearing research has long been performed using rodent or guinea pig models, and many approaches, such as gene therapy and regenerative therapy, have been suggested as a new treatment for hearing loss. However, there is a large difference between humans and rodents in terms of the auditory system, and it is very difficult to translate animal models to human applications. The common marmoset (C. jacchus), a New World monkey originating from the Amazon, has become an attractive non-human primate model for various basic research studies for several reasons. First, its anatomy and physiology are more similar to those of the humans rather than rodents. Second, the basic biological information about this species, including diseases, neural networks, behavior, and the genome, is well characterized. Auditory and vocal processing, the cortical coding of periodicity, the representation of pitch, and the auditory-vocal interactions of common marmosets have also been reported4,5,6,7,8,9,10. In recent histological studies, researchers identified distinctive expression patterns of 20 deafness genes and anion exchangers in the cochlea of the common marmoset and found that five genes that are causative of progressive deafness, and three anion exchangers, had different expression patterns from those of rodents11,12. These profiles lead us to believe that the common marmoset is a powerful tool for hearing research.
The most marked attributes of the common marmoset as an experimental animal are as follows:
<ul>
	<li>Easy handling with a small body size compared to some species of Old World monkeys: Adult marmosets weigh 300 - 400 g and are approximately 60 cm in height, which is similar to rats.</li>
	<li>Highly reproductive primate: Marmosets attain sexual maturity at an age of 18 months and are able to bear twice a year and produce 4 to 6 offspring annually.</li>
	<li>Genetic modifications are available: Sasaki E et al. succeeded in creating transgenic13 and knock-out14 primates using marmosets by genetic modifications implicated in neurological disorders (e.g., Parkinson&#39;s disease and Alzheimer&#39;s disease).</li>
	<li>Both embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been established15,16. Though it is rather difficult to keep the same number of marmosets compared to rodents, the availability of stem cells or iPS cells provides in vitro assays which will reduce the number of in vivo assays required.</li>
</ul>
To facilitate better translational research in the field of deafness and its potential therapy, we established an imaging study protocol using CT and MRI, general anesthesia and hearing tests using auditory brain stem responses (ABRs). These experimental systems may provide us with better opportunities to obtain pre-clinical proof of concept studies which are essential for bridging the gaps between rodent studies and clinical trials. Here, we report a surgical method to administrate drugs to the inner ear of the common marmoset through the round window membrane. To obtain a clear view around the round window, without manipulating the tympanic membrane which might cause a conductive hearing loss, it is useful to approach from the mastoid cavity. Clinically, this approach, referred to as &#34;posterior tympanotomy&#34; is well established and usually used for cochlear implantation and cholesteatoma surgery. We believe that posterior tympanotomy allows us to perform the accurate administration of drugs without inducing hearing loss.

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			<td height="21" style="height:21px;width:216px;">common marmoset</td>
			<td style="width:92px;">CLEA Japan</td>
			<td style="width:160px;">EDM:C.Marmoset(Jic)</td>
			<td style="width:207px;"></td>
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			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:92px;">Phizer</td>
			<td style="width:160px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">medetomidine</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">midazolam</td>
			<td style="width:92px;">astellas</td>
			<td style="width:160px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">butorphanol</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
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			<td height="24" style="height:24px;width:216px;">ampicillin</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lidocaine hydrochloride</td>
			<td style="width:92px;">AstraZeneca</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;6-0 absorbent thread</td>
			<td style="width:92px;">ETHICON</td>
			<td style="width:160px;">RD-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">atipamezole hydrochloride</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phosphate buffered saline</td>
			<td style="width:92px;">Wako</td>
			<td style="width:160px;">045-29795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surge Wave</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">TR-900-OR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 006</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 010</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">intensive care unit</td>
			<td style="width:92px;">Menix</td>
			<td style="width:160px;">P-100</td>
			<td></td>
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a surgical procedure for the administration of drugs to the inner ear in a non-human primate common marmoset (callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

Posterior tympanotomy was performed without any complication such as surgical site infection, bleeding with vascular injury, or vestibular dysfunction, which sometimes occurs with middle ear manipulation. We performed posterior tympanotomy on the right ear of 7-year-old common marmoset and administered 1 &#181;L of phosphate buffered saline to the round window niche. ABRs were measured before and two months after surgery (Figure 5A-B). Wavefo...

Watch this Scientific Journal Video about A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus at JoVE.com

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

The overall goal of this surgical procedure is to evaluate the effects of specific drugs of interest on the hearing function of non-human primate common marmoset through their safe administration into the inner ear without inducing hearing loss. This method can help answer key questions about inner ear drug delivery, including how to open the master cavity and where to deliver the drug. The main advantage of this technique is that the inner ear can be a plus without complications, which is critical for evaluating the efficiency of inner ear treatments in primates.The implications of this technique extend towards the study of potential therapies for human deafness, as the auditory system of the marmoset is quite similar to that of humans. The masteration of this method is critical and the posture and anatomy is difficult to learn and require that small window to be created in the narrow space without damaging the facial knob or the ossicular chain. Confirm the appropriate level of sedation by toe pinch and place the animal in the supine position on a heating mat with the head and neck extended.Fix maxilla to the table with nylon thread and pull out the tongue. Insert an endotracheal tube along the laryngeal surface of the epiglottis. Check the entitle carbon dioxide level in the endotracheal tube to confirm that the tube has been properly placed within the trachea and secure the tube to the animal's face.Incline the head to the 30 to 45 degree angle toward the non-operative side and shave the postauricular region of the operative side of the animal Then disinfect the postauricular region and the external auditory canal with povidone iodine and cover the animal with a surgical drape sheet with a round opening over the surgical area. Before opening the skin inject 1%of lidocaine hydrochloride subcutaneously to the incision site. Then, using a surgical microscope and a number 15 round shaped blade, make an incision into the postauricular skin and apply pressure to the bleeding area with a gauze soaked in one to 5, 000 diluted epinephrine.Open the soft tissue of the postauricular region so that the auricular cartilage and the postauricular muscle can be identified and expose the external auditory canal bone deep within the cartilage. To confirm the depth of the tympanic membrane, expose the bony part of the tympanic rim and peel the postauricular muscles toward the caudal aspect to separate the muscles from the temporal bone. Expose the lateral surface of the temporal bone and use a one millimeter diamond burr to make a hole approximately five millimeters behind the external auditory canal.Drill further to enlarge the hole as appropriate and locate the horizontal semi-circular canal, which bulges toward the mastoid cavity. Then drill and remove any bone septa as necessary to obtain a clear view of the facial recess, located at the center of the triangular area between the facial nerve and the corded tympany. Using a 0.6 millimeter diameter diamond burr at 1, 000 rpm, drill a small hole between the vertical section of the facial nerve, which appears as a pink line through the thinned bone and the tympanic rim.Stop the drilling just before the nerve is exposed as a white line structure and confirm that the round window niche can be visualized through the hold. Using a 25 gauge or thinner needle, deliver the drugs of interest into the niche and cover the niche with an appropriate miscues solution to ensure the drug is retained. Confirm that hemostasis is securely controlled and use six o absorbent threads to close the epidermal and dermal layers with simple, interrupted sutures.Then place a piece of gauze onto the surgical wound, fix the gauze in place with a bandage, and allow the marmoset to recover in an intensive care unit at 37 degrees Celsius in 30%oxygen until complete arousal. In these graphs, the auditory brain stem responses of a seven year old common marmoset before and two months after the delivery of one microliter of PBS to the round window niche, as just demonstrated, are shown. Comparison of these wave forms and thresholds to a previous study demonstrated no changes in these parameters before or after the surgery, confirming that posterior tympanotomy is a safe and useful procedure for drug administration to the round window niche of the common marmoset.Once mastered, this technique can be completed in an hour, including the anesthesia and induction, if it's performed properly. While attempting this procedure, it's important to remember not to damage the ossicle or the facial knob which can result in conductive hearing loss or facial paralysis respectively. After its development, this technique paved the way for researchers in the field of the translation research to test potential new therapies for hearing loss.After watching this video, you should have a good understanding of how to approach the inner ear of the common marmoset safely and to administer the drug into round window niche properly.

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Research

JoVE Journal

Medicine

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic effects of Zadi-5 against depression have been indicated in previously reported clinical studies, the identity and impact of the active pharmaceutical compounds present in the drug have not been fully elucidated.&#160;This study used network pharmacology to predict the drug composition and identify the therapeutically active compounds in Zadi-5 pills. Here, we established a rat model of chronic unpredicted mild stress (CUMS) and conducted an open field test (OFT), Morris water maze (MWM) analysis, and sucrose consumption test (SCT) to investigate the potential therapeutic efficacy of Zadi-5 in depression. This study aimed to demonstrate Zadi-5&#39;s therapeutic effects for depression and predict the critical pathway of the action of Zadi-5 against the disorder. The vertical and horizontal scores (OFT), SCT, and zone crossing numbers of the fluoxetine (positive control) and Zadi-5 groups were significantly higher (P &#60; 0.05) than those of the CUMS group rats without treatment. According to the results of network pharmacology analysis, the PI3K-AKT pathway was found to be essential for the antidepressant effect of Zadi-5.

The present protocol describes a method for the behavioral test validation and bioinformatical prediction of the therapeutic efficacy of Zadi-5, a traditional Mongolian medicine, in depression.

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic ...