Supported by National Institutes of Health grants R01-AA015614, R01-AA014973, T32-AA014127 and K12-GM088021. The authors thank Samantha L. Blomquist for technical assistance and Drs. Kevin Caldwell and Donald Partridge for critically evaluating the manuscript and video.

All Animal procedures were approved by the University of New Mexico-Health Sciences Center Institutional Animal Care and Use Committee.
1. Vapor Chamber Assembly
<ol>
	<li>Cut polycarbonate sheets with a circular saw or jigsaw to the dimensions provided in the video for the top, bottom, front, back, sides, and door (Figure 1 and Table 1).</li>
	<li>With a circular saw or jigsaw, cut an opening 8 inches high by 16 inches wide in the middle of the front panel.</li>
	<li>Measure and mark the holes for the piano hinge onto the 18 inch by 10 inch polycarbonate sheet that will become the door of the chamber.</li>
	<li>In the door, drill counter sink holes with a 5/16 inch drill bit, and with a 3/16 inch drill bit, drill the hole for the screws. Make sure to drill counter sink holes on the inside of the door for the screw heads.</li>
	<li>Prepare the polycarbonate sheets for welding.</li>
	<li>Assemble front, back, and side panels onto the bottom panel using Weld-on #16.</li>
	<li>Seal any gaps between panels with Weld-on #16.</li>
	<li>Attach the top panel with Weld-on #16.</li>
	<li>Use bar clamps or heavy text books to hold everything in place while the welds cure.</li>
	<li>Attach a 1 inch by a 12 inch piece of polycarbonate onto the front panel 1 inch below the opening for the attachment of the door hinge.</li>
	<li>Allow at least 24 hr&#160;for Weld-on to cure.</li>
	<li>Cut 2 pieces of PEX tubing 12 inches long and 1 piece of 1 inch long PEX tubing.</li>
	<li>With a 5/16 drill bit, drill a hole in each of the 12 inch PEX tubing approximately 1 to 2 inches from one end.</li>
	<li>Attach 2 x 12 inch PEX tubing to a 3/8 inch T-connector with the holes located away from the connector.</li>
	<li>Cap the open ends of the PEX tubing with 3/8 inch plugs.</li>
	<li>Attach the piano hinge to the front panel and door using 4-40 machine screws and nuts.</li>
	<li>Using the door as a guide, mark the holes needed for the piano hinge onto the 1 inch spacer on the front panel of the chamber.</li>
	<li>With a 3/16 inch drill bit, drill the holes into the front panel for the piano hinge.</li>
	<li>Attach the door and piano hinge to the front panel using 4-40 machine screws and nuts.</li>
	<li>Assemble the toggle clamps as shown in the video with a washer and nut on either side of the side arm.</li>
	<li>Mark and drill 3/16 inch holes for the toggle clamps on the front panel and attach toggle clamps with 4-40 machine screws and nuts.</li>
	<li>Add 3/8 inch rubber bulb seals to the inside of the door.</li>
	<li>With a 5/8 inch boring drill bit, drill a hole in the center of the top panel for the inlet port.</li>
	<li>Drill a 1/2 inch hole in bottom / center of the back panel for the exit port.</li>
	<li>Assemble the exit port by pushing the threaded part of the 3/8 inch through-wall adaptors through the back wall of the vapor chamber in the exit port hole. Attach the nut from the inside of the chamber to hold in place.</li>
	<li>Remove the protective covering from polycarbonate sheets.</li>
	<li>Attach the 1 inch piece of PEX tubing to the T-connector and force the PEX tubing through the hole in the top of the chamber from the inside.</li>
	<li>Attach a 3/8 inch 90 elbow to the top of the 1 inch PEX tubing from the outside. 
	NOTE: Drill a 1/2 inch hole in the door and insert a 1/2 inch seal septa into the hole.</li>
	<li>Repeat steps 1-22 for the air only control chamber.</li>
</ol>
2. Rack and Air Delivery Assembly
<ol>
	<li>If placing the chambers on the cart/rack mentioned in the material list, assemble the cart according to the manufacturer&#8217;s instructions.</li>
	<li>With spare nuts and bolts, attach a scrap piece of polycarbonate to the rack to hold the airflow regulators.</li>
	<li>Anywhere on the scrap piece of polycarbonate, mark and drill 3/4 inch holes for the airflow regulators and attach regulators with the nuts provided.</li>
	<li>Attach 3/8 inch through-wall adapter to the inlet and outlet ports of the air flow regulators.</li>
	<li>Assemble the ethanol flask with the aeration stone, #8 stopper and quick release in-line connectors.</li>
	<li>Using 3/8 inch TYGON tubing, connect the flow regulators to the air pump and ethanol flask as shown in Figure 1.</li>
	<li>For the air only chamber, attach the exit port of the airflow regulator to the inlet 3/8 inch 90 elbow with 3/8 inch TYGON tubing as shown in Figure 1.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51839/51839fig1highres.jpg" width="500" /> 
Figure 1. Schematic representation of the configuration of the vapor chambers. A T-connector is attached to the low noise air pump. One side of the T-connector is directly connected to an airflow regulator for the air only vapor chamber. The other side is split again and attached to 2 different airflow regulators, one for air and one for ethanol. The air chamber is directly connected to the regulator as shown. For the ethanol vapor chamber, one airflow regulator is connected to the aerator stone immersed in liquid ethanol in the filter flask. The side arm port of the filter flask is connected with the output of the air regulator as indicated. The merged ethanol vapor and air is then connected to the inlet of the ethanol vapor chamber. The outlet tubes (not shown) are connected to a ventilation outlet in the room.
3. Test Vapor Chambers and Adjust Ethanol Levels
<ol>
	<li>Add 600 ml&#160;of 190 proof ethanol to the filter flask, insert the aeration stone, and hook up to inlet tube to the side arm of the flask.</li>
	<li>Close chamber doors and turn on air pump.</li>
	<li>Adjust airflow regulators so that approximately half as much air flows through the liquid alcohol as the air it is mixed with. Adjust the air only airflow to the combined flow of the alcohol and air of the ethanol chamber.</li>
	<li>Allow chambers to equilibrate for at least 30 min&#160;before measuring the air ethanol concentration.</li>
	<li>Measure air ethanol concentration by extracting 5 ml&#160;of air with an 18G&#160;needle and a 60 ml&#160;syringe through the septum. Dilute that sample with room air by drawing the plunger back to 60 ml&#160;(1:12 dilution). Measure air ethanol level using a breathalyzer in accordance to manufacturer&#8217;s directions. The dilution of the chamber air is needed to achieve alcohol vapor levels within the detection range of the breathalyzer. 
	NOTE: If using different levels of alcohol exposure the dilution may need to be adjusted.</li>
	<li>As a starting point, adjust the airflow regulators as needed to achieve an air alcohol concentration of approximately 4.5-5 g/dl&#160;(g of vaporized alcohol per dl&#160;of air).</li>
</ol>
4. Animal Breeding
<ol>
	<li>Group house females C578BL/6 mice (2-3 months old) for at least 1 week to synchronize ovarian cycles.</li>
	<li>Individually house male C57BL/6 mice (2-5 months old) for at least 2 weeks.</li>
	<li>After synchronizing, put a single female in with a single male for 5 days to allow for mating.</li>
	<li>Following mating, remove males and individually house females and place them into chambers.</li>
</ol>
5. Pre- and Post-natal Ethanol Vapor Exposure
<ol>
	<li>Expose pregnant dams to ethanol vapor for 4 hr&#160;per day starting at 10 am during the light cycle (lights on from 6 am-6 pm) except the day of birth to prevent pup death.</li>
	<li>Weigh dams on gestational day (G) 5, G13-G14, and G18-G20 to monitor pregnancies; bedding was changed on days that females were weighed to minimize handling.</li>
	<li>Each day replace the food for the ethanol exposed groups to avoid consumption of pellets with any ethanol absorbed into them.</li>
	<li>On the day of birth do not expose animals. After birth expose the dams and pups for 4 hr&#160;per day starting at 10 am from P1-P12.</li>
	<li>Weigh pups on P2, P8, P12, and P25; and change bedding on P8 and P12 to minimize additional handling.</li>
	<li>Immediately after the last exposure (P12), transfer the cages to a standard animal housing room.</li>
</ol>

The authors have nothing to disclose.

Here, we describe in detail the methods for the construction of vapor inhalation chambers. The materials and tools required to build the chambers are readily available from a number of commercial suppliers and the steps for the construction of the chambers are relatively straightforward. The system that we describe here does not contain any in-line check valves to prevent back-flow and mixing. We were unable to measure any detectable ethanol in the air only chambers suggesting that we do not have any mixing or back flow ...

Drinking during pregnancy can harm the fetus, causing persistent alterations in many organs and systems that significantly decrease the quality of life for the affected individuals and their families. It is estimated that approximately 10-30% of women drink during pregnancy in the U.S., with 1-8% drinking in a binge pattern1,2. The range of effects produced by ethanol exposure during fetal development is collectively known as fetal alcohol spectrum disorders (FASDs). Recent estimates indicate that FASDs are a major public health problem with a prevalence as high as 2-5% in the U.S.3. The more severe manifestation of FASDs is Fetal Alcohol Syndrome (FAS), which is characterized by growth retardation, craniofacial abnormalities, and neurobehavioral deficits, including learning disabilities. The prevalence of FAS has been estimated to be 0.2-0.7% in the U.S.3. The currently available treatments for FASDs are only partially effective and development of more effective treatments is limited by the poor understanding of the cellular and molecular underpinnings of this complex spectrum of disorders.
Data from the National Birth Defects Prevention Study (NBDPS) indicate that pregnant women most commonly drink during the 1st trimester, before pregnancy has been detected, followed by abstinence during later stages of gestation 2. The NBDPS also found that the second most common pattern of ethanol consumption during gestation involves drinking throughout all trimesters of pregnancy2. The reasons for this include lack of awareness about the potentially harmful effects of fetal ethanol exposure (even at low doses), limited access to prenatal care, positive history for neuropsychiatric disorders, and abuse of or dependence on ethanol4. Interestingly, the NBDPS reported that the third most common pattern of consumption involved abstinence during the 1st and 2nd trimesters followed by consumption during the 3rd trimester, when it is often assumed that drinking is safe because organogenesis has been mostly completed. However, the 3rd trimester is a period of high susceptibility to ethanol-induced nervous system damage because this is a period when neuronal circuits undergo profound refinement2. The NBDPS also identified other, less frequent patterns of alcohol consumption that occur during pregnancy, including consumption throughout the 1st and 2nd trimesters followed by abstinence during the 3rd trimester2.
In an attempt to model the different patterns of ethanol consumption observed in pregnant women, a number of developmental ethanol exposure paradigms have been established using diverse animal species, with rats and mice being most common5,6. The duration of pregnancy in these animals typically lasts approximately 3 weeks, which corresponds to the 1st and 2nd trimesters of human pregnancy. Many rodent studies have assessed the impact of various doses and patterns of ethanol exposure during this period. Examples of the methods frequently used to administer ethanol to pregnant mice and rats include administration via liquid diets7,8, addition of ethanol to the drinking water9,10, voluntary drinking of saccharin-sweetened solutions11, gastric gavage12, vapor inhalation13, and subcutaneous or intraperitoneal injection14. Results of these studies have recapitulated several of the deficits observed in humans with FASDs, demonstrating that exposure during early stages of pregnancy is sufficient to damage neuronal circuits across the brain (reviewed in 6,15).
Experiments with rodents have also demonstrated that exposure during the equivalent to the 3rd trimester of human pregnancy, which approximately corresponds to the first 1-2 weeks of postnatal life in rats and mice, can significantly impair brain development. Exposure during this period has been modeled by administering ethanol to neonatal rats or mice. Ethanol has been administered to these animals using a variety of methods, including feeding via gastrostomy in artificially-reared animals16-18, intragastric intubation19, subcutaneous injection20, and vapor inhalation21,22. These studies have convincingly demonstrated that the brain growth spurt is a period of high vulnerability to the developmental effects of ethanol6.
As mentioned above, drinking during all trimesters of pregnancy is a common pattern of ethanol consumption in women2. However, comparatively few studies have assessed the impact of this pattern of exposure using animal models. Some of these studies have taken advantage of large animals where the 3rd trimester-equivalent occurs in utero rather than the neonatal period as in the case of rats and mice. These animal models include non-human primates23,24 and sheep25-27. However, these animal models have not been widely used in FASDs research, in part, because of high cost and the need for specialized care facilities. Rodents have been more commonly used to characterize the effect of all-trimester ethanol exposure on fetal development5. Guinea pigs have been particularly advantageous in this regard given their extensive prenatal development and similarities in brain maturation to that of humans28,29. With guinea pigs, it has been possible to characterize the effect of ethanol exposure in utero that includes the equivalent development period of the human 3rd trimester. The comparatively high cost of these animals, as well as the relatively long duration of their pregnancy (~67 days), has limited their use to a few laboratories working on FASDs research.
Because of their cost-effectiveness and wide use in biomedical research, investigators have used rats to model exposure to ethanol during all trimesters of pregnancy. In initial studies, rats were exposed during pregnancy via liquid diets followed by administration of ethanol via gastrostomy to artificially reared neonates (postnatal days (P) 1-10) resulting in peak blood ethanol levels (BEC) in the dams of 0.08 g/dl&#160;and in the pups 0.16 g/dl. This paradigm caused long-lasting alterations in optic nerve myelination and reduced the number of Bergmann glia fibers in the cerebellum30-32. Similarly, Maier and collaborators using artificial rearing conditions administered ethanol to pregnant rat dams in a binge-like manner via intragastric intubation followed by neonatal administration during part of the 3rd trimester equivalent (P4-9)33,34. Peak maternal and pup BECs were 0.3 g/dl at both gestational day 20 and P6. This all-trimester exposure paradigm resulted in growth retardation that was significantly greater than that observed in pups exposed during selected periods of gestation33. In addition, rats exposed to ethanol during the equivalent to all trimesters exhibited a reduction in the number of cerebellar Purkinje and granule cells that was greater than that observed in animals exposed during other periods34. Reductions in hippocampal cell numbers were also reported with this paradigm, but these effects appear to be primarily a consequence of exposure during the 3rd trimester-equivalent35. A method that involves ethanol administration via intragastric gavage to both pregnant rats and neonatal mice has also been used to model all trimester exposure36. This method, which yielded BECs of 0.13 g/dl in the dams (gestational day 17) and 0.24 g/dl in P6 pups, induced long-lasting alterations in monoamine neurotransmitter levels in the hippocampus and hypothalamus, and increased expression of DNA methyltransferases and methyl CpG binding protein 2 in the hippocampus37,38. Using a similar exposure paradigm (BEC = 0.14-0.2 g/dl in dams and 0.2 g/dl in pups), Gil-Mohapel et al.39 detected an increase in the number of new immature neurons in the dentate gyrus of adult rats that may represent a compensatory mechanism to ethanol-induced neuronal damage or an alteration in the maturation of adult-born neurons. Investigators have also attempted to model all trimester ethanol exposure by exposing dams via liquid diets or drinking water during both pregnancy and lactation9,40. However, the utility of exposing the pups via their mother&#8217;s milk is limited because it typically results in low pup BECs (e.g., 0.002-0.05 g/dl; 41,42).
Mice have also been used extensively to characterize the effects of developmental ethanol exposure. This animal model shares many of the strengths described above for the rat animal model, with the additional advantage that many genetically modified mouse strains are available5. Mice have been successfully used to characterize the effects of ethanol during the 1st, 2nd or 3rd trimesters of pregnancy43,44. However, the impact of all trimester exposure on these animals has not been well characterized because it is technically more difficult to expose mice during the equivalent to all trimesters of human pregnancy. For instance, artificial rearing and gastric gavage, which have been used successfully in rats, require more specialized procedures in mice45. To the best of our knowledge, only one study to date has attempted to study the effect of all trimester ethanol exposure using mice; these animals were exposed to ethanol solution in drinking water during pregnancy and lactation46. Maternal BECs were 0.07 g/dl and pup BECs were not determined, but expected to be a fraction of those in dams.
Here, we describe a new model for all-trimester ethanol exposure of mice where alcohol is administered to both pregnant dams and neonates via vapor inhalation chambers. Vapor chambers were built based on a previous design47. We provide detailed instructions on how to build the inhalation chambers and carry out the exposure procedures. We also provide information on the BECs that can be achieved and the impact of exposure on pup survival and growth.

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construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

Figure 2A shows that both pregnant mice and neonatal offspring were exposed to relatively stable ethanol vapor concentrations in the chambers. These ranged between 4-6 g/dl. Figure 2B shows the BECs achieved in the pregnant mice as a function of time. BECs were measured using a standard alcohol dehydrogenase based assay48. At G5, BECs quickly rose to ~60 mM 2 hr after the start of exposure and peaked at the end of the 4 hr exposure period. BECs gradually decreased to ~12 mM af...

Watch this Scientific Journal Video about Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development at JoVE.com

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

construction-vapor-chambers-used-to-expose-mice-to-alcohol-during

Research

JoVE Journal

Medicine

14.6K Views.  University of New Mexico Health Sciences Center. We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3 trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Video: Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

Psychophysical Tracking Method to Assess Taste Detection Thresholds in Children, Adolescents, and Adults: The Taste Detection Threshold (TDT) Test

This paper describes a two-alternative, forced-choice, staircase, tracking procedure, called the Taste Detection Threshold (TDT) test, that provides a reliable measure of sweet, salty, and umami taste detection thresholds from childhood to adulthood. Advantages of the method include procedures that are identical for children and adults, thus allowing the determination of age-related and individual differences in taste perception, if any, and tasks that can be completed in a relatively short time frame, do not rely on continuous attention or require memorization, control for subjective response biases, and minimize the impact of language development. After a 1 hour fast, participants are presented with pairs of solutions; in each pair, one solution is water, and the other solution contains varying concentrations of the tastant.
Using a whole-mouth tasting method, participants taste each solution (without swallowing and with rinsing between tastings) and then point to the solution with a taste or that tastes different from water. The concentration of the stimulus in the subsequent pair increases after a single incorrect response and decreases after two consecutive correct responses. A reversal occurs when the concentration sequence changes direction. The task is deemed completed after the occurrence of four reversals, provided there are a maximum of two dilution steps between two successive reversals, and the series of reversals do not form an ascending pattern. These additional criteria ensure greater reliability in outcomes. The TDT is then calculated as the geometric mean of the concentrations of the four reversals. This method has real-world relevance as it provides information on a dimension of taste perception that is independent of hedonics, and that can change with aging and certain disease states, making it a valuable psychophysical test.

Psychophysical Tracking Method to Assess Taste Detection Thresholds in Children, Adolescents, and Adults: The Taste Detection Threshold TDT Test

Psychophysical tools measure the functionality of the taste system for both research and health assessment purposes. This paper describes a method to measure taste detection thresholds that can determine the lowest concentration of sucrose, sodium chloride, or monosodium glutamate that can be tasted by individuals as young as 6 years.

This paper describes a two-alternative, forced-choice, staircase, tracking procedure, called the Taste Detection Threshold (TDT) test, that provides a ...

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Enhancing Stroke Rehabilitation with Virtual Reality-Based Occupational Training

Cognitive Function and Upper Limb Rehabilitation Training Post-Stroke Using a Digital Occupational Training System

Stroke rehabilitation often requires frequent and intensive therapy to improve functional recovery. Virtual reality (VR) technology has shown the potential to meet these demands by providing engaging and motivating therapy options. The digital occupational training system is a VR application that utilizes cutting-edge technologies, including multi-touch screens, virtual reality, and human-computer interaction, to offer diverse training techniques for advanced cognitive capacity and hand-eye coordination abilities. The objective of this study was to assess the effectiveness of this program in enhancing cognitive function and upper extremity rehabilitation in stroke patients. The training and assessment consist of five cognitive modules covering perception, attention, memory, logical reasoning, and calculation, along with hand-eye coordination training. This research indicates that after eight weeks of training, the digital occupational training system can significantly improve cognitive function, daily living skills, attention, and self-care abilities in stroke patients. This software can be employed as a time-saving and clinically effective rehabilitation aid to complement traditional one-on-one occupational therapy sessions. In summary, the digital occupational training system shows promise and offers potential financial benefits as a tool to support the functional recovery of stroke patients.

Author Spotlight: Rehabilitation of Stroke Patients With a Digital Occupational Training System

The current protocol outlines how the VR-based digital occupational training system enhances the rehabilitation of patients with cognitive impairment and upper limb dysfunction following a stroke.