This work was supported in part through the National Institutes of Health (DK097335 &#38; T32AI007260) and the University of Iowa Stead Family Department of Pediatrics.

All animal procedures were approved by the University of Iowa Institutional Animal Care and Use Committee (Protocol #8041401). All animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) approved vivarium at the University of Iowa. All mice were wild type strain C57Bl/6J.
1. Establishment of FEMI in pregnant mice
<ol>
	<li>LPS preparation
	<ol>
		<li>Use LPS derived from Escherichia coli O55:B5 (stock concentration 2 mg/mL).</li>
		<li>Dilute LPS stock concentration 1:100 with sterile saline for a working concentration of 20 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Maternal LPS injection
	<ol>
		<li>Inject pregnant dams at gestation day e15. This timepoint is approximately 75% through the murine pregnancy, making this model developmentally similar to the early third trimester of human pregnancies, which is when the majority of preterm births due to chorioamnionitis occur.</li>
		<li>Weigh pregnant mice immediately prior to injection to determine appropriate LPS dosing.</li>
		<li>Calculate the dose of working concentration by using the following formula: 5 &#181;L x gram body weight (gbw), for a total dose of LPS of 100 &#181;g/kg. For control animals, use an equivalent volume of normal saline for injection.</li>
		<li>Vortex LPS solution three times for 15 seconds on high prior to each injection.</li>
		<li>Draw up LPS volume into a 1 mL syringe.</li>
		<li>Restrain pregnant mouse with scruffing technique. Hold in dorsal recumbency position and perform the injection.
		<ol>
			<li>Insert a 30 gauge 8 mm needle bevel up overlying the right lower quadrant of the abdomen (to avoid bladder and abdominal vessels) at a 30&#8210;40&#176; angle. Insert about &#188; to &#189; the length of the needle.</li>
			<li>Pull back on the syringe plunger to ensure negative pressure prior to injecting. Proceed with injection if negative pressure is present.</li>
			<li>Following injection, monitor mice for approximately 30 min and then return to cages for the remainder of the pregnancy.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Delivery and care of offspring, and intestinal harvesting
<ol>
	<li>Deliver pups normally via vaginal delivery at e20. 
	NOTE: This model does have an expected dose-dependent fetal loss rate that can be seen in Figure 1 and is discussed in the results below.</li>
	<li>Allow pups to remain with mothers and receive ad libitum feeds.</li>
	<li>On the day of harvest, typically postnatal day 14 (P14), euthanize pups via cervical dislocation in compliance with Institutional Animal Care and Use Committee protocols.</li>
	<li>Using scissors and forceps, make a vertical incision down the midline of the abdomen, through the skin and peritoneum, for the entire length of the abdomen. Excise the small intestine from the stomach to the cecum with scissors and remove the mesentery with forceps.&#160;</li>
	<li>Isolate and keep the distal 1/3 of the small intestine (section representative of human ileum), discarding the proximal small intestine, the cecum, and the colon.</li>
	<li>Divide the ileum portion in half using scissors.</li>
	<li>Place the proximal half in an RNA stabilization solution for later RNA quantification.</li>
	<li>Place the distal half in 10% neutral buffered formalin for slide preparation.</li>
</ol>
3. Intestinal injury scoring
<ol>
	<li>Section paraffin embedded tissue into 5 &#181;m thick slices and mount on glass slides. 
	 
	NOTE: We send the specimens to a histology core for paraffin embedding, sectioning, and mounting onto slides.</li>
	<li>Deparaffinize slides according to standard procedures.</li>
	<li>Stain sections with hematoxylin and eosin according to standard procedures.</li>
	<li>Score sections on a 3-point scale for intestinal injury as previously described42,43.
	<ol>
		<li>Using light microscopy, assess generalized intestinal injury by two separate blinded investigators on a 3-point scale evaluating villus integrity and separation from the basement membrane43 (Supplementary Figure 1). Intestinal injury is best assessed at 20x magnification and numerical aperture 0.50.</li>
		<li>Assign a score of 0 to describe normal mucosa.</li>
		<li>Assign a score of 1 describe mild injury which encompasses the development of subepithelial Gruenhagen&#8217;s space, vacuolization or subepithelial lifting limited to the lamina propria or tips of villi.</li>
		<li>Assign a score of 2 to describe severe injury, indicated by epithelial lifting and vacuolization greater than half of the villi, villi distortion, or mucosal ulceration and disintegration of the lamina propria.</li>
	</ol>
	</li>
</ol>
4. Quantification of Paneth and goblet cells
<ol>
	<li>Following deparaffinization, stain slides of tissue sections from Step 2.8 with Alcian Blue/Periodic Acid Schiff stain to denote both goblet and Paneth cells as previously described44,45 according to the following steps. 
	NOTE: While Alcian Blue/Periodic Acid Schiff stain is not specific to either Paneth or goblet cells, in our experience, blinded experienced investigators have equivalent cellular quantification using this stain compared to cellular targeted antibodies, with significantly less background staining46.</li>
	<li>Deparaffinize, stain, and dehydrate slides as follows.
	<ol>
		<li>Submerge slides in xylene for 10 min twice. 
		CAUTION: Xylene should be used in a fume hood.</li>
		<li>Rinse with 100% EtOH.</li>
		<li>Submerge slides in 100 % EtOH for 3 min, then in 90% EtOH for 3 min, followed by 70% EtOH for 3 min, and lastly submerge slides in 50% EtOH for 3 min.</li>
		<li>Wash under running tap water for 5 min. 
		CAUTION: Point the section away from the running water to prevent loss of tissue sample.</li>
		<li>Filter Alcian blue stain solution with a standard coffee filter.</li>
		<li>Stain slides in Alcian blue stain for 15 min and then wash under running tap water for 2 min.</li>
		<li>Dilute 1 mg of periodic acid in 200 mL of double distilled water. Submerge slides in this solution for 5 min. Then wash under running tap water for 1 min.</li>
		<li>Stain with Schiff&#8217;s reagent for 10 min. Wash under running tap water for 5 min.</li>
		<li>Stain the slides with hematoxylin for 1 min and then wash under running tap water for 2 min.</li>
		<li>Submerge them in acid alcohol (1 mL of hydrochloric acid mixed in 99 mL of 70% EtOH) for 1 min.</li>
		<li>Submerge in Scott&#8217;s tap water (0.1% concentration of NaHCO3 in tap water) for 1 min and then wash under running tap water for 1 min.</li>
		<li>Dehydrate the slides.
		<ol>
			<li>Dip each slide 10 times in 70% EtOH, then dip 10 times in 90% EtOH, and 10 times in 100% EtOH.</li>
			<li>Submerge slides in 100% EtOH for 10 min, followed by submerging twice in fresh xylene for 3 min each.</li>
		</ol>
		</li>
		<li>Place a drop of mounting media on the specimen and place a coverslip over it.</li>
	</ol>
	</li>
	<li>Goblet cell counting
	<ol>
		<li>Using light microscopy, count goblet cells (Supplementary Figure 2). For each piece of intestinal tissue, count the number of goblet cells and 500 epithelial cells and express goblet cell ratio as a ratio per 100 epithelial cells. Goblet cells are best counted at 20x magnification and numerical aperture 0.5.</li>
	</ol>
	</li>
	<li>Paneth cell counting
	<ol>
		<li>Using light microscopy, count Paneth cells (Supplementary Figure 2). For each piece of intestinal tissue, express as a ratio of Paneth cells per intestinal crypt. Count 100 intestinal crypts per each piece of intestinal tissue. Paneth cells are best counted at 20x-60x magnification and numerical aperture 0.50-1.30.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Chorioamnionitis impacts 2&#8210;4% of term and 25&#8210;30% of preterm deliveries8,9. However, the impact of chorioamnionitis can extend long past birth as it has been shown to have significant effects on the fetus and neonate10,11,12,13,14,15,<sup class="...

The chorionic membranes play an integral role in mammalian pregnancy. They include the chorion and amnion, which serve multiple functions. They surround and protect the fetus, facilitate paracrine signaling between the maternal and fetal compartments1, and create local feedback loops within the chorionic membranes, which may be involved in initiating parturition1. Current understanding of the membranes indicates that the amnion provides structural barrier function, and the chorion provides an immunological buffer primarily to protect the developing fetus from the maternal immune system2. Inflammation of these membranes is known as chorioamnionitis. Historically, the diagnosis of clinical chorioamnionitis was made following the presence of maternal fever plus one or more fetal or maternal clinical findings3,4. However, while this definition is clinically useful, its lack of precision has made chorioamnionitis research challenging. In 2015, in an attempt to clarify the diagnosis, an expert panel workshop by the Eunice Kennedy Shriver National Institute for Child Health and Human Development defined chorioamnionitis as intrauterine inflammation, or infection, or both (triple I)3. This clarification is important because while microbial induced infection is an important cause of uterine/amniotic inflammation, it occurs less commonly than sterile uterine/amniotic inflammation5,6,7. Overall, chorioamnionitis remains a significant public health problem, as it is seen in 2&#8210;4% of term deliveries and 25&#8210;30% of preterm deliveries8,9.
Chorioamnionitis can have significant effects on the fetus and neonate. It has been well documented in the literature that chorioamnionitis is associated with increased risk of many of the morbidities of prematurity, including bronchopulmonary dysplasia10, cerebral white matter injury11, intraventricular hemorrhage12, retinopathy of prematurity13, and both suspected and confirmed early onset neonatal sepsis14,15. As we are interested in injury and repair mechanisms of the immature intestinal tract, it is important to note that chorioamnionitis is also associated with later development of necrotizing enterocolitis (NEC)15,16. NEC is a devastating gastrointestinal disease of preterm infants that results in a dysregulated host response to inflammation and subsequent intestinal necrosis17. Each year, NEC affects over 4000 infants in the United States, and up to one third of these infants die from the disease18. The pathogenesis of NEC likely involves a combination of intestinal immaturity, dysregulation of the immature immune system, intestinal inflammation, and bacterial translocation19, culminating in a final common pathway of intestinal necrosis. Importantly, the onset of NEC often occurs weeks after birth and potential exposure to chorioamnionitis, making the mechanistic link between chorioamnionitis and subsequent development of NEC unclear20. One potential mechanism by which chorioamnionitis may contribute to the pathophysiology of NEC is through upregulation of the maternal immune system, subsequently producing a strong fetal inflammatory response which may disrupt normal fetal developmental patterns21,22,23.
Multiple mammalian models of chorioamnionitis exist in rodents and sheep24,25,26,27,28,29,30,31,32. However, few data exist concerning the development of the intestinal tract beyond the initial newborn period following chorioamnionitis-induced fetal exposure to maternal inflammation (FEMI). In order to explore the relationship between FEMI and subsequent development of injury of the immature intestinal tract, we have adapted the lipopolysaccharide (LPS)-induced FEMI model. Lipopolysaccharides are a major component of the extracellular surface on gram negative bacteria and are a potent stimulant of the innate immune system of multiple eukaryotic species, including humans33. Maternal LPS injection results in a sterile inflammatory cascade without the confounding effects of live bacteria, and it is a well-established model for induction of preterm birth34, as well as a model of acute chorioamnionitis and the fetal inflammatory response syndrome (FIRS), which is the most severe form of chorioamnionitis24,35. It has also been shown to induce both cerebral white and gray matter injury in a sheep model36 and a murine model37,38,39,40. However, to our knowledge, we are the first to use this model of chorioamnionitis and FEMI to investigate its effects on the development of the gastrointestinal tract past birth, as well as to investigate a possible mechanistic link between chorioamnionitis and later development of NEC41,42.

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>Newcomer supply</td>
			<td>1003A</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl6/J mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin stain</td>
			<td>Leica</td>
			<td>381562</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>2CE-MQVJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid</td>
			<td>ACROS</td>
			<td>H5106</td>
			<td>CAS# 10450-59-9</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermofisher</td>
			<td>Am7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#39;s reagent</td>
			<td>Sigma</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Secor Imager 2400</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-Plex Assay</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

Exposure to FEMI on embryonic day 15 leads to a dose-dependent loss of pregnancy and a dose dependent rate of preterm labor (Figure 1)42. For the experiments, we chose to use an LPS dose of 100 &#181;g/kg to minimize pregnancy loss and prematurity (50% loss between both prematurity and intrauterine fetal demise) while exposing the fetuses to a significant inflammatory insult.
Using this approach, we next examined the effects of FEMI on subs...

Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

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3.7K Views.  University of Iowa. We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Video: A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

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 ...