The authors are grateful to Dan Firl, Chris Corbett and Jesse Denson, PhD. Funding was provided by NIH NINDS R01 NS060765 to SR, the P30 CoBRE Pilot Program to LJ and the Child Health Signature Program to LJ at the University of New Mexico.

Institutional Care and Use Committees at both Boston Children&#8217;s Hospital and the University of New Mexico Health Sciences Center approved all experimental procedures.
NOTE: Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgical drapes. Additionally, prepare post-operative medications in sterile vials including 0.125% bipivucaine and 0.1 mg/kg buprenorphine. Also prepare the lipopolysaccharide (LPS) solution sterilely: 0.04 mg/ml LPS (0111:B4) in sterile saline containing dilute Evan&#8217;s blue dye.
1. Anesthesia
<ol>
	<li>Induce anesthesia in an embryonic day 18 (E18) pregnant Sprague Dawley rat with a mixture of 3% isoflurane balanced 70% nitrogen and 30% oxygen.</li>
	<li>Remove rat from the induction chamber and place the rat supine on draped surgical circulating water blanket set at 37 &#176;C. Transfer anesthesia to nose cone and reduce isoflurane level to 2%.</li>
	<li>Gently apply ophthalmic ointment to each eye to prevent corneal drying. During the procedure continually monitor temperature, respiration rate and heart rate of the animal. Maternal physiology should remain stable throughout the procedure.</li>
</ol>
2. Surgical Prep and Scrub
<ol>
	<li>Using small animal clippers remove all hair in the lower abdominal region. Shave in a rectangular pattern with care to avoid nicking the nipples or generating razor rash that can be irritating for future nursing of live born pups.</li>
	<li>Prepare abdominal skin by alternating application of povidone-iodine and 70% ethanol scrub with sterile cotton swabs. Repeat the scrub such that povidone-iodine and 70% ethanol are each applied 3x in alternating fashion. Let dry.</li>
	<li>Confirm depth of anesthesia via absence of toe-pinch reflex. In the absence of reflex and stimuli to pain, reduce isoflurane level to 1%.</li>
	<li>Using sterile surgical towels, drape the animal. Take care to place the drapes at an appropriate angle such that they maximize the amount of irrigation fluid they absorb whilst not obstructing blood flow to the uterine horns.</li>
</ol>
3. Abdominal Laparotomy
<ol>
	<li>Using a scalpel make a 3 cm midline incision in the prepared abdominal skin. Bluntly dissect the skin layer from the abdominal fascia with scissors. Using forceps and surgical scissors, elevate the abdominal fascial layer and make an incision of the avascular linea alba to gain access to the peritoneal cavity.</li>
	<li>Place surgical gauze on the exterior of the incision and moisten with sterile saline. Using blunt forceps and external pressure on the abdomen, gently remove the uterine horns from the peritoneal cavity and arrange on the moistened gauze.</li>
	<li>Carefully avoid entanglement and contact with intestines. Arrange fetuses using forceps by contacting only the muscular tissue in between individual amniotic sacs. Expose and isolate the 4 uterine arteries using blunt dissection. 
	NOTE: Care must be taken to dissect the uterine arteries. Surrounding tissue and vessels themselves are extremely delicate. Damage to maternal vessels can cause bleeding, and in severe cases, fetal and maternal death.</li>
</ol>
4. Placement of Aneurysm Clips
<ol>
	<li>Place a rat 30 G aneurysm clip on each uterine artery. Ensure cessation of blood flow, including proximal and distal pulses, and darkening of the uterine vessels including individual placentas. Cover the exposed horns and entire surgical field with gauze and irrigate with sterile saline. Take care to keep the field moist with irrigation approximately every 10 min.</li>
	<li>After 60 min, remove the gauze and irrigate the field. Ensure that the uterine horns and vessels are adequately moistened for successful clip removal. Gently remove each aneurysm clip using forceps. Take care not to cause trauma to the vessel, and maintain tissue integrity during removal.</li>
	<li>Thoroughly irrigate the uterine horns and field, taking care to remove any stray threads of gauze from the amniotic sacs.</li>
</ol>
5. Injection of Lipopolysaccharide in to Amniotic Sacs
<ol>
	<li>At the base of each individual amniotic sac, just anterior to the placental plate, inject 100 &#181;l of LPS (4 &#181;g/sac) with diluted Evan&#8217;s blue dye in to the amniotic fluid. Use blunt forceps to stabilize and rotate each amniotic sac in to an optimal position for injection. Dilute Evan&#8217;s blue dye is a contrast agent that is helpful in confirming proper syringe placement and injection. 
	NOTE: Use only an ultra-fine 0.3 ml insulin syringe with attached 8 mm 31 G needle for the intra-amniotic injections. Using larger gauge needles will result in chronic amniotic fluid loss, fetal death and reabsorption of the pregnancy. Small amounts of amniotic fluid leakage upon removal of the syringe can be mitigated by direct pressure to the amniotic sac. Some rat fetuses may tolerate a degree of oligohydramnios. However, acute amniotic fluid loss from puncture with large gauge needles, or accidental puncture resulting in chronic fluid leakage, results in fetal loss and in severe cases, loss of neighboring pregnancies.</li>
	<li>Irrigate the uterine horns 3x with sterile saline.</li>
</ol>
6. Closing the Laparotomy
<ol>
	<li>Using forceps, carefully return the uterine horns to the peritoneal cavity. Ensuring adequate space between the amniotic sacs and the midline incision, re-approximate the musculofascial layer edges using a running 3-0 silk suture. Be aware of the placement of the amniotic sacs while closing the muscle incision. Be careful to not to suture into or through a sac.</li>
	<li>Re-approximate the skin layer, using a running 3-0 silk suture, closing the skin layer. 
	NOTE: The laparotomy should be closed in two layers of continuous sutures to allow for skin and muscular expansion with increasing gestation. Continuous sutures allow for evenly distributed wound tension. Interrupted sutures are less desired as multiple knots are irritating and can be easily chewed by the rat upon recovering from anesthesia. Surgical staples are not desired. Tails of the surgical knots should be cut very short (&#60;3 mm).</li>
	<li>Inject 1 ml of 0.125% bupivacaine&#160;subcutaneously around wound edges using a 26 G needle. Administer one dose of 0.1 mg/kg buprenorphine subcutaneously at the nape of the neck.</li>
	<li>Turn off isoflurane and towel dry rat as necessary. Place in clean home cage and monitor recovery from anesthesia. Ensure rat does not become hypothermic.</li>
</ol>
7. Postoperative Recovery and Care
<ol>
	<li>Monitor the rat every 8-12 hr for 72 hr and then daily until pups are born (approximately E22 or E23). Administer additional doses of buprenorphine q8-12 hr/72 hr or prn as dictated by the IACUC.</li>
	<li>Monitor rats for signs of pain, discomfort, vaginal bleeding or bleeding from the surgical site. Inspect the sutures and incision and take care to ensure rat is not chewing or removing their sutures prematurely. Although exceptionally rare, rats that compromise their sutures are at risk for wound dehiscence. 
	NOTE: Occasionally rats may ingest excessive amounts of bedding or non-food items, termed pica, as a side effect of buprenorphine administration. Although very uncommon, rats must be monitored for pica and potential subsequent bowel obstruction.</li>
</ol>
8. Tissue Processing and Cryosectioning
<ol>
	<li>To prepare for Hematoxylin &#38; Eosin (H&#38;E) staining of the postnatal brain, remove pups from their home cage on postnatal day 2 (P2). Using surgical scissors decapitate rat pups and gently remove the brain from the skull.</li>
	<li>Drop fix brain in a 15 ml conical tube containing 7 ml of 4% paraformaldehyde in phosphate buffered saline (PBS). Place brain at 4 &#176;C and fix for 72 hr.</li>
	<li>After 72 hr, transfer brains to a sterile PBS solution containing 30% sucrose (w/v) and return to 4 &#176;C. 
	NOTE: Once brains drop in the sucrose solution they are ready to be sectioned on a cryostat.</li>
	<li>Rapidly freeze brains and mount on cryostat pestle for acquisition of frozen coronal sections. Cut 20 &#181;m frozen coronal sections and mount on slides. Ensure sections are collected serially.</li>
	<li>Allow slides to dry at room temperature overnight. Store slides at -20 &#176;C.</li>
</ol>
9. Hematoxylin &#38; Eosin Staining
<ol>
	<li>Take slide mounted frozen sections and warm to room temperature. 
	NOTE: Prepare all solutions fresh.</li>
	<li>Place slides on a slide warmer set to 50 &#176;C for 2 hr.</li>
	<li>Transfer slides to a staining rack. Dip slides 10x in double-distilled deionized water (ddH2O).</li>
	<li>Incubate slides in 100% hematoxylin for 5 min. Time in hematoxylin can be optimized depending on degree of purple staining.</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Dip slides 15x in acid alcohol (250 ml 70% ethanol + 1 ml concentrated hydrochloric acid).</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Incubate in 1% lithium carbonate for 2 min.</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Incubate in 95% ethanol for 1 min.</li>
	<li>Dip slides 7x in 100% Eosin. Number of dips in Eosin can be modified depending on degree of&#160;pink staining.</li>
	<li>Dip slides 5x in 95% ethanol.</li>
	<li>Dip slide 5x in a fresh change of 95% ethanol.</li>
	<li>Incubate slides in 100% ethanol for 1 min.</li>
	<li>Incubate slides in a fresh change of 100% ethanol for 1 min.</li>
	<li>Incubate slides in 100% xylene for 15 min. 
	NOTE: Xylene steps and coverslipping should occur in a fume hood.</li>
	<li>Incubate slides in fresh changes of xylene for 15 min.</li>
	<li>Coverslip with Permount and let dry in fume hood.</li>
	<li>Image slides with a light microscope. &#160;</li>
</ol>

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The authors have nothing to disclose.

Encephalopathy of prematurity is difficult to model in animals because of the complex interaction of etiologies, neurodevelopmental time course, intricacy of human cerebral network formation, overlapping mechanisms of injury, and the diverse phenotypes of CNS insults manifest in human preterm infants. EoP is associated with specific cell-type vulnerabilities (i.e. immature oligodendrocytes)21, as well as diverse developmentally-regulated pathways (i.e. subplate, membrane transporters and rece...

With over 12% of infants born in the United States before 37 weeks estimated gestational age1, perinatal brain injury (PBI) from prematurity is a significant cause of permanent disability. PBI from prematurity, also termed encephalopathy of prematurity (EoP), affects the entire central nervous system (CNS). CNS injury often commences in utero, and is exacerbated by antenatal processes including chorioamnionitis and postnatal complications such as hypoxia and sepsis. PBI from systemic insults alters neurodevelopment and leads to cerebral palsy, epilepsy, cognitive delay and numerous neuropsychiatric disorders affecting emotional regulation, memory and executive function1,2. Although much progress has been made, a limited understanding remains of how the cellular and molecular consequences of CNS injury from preterm birth translate to the multitude of neurological sequelae in children who are born preterm. This lack of knowledge hinders real-time diagnosis of CNS injury severity and informed dosing of emerging interventions. Additionally, age-appropriate therapeutic strategies for this vulnerable patient population remain elusive.
Intrauterine inflammation is very common in extreme prematurity and involves a complex fetal-maternal-placental inflammatory cascade3. Intrauterine infection is often subclinical. Specific placental findings consistent with acute inflammation, or histologic chorioamnionitis, are major determinants of the fetal inflammatory response and are coincident with brain injury associated with preterm birth3-5. Indeed, the fetal inflammatory response has distinct clinical implications for long-term outcomes from preterm birth. Infants who are small for gestational age (SGA) or who experience infection are exceptionally vulnerable to neurological deficits3,4. Chorioamnionitis is a typical pathological diagnosis following preterm birth6,7, and histological examination reveals signs of inflammation in 70% of placentas from infants born very preterm4. Further, chorioamnionitis is associated with cognitive impairment at two years8. Evidence of maternal vascular underperfusion in the placenta of infants born extremely preterm is also associated with cerebral palsy in childhood9. The synergistic impact of chorioamnionitis and placental perfusion defects is well illustrated by the remarkably high risk of abnormal neurologic outcomes in this patient population at two years of age10,11.
To mimic human systemic placental perfusion defects and chorioamnionitis associated with pathogen-induced inflammation, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS) in rats. Our goal was to adapt our model of TSHI alone in rats12-16 to include intrauterine inflammation, to facilitate preclinical modeling of CNS injury associated with preterm birth. TSHI alone has revealed persistent loss of oligodendroglial lineage cells, cortical neurons, increased cell death, and elevated pro-inflammatory cytokine levels, with progressive ischemic intervals leading to a graded pattern of injury consistent with prenatal brain injury16. Modifications to the ischemic components of this model have also demonstrated deficits in memory encoding, short and long-term memory and mild musculoskeletal alterations in rats as they age17-19. Indeed, we have previously demonstrated that the combination of TSHI+LPS recapitulates the pathophysiological hallmarks of EoP, including oligodendrocyte and neuronal loss, axonal injury, cellular inflammation and functional abnormalities20.

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modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Following TSHI+LPS at E18, hematoxylin and eosin staining reveals significant histopathological abnormalities in both the placenta (Figure 1) and in the brain (Figure 2). Placentas examined on E19 and E21 are grossly edematous with micro-hemorrhage, and necrosis throughout the decidua and labyrinth. Significant inflammatory infiltrate and increased vascularity is also observed. Brains examined on P2 reveal ventriculomegaly, as well as white matter and subplate neuron loss compared to sha...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.2K Views.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Video: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

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Chapters in this video

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with ¹⁸F-FDG

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Modeling Chemotherapy Resistant Leukemia In Vitro