Name: air-inflation of murine lungs with vascular perfusion-fixation
Uploaded: 2021-02-02T14:00:00
Description: Watch this Scientific Journal Video about Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation at JoVE.com

This work was funded by the National Heart, Lung, and Blood Institute (NHLBI) grants HL140039 and HL130938. The authors would like to thank Shannon Hott and Jazalle McClendon for their technical expertise.

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of National Jewish Health.
NOTE: The protocol is organized into three components. The first component details the construction of the air-inflation with perfusion/fixation equipment. A second section describes how to set up the equipment for an experiment. The final section describes how to prepare the animal and perform the experiment.
1. Construction ...

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Although commonly used, intratracheal-based fixation methods displace leukocytes from the airways and can alter normal lung architecture. The method of air-inflation with vascular perfusion-fixation that is provided in this protocol overcomes these pitfalls and more accurately preserves lung anatomy. The keys to obtaining high-quality tissue from the vascular perfusion-fixation method include careful monitoring of air-inflation pressures, avoidance of air leaks, and ensuring adequate perfusion of fixative into the vascul...

Lung histology represents the gold standard for assessing lung architecture during health and disease and is one of the most commonly used tools by pulmonary researchers1. One of the most critical aspects of this technique is the proper isolation and preservation of lung tissue, since variability in this step can lead to poor tissue quality and erroneous results1,2,3. In living animals, lung volume is determined by the balance between inward elastic recoil of the lung and outward forces transmitted from the chest wall and diaphragm by surface tension. Accordingly, when the thorax is entered, outward forces are lost and the lung collapses. Histologic sections prepared from collapsed lungs have a crowded appearance and boundaries between anatomic compartments (i.e., airspaces, vasculature, and interstitium) can be difficult to distinguish. To circumvent this challenge, researchers often inflate the lungs during chemical fixation so that airspace size and architecture is maintained.
Lungs can be inflated with air or liquid. The pressure necessary to inflate the lungs to the same volume differs between air- and liquid-inflation due to intermolecular forces at the air-liquid interface. Higher pressure (e.g., 25 cmH2O) is required during air-inflation than liquid inflation (e.g., 12 cmH2O) to overcome surface tension and open the collapsed alveoli4. Once alveoli have been recruited, a lower pressure can keep the alveoli open to the same volume as the pressure-volume curve plateaus, and pressures equalize throughout the lung according to Pascal&#39;s law4,5,6,7,8.
Two main methods of lung inflation and fixation exist to preserve murine lungs for histology. Most commonly, the airspaces are instilled with liquid - often containing a fixative. The main advantage of this approach is that it is relatively easy and requires little training. While intratracheal instillation of fixative may be preferred in studies that focus on the vasculature, liquid that is instilled via the trachea tends to push proximal airway cells and mucins into more distal airspace regions while air inflation does not1,3,4,9,10,11. Moreover, inadvertent detachment of leukocytes from the epithelium during liquid inflation alters their morphology, artifactually giving them a simple, rounded appearance4,10,11,12. Finally, inflation of the lungs with liquid can unintentionally compress the interstitium4,10,11. Together, these factors can distort the normal anatomy and cellular distributions within the preserved lungs, thus limiting the technique.
An alternative method of tissue preservation is vascular perfusion-fixation. In this method, fixative is perfused into the pulmonary vasculature via the vena cava or the right ventricle. This method preserves the location and morphology of cells in the airspace lumen. However, unless the lungs are inflated during perfusion-fixation, the lung tissue is likely to collapse.
Air-inflation with vascular perfusion-fixation harnesses strengths from each of the above fixation techniques. Herein we provide a protocol for this technique. The materials and equipment that are required are relatively inexpensive and can be easily obtained and assembled. The completed setup, shown in Figure 1A, provides constant airway pressure to the lungs by way of an adjustable, fluid-filled column while a peristaltic pump delivers fixative via the right ventricle. Lungs with preserved morphology can then be further processed for structure-function analyses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>00117XF-Stopcock 1 way 100/PK M Luer</td>
			<td>Cole-Parmer</td>
			<td>Mfr # VPB1000050N &#8211; Item # EW-00117-XF</td>
			<td>Stopcock</td>
		</tr>
		<tr>
			<td>BD 60 mL syringe, slip tip</td>
			<td>BD</td>
			<td>309654</td>
			<td>Syringe used to construct the water column</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 25G x 5/8</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td>Needle for vascular perfusion/fixation</td>
		</tr>
		<tr>
			<td>Female Luer Thread Style Panel Mount 1/4-28 UNF to Male Luer</td>
			<td>Nordson Medical</td>
			<td>FTLLBMLRL-1</td>
			<td>Female Luer</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td>Heparin solution.</td>
		</tr>
		<tr>
			<td>Luer-Stub Adapter BD Intramedic 20 Gauge</td>
			<td>BD Biosciences</td>
			<td>427564</td>
			<td>Luer-Stub Adapter</td>
		</tr>
		<tr>
			<td>Male Luer (2) to Female Luer Thread Style Tee</td>
			<td>Nordson Medical</td>
			<td>LT787-9</td>
			<td>Male Luer</td>
		</tr>
		<tr>
			<td>Nalgene 180 Clear Plastic PVC Tubing</td>
			<td>ThermoFisher Scientific</td>
			<td>8000-9020</td>
			<td>Tubing</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Aqueous Solution - 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Fixative solution. Diluted to 4% with phosphate buffered saline</td>
		</tr>
		<tr>
			<td>Permatex Ultra Blue Multipurpose RTV Silicone Gasket Maker</td>
			<td>Permatex</td>
			<td>81724</td>
			<td>Silicone Gasket Maker for air-tight sealing of chambers</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, 1x Without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Bottle used to construct the air-inflation chamber, and buffer used for heparin and fixative solutions</td>
		</tr>
		<tr>
			<td>Sterilite Ultra Seal 16.0 cup rectangle food storage container</td>
			<td>Sterilite</td>
			<td>0342</td>
			<td>Animal processing container</td>
		</tr>
	</tbody>
</table>

air-inflation of murine lungs with vascular perfusion-fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

In an intact thorax, the lungs are held open by outward forces applied by the chest wall via the pleural space6,14. When the diaphragm is entered during dissection, the integrity of the pleural space is abolished and the lungs should collapse (Figure 2A, 2B). To re-expand the lungs, air inflation is performed. As a first step, 25 cm of water pressure is applied to ensure recruitment of collapsed airspaces. Accordingl...

Watch this Scientific Journal Video about Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation at JoVE.com

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...

This is an inexpensive method that combines the advantages of air inflation and vascular perfusion fixation for preserving lungs for structural and functional analyses. The main advantage of this technique is the preservation of both cell morphology and location within the airspaces of the lung. Care should be taken while placing the lure stub adapter into the trachea and the perfusion needle into the right ventricle to ensure adequate inflation and fixation.To set up the air-inflation apparatus, place a syringe for the water column into a ring holder and measure a vertical height of 25 centimeters from the animal platform to 25 centimeters on the water column. Attach to the end of the water column tube to the stopcock on the air chamber and attach a tube from the female lure of the air chamber to the stopcock on the animal processing container. Confirm that the cap to the air chamber and the stopcock on the outside of the animal processing container are closed, and that the stopcock on the tubing leading from the water column to the air inflation chamber is open.Then fill the syringe with water to the 25 centimeter mark. Water will flow through the syringe and tubing into the air chamber. Once the pressure is equalized, the water will stop flowing.To prepare the lungs for inflation make one lateral incision in the abdominal wall below the rib cage of the euthanized mouse, and a second lateral abdominal wall incision above the hips. Cut along the midline from the interior incision toward the superior incision, and to use blunt scissors to carefully make an incision into the lateral side of the diaphragm. The lungs should collapse as soon as the diaphragm is punctured.Cut transversely along the diaphragm to open the thoracic cavity and cut superiorly along the sternum from the xiphoid process to the juggler notch, and laterally above the rib cage to fully expose the heart and lungs. Pin down the sides of the rib cage and make a midline incision in the neck, above the trachea. Remove the skin, muscle, thyroid gland, and connective tissue surrounding the trachea and use curved forceps to slide two pieces of suture under the posterior trachea.Use one piece of suture to hold the inflation lure stub adapter in place and use an 18 gauge by one inch needle to make a small hole in the trachea. Insert a 20 gauge lure stub adapter into the hole and tie the suture around the trachea immediately distal to where the lure stub adopter enters the hole. Then transfer the animal to the animal processing container and attach the lure stub adapter to the female lure on the inside of the animal processing container.To inflate the lungs, place the 25 gauge by 5/8th inch needle attached to the perfusion apparatus tubing into the right ventricle of the heart and cut the abdominal aorta to allow blood to drain from the heart and to promote the flow of perfusate through the lungs. Open the stopcock on the outside of the animal processing container and inflate the lungs at 25 centimeters of water for five minutes, while taking care that the water level in the syringe does not decrease too quickly. During the last minute of the lung inflation, turn on the peristaltic pump to a flow rate of 10 milliliters per minute.Heparin solution should flow from the bottle through the tubing into the animal. After inflating the lungs for five minutes, turn off the pump and switch the perfusate from heparin to fixative. Lower the water column syringe to 20 centimeters.It's normal for air bubbles to move within the water column as the pressure changes. Check the water level in the syringe. It should be at the 25 centimeter mark.Then wait one minute to allow the lungs to deflate from 25 to 20 centimeters of water pressure before restarting the pump at a 6.5 milliliter per minute flow rate for 10 to 15 minutes, vascular fixative perfusion. For extraction of the inflated and fixed lung tissue, tightly tie the second piece of thread around the trachea, distal to the lure stub adapter, and remove the lure stub adapter from the trachea. Remove the needle from the heart and use blunt scissors to cut the connective tissue, posterior to the mediastinum, to free the lungs and heart from the thoracic cavity taking care to avoid puncturing the lungs.Carefully remove the heart from the lungs and place the lungs into 20 to 25 milliliters of fixative in a 50 milliliter conical tube. Pull the suture holding the trachea through the opening in the conical tube and secure the suture by the threads of the cap. Then invert the tube to ensure that the buoyant, air-inflated lungs remain fully submerged in fixative and process the lungs for histologic studies according to standard protocols.When the diaphragm is entered during dissection, the integrity of the pleural space will be abolished and the lungs should collapse. Upon the application of 25 centimeters of water pressure, air will enter the lungs via the trachea and inflation should be easily observed. Once the lungs have fully expanded, the inflation pressure can be decreased to 20 centimeters to keep the lungs inflated without overdistension.The lungs should remain inflated after tracheal ligation and removal from the thorax. Deflation can occur if the lungs are punctured during the animal preparation or lung extraction. Histological analysis of inflated lung sections reveals that very few immune cells are present in the airway lumen of tissues fixed using traditional liquid-based inflation.In contrast, inflammatory cells are preserved within the airspaces of tissues fixed via vascular perfusion with air inflation. Following air inflation with profusion fixation, lung tissue may be embedded in formalin for tissue sectioning and subsequent analysis via histology techniques, including staining, immunohistochemistry, and immunofluorescent imaging.

air-inflation-of-murine-lungs-with-vascular-perfusion-fixation

Research

JoVE Journal

Neuroscience

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...