The authors would like to thank the researchers belonging to GRIVAS Health for their valuable input. Also, midwives and clinical staff from the Obstetrics and Gynecology Service belong to the Hospital de Chillan, Chile. Founded by Fondecyt Regular 1200250.

The research was carried out following the principles expressed in the Declaration of Helsinki and under the authorization of the respective Ethical Review Boards. All human participants gave their informed consent before sample collection, as reported previously25. Additionally, the Bioethics and Biosafety Committee of the B&#237;o-B&#237;o University approved this project (Fondecyt grant 1200250). The animal work was conducted in accordance with the cardinal principles of the three R&#39;s in the use of animals in experimentation35, and according to the recommendations of the guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health. Animals were kept in appropriate environments at the Vivarium of the University of B&#237;o-B&#237;o. Fresh placentas (n = 4) were obtained within 1 h after elective cesarean section from mothers (ages 28-31 years old) with normal pregnancies at term (38 to 41 weeks of gestation). Cesarean sections were performed at the Herminda Martin Clinical Hospital, Chillan, Chile, as previously reported25. To apply the in vivo model, 4-6-month-old female nonpregnant mice (strain C57BLACK/6) were used. They were divided into three experimental groups: (1) control (without treatment), (2) treated with sEVs from normoxia (sEVs-Nor), and (3) treated with sEVs from hypoxic cultured placentas (sEVs-Hyp), which were used to evaluate the disruption of the BBB in vivo25. All the injected solutions were sterile. Also, the preparation of sEVs was performed in aseptic conditions and under a class II biosafety hood to avoid contamination.
1. Placental culture explants
<ol>
	<li>To extract explants of normal pregnancy placentas, check the method published by Miller et al. 200536.</li>
	<li>Carefully remove the clots from the basal plate of the human placenta using sterilized forceps. Extract small explants to obtain a final tissue extraction of 10 g.</li>
	<li>Ensure that placental explants are removed from the four quadrants of the maternal portion of the placenta. Ensure that devitalized tissue and calcified areas are excluded.</li>
	<li>To manipulate placental tissue, do so on ice, under sterile conditions, and within a biosafety cabinet.</li>
	<li>Wash the explants (at least three times) with copious amounts (five volumes of the explants) of cold (4 &#176;C) phosphate buffer solution (PBS 1x, pH 7.4) to remove as much blood as possible. Centrifuge (252 x g for 10 min) at room temperature between washes.</li>
	<li>Resuspend 10 g of washed explants in 20 mL of culture medium supplemented with 2% fetal bovine serum, 100 IU/mL penicillin, and 100 &#181;g/mL of streptomycin (see Table of Materials).</li>
	<li>Put the suspension in a 100 mm culture dish. Leave the explants for 2 h in an incubator at 37 &#176;C with 21% oxygen and 5% CO2.</li>
	<li>After that, rewash the explants with 1x PBS at 37 &#176;C (three times). Centrifuge (252 x g for 10 min) at room temperature between washes.</li>
	<li>Further, resuspend the tissue in 20 mL of culture medium previously depleted of nanoparticles. 
	NOTE: For nanoparticle depletion, perform ultracentrifugation (120,000 x g for 18 h at room temperature) and microfiltration (0.22 &#181;m filter).</li>
	<li>Split the resuspended explants into two cultures dishes (100 mm). Each culture dish must include 10 mL of resuspended explants.</li>
	<li>After that, place one of the dishes containing placental explants in standard culture conditions (37 &#176;C with 8% oxygen and 5% CO2), while placing the other one in a hypoxic chamber with 1% O2. 
	NOTE: (OPTIONAL) Analysis of tissue viability can be performed using the same tissue extractions and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) as described elsewhere37,38. Use total protein concentration measured in the explants homogenates to normalize MTT values.</li>
	<li>After 18 h in culture, harvest the conditioned media of the explants into a new 15 mL tube. 
	&#8203;NOTE: At this step, conditioned media can be frozen (-80 &#176;C) for an extended period (weeks).</li>
</ol>
2. Placental-derived sEVs isolation
<ol>
	<li>Isolate sEVs from the conditioned medium using the differential centrifugation and microfiltration protocol following previously published reports25,39.</li>
	<li>Perform sequential centrifugation for the harvested conditioned medium. Sequential steps of centrifugations are (1) 300 x g for 10 min, (2) 2000 x g for 10 min, (3) 10,000 x g for 30 min, and (4) 120,000 x g for 2 h. Perform the centrifugations room temperature.</li>
	<li>In all these centrifugations, using a 20-100 &#956;L pipette, carefully collect the supernatant and discard the pellet.</li>
	<li>Once done, pass the last collected supernatant through a 0.22 &#181;m filter (see Table of Materials). Subsequently, perform one additional centrifugation at 120,000 x g for 18 h at room temperature.</li>
	<li>After that, discard the supernatant while resuspending the pellet (which contains placental sEVs) in 500 &#181;L of PBS (pH 7.4) and again pass through a 0.22 &#181;m filter. Finally, perform one last centrifugation at 120,000 x g for 3 h at room temperature.</li>
	<li>Next, resuspend the pellet (which contains placental sEVs) in 500 &#181;L of PBS (pH 7.4, previously depleted of sEVs) and pass through a 0.22 &#181;m filter.</li>
	<li>Label the samples as stock of sEVs-Normoxia (sEVs-Nor) or sEVs-Hypoxia (sEVs-Hyp). 
	NOTE: Prepare 50-100 &#181;L aliquots of the isolated sEVs for further experimentation. Small EVs can be stored at -80 &#176;C for an extended period (months).</li>
	<li>Characterize the placental sEVs by size, count, and sEVs protein marker, as reported previously25. The ideal mean particle diameter of sEVs is 50-150 nm. 
	NOTE: sEVs characterization includes positive detection of CD63, Tsg101, Alix, and HSP70 (i.e., to characterize sEVs population enriched in exosomes). Also, use PLAP as a marker of placental origin25.</li>
	<li>Measure the total amount of protein in the sEVs solution using the BCA protein assay kit following the manufacturer&#39;s instructions (see Table of Materials). 
	&#8203;NOTE: Figure 1 shows an overview of the placental explant culture and extracellular vesicle isolation.</li>
</ol>
3. Mice injection
<ol>
	<li>Anesthetize the animal in an anesthetic induction chamber with 5% isoflurane distribution (see Table of Materials). Verify the level of anesthesia via a toe pinch.</li>
	<li>After ~40 s, put the mouse into the anesthesia system using a facemask and maintain an anesthesia level of 2% isoflurane.</li>
	<li>Next, position the animal on a heating platform (see Table of Materials) at a room temperature of 28 &#176;C. Keep the eyes lubricated with artificial tears.</li>
	<li>Before injecting, dilute placental sEVs (200 &#956;g of total protein) with phosphate buffer (pH, 7.4) until reaching a final volume of 70 &#181;L.</li>
	<li>Disinfect the injection area using 70% ethanol and cotton swabs. Ensure to use an insulin syringe with a 30 G needle to inject the sEVs solution. 
	NOTE: The syringe can be positioned on the heating platform for 5 min to acquire a physiological temperature.</li>
	<li>Then, inject the solution into the external jugular vein. This step needs special training. 
	NOTE: The procedure must be performed cautiously, slowly retracting the needle and gently aspirating to evidence of blood reflux.</li>
	<li>After injection, apply light pressure to the injected area for ~15 s with a dry cotton swab. After that, return the animals to their respective cages.</li>
	<li>Ensure to evaluate consciousness and behavior for 15 min. An indicator of consciousness achievement is the animal recovery of total motor activity. 
	&#8203;NOTE: Ensure to keep a warm temperature in animal&#39;s cages.</li>
</ol>
4. Rapid murine coma and behavior scale (RMCBS)
<ol>
	<li>Use the RMCBS to evaluate the neurological and well-being parameters of the animals40. 
	NOTE: RMCBS allows a fast evaluation (~ 3 min) with minimal animal stress due to operator intervention. RMCBS has ten parameters (each score is 0-2).</li>
	<li>Evaluate mouse well-being with the RMCBS scale at 0 h (before injecting sEVs) and 3 h, 6 h, 12 h, and 24 h after injection. 
	&#8203;NOTE: Perform analysis of claudin-5 (at 6 h after sEVs injection) and Evan&#39;s blue extravasation (at 24 h after sEVs injection) to evaluate the disruption of the BBB (Figure 2).</li>
</ol>
5. Analysis of Evan&#39;s blue extravasation
<ol>
	<li>After placental sEVs injection (24 h), anesthetize the mice as described in step 3.1.</li>
	<li>Prepare Evan&#39;s Blue solution (2%, diluted in phosphate buffer solution, 1x) (see Table of Materials).</li>
	<li>Using retro-orbital access25, inject Evan&#39;s Blue solution at 2 mL/kg.</li>
	<li>Allow Evan&#39;s blue solution to circulate for 20 min under maintenance of anesthesia.</li>
	<li>Next, perform intracardiac perfusion following previously described protocol41 with saline solution (~3 mL, 0.9%, w/v) to remove Evan&#39;s blue dye from the circulation. This step is mandatory. 
	NOTE: For this procedure, increase anesthesia to 5% and verify a deep plane of anesthesia (i.e., drastic reduction in respiratory rate).</li>
	<li>Expose the heart via medial thoracotomy41 (Figure 3).</li>
	<li>After that, fix the whole animal with an intracardial infusion of paraformaldehyde solution (4% in PBS, v/v). 
	NOTE: Check the rigidity of the tail for monitoring the appropriate fixation.</li>
	<li>Further, once fixation is complete, carefully extract the brain, weigh it, and photograph it41. Take pictures with the whole brain close to a ruler42.</li>
	<li>Next, put the brain in a brain mouse slicer (see Table of Materials).</li>
	<li>Dissect the brain into nine sections, crane-caudal, including the cerebellum (100 &#956;m each section).</li>
	<li>After dissection, visualize and capture images of the brain slices in a stereo-zoom microscope (see Table of Materials).</li>
	<li>Use the captured images to quantify Evan&#39;s blue extravasation using ImageJ software (see Table of Materials). 
	NOTE: To analyze the presence of Evan&#39;s blue dye, set Evan&#39;s blue values using a positive control of the brain section (from an animal control animal injected with dye but without intracardial perfusion). Evan&#39;s blue values reached from positive control are generally between 75 and 110 in the intensity histogram of the blue channel (Figure 4).</li>
	<li>Ensure that brain images are analyzed blindly by the respective experimental group to avoid observer bias. Alternatively, quantify Evan&#39;s blue extravasation by brain homogenization followed by spectroscopy43. 
	NOTE: In separate experiments, using freshly isolated brains from sEVs injected mice (6 h), analyze the protein levels of claudin 5 (CLND-5) (see Table of Materials), a critical tight junction involved in the tightness of the BBB44 (Figure 5).</li>
</ol>

Evaluating Murine BBB Disruption Induced by Hypoxic Human Placenta-Derived sEVs

The authors do not have any conflict of interest to declare.

This study unveils fresh insights into potential harm resulting from sEVs isolated from placental explants cultured in hypoxic conditions on the disruption of the rodent blood-brain barrier. The pathological mechanism involves a reduction in CLND-5 in the posterior brain region25.
Prior investigations have revealed that plasma-sEVs from individuals with preeclampsia induce endothelial dysfunction in various organs using in vitro models46...

Approximately 70% of maternal deaths due to preeclampsia, a hypertensive pregnancy syndrome characterized by impaired placentation processes, maternal systemic endothelial dysfunction, and, in severe cases, multi-organ failure1,2, are associated with acute cerebrovascular complications3,4. Most maternal deaths occur in low and middle-income countries5. However, the underlying mechanisms are still unclear despite the clinical and epidemiological relevance of cerebrovascular complications associated with preeclampsia.
On the other hand, extracellular vesicles (EVs) (diameter ~30-400 nm) are essential mediators of intercellular communication among tissues and organs, including maternal-placental interaction6. In addition to proteins and lipids on the external surface, EVs carry cargo within (proteins, RNA, and lipids). EVs can be categorized into (1) exosomes (diameter ~50-150 nm, also named small EVs (sEVs)), (2) medium/large EVs, and (3) apoptotic bodies, which differ by size, biogenesis, content, and potential signaling function. The composition of EVs is determined by the cells from which they originate and the disease type7. Syncytiotrophoblast-derived EVs express placental alkaline phosphatase (PLAP)8,9, which detects placentae-derived circulating small EVs (PDsEVs) in pregnancy. Also, PLAP helps discern changes in the PDsEVs cargo and their effects in preeclampsia versus normotensive pregnancies10,11,12,13,14,15.
The placenta has been recognized as the necessary component in the pathophysiology of preeclampsia16 or cerebral complications associated with this disease17,18,19. However, how this distant organ might induce alterations in brain circulation is unknown. Since sEVs play pivotal roles in cell-to-cell communication due to their capacity to transfer bioactive components from donor to recipient cells6,20,21, a growing number of studies have associated placental sEVs with the generation of maternal endothelial dysfunction21,22,23,24, including brain endothelial cells25,26in women with preeclampsia. Thus, the compromise of brain endothelial function may lead to disruption of the blood-brain barrier (BBB), a critical component in cerebrovascular complications associated with preeclampsia3,27.
Nevertheless, preclinical findings using rat cerebral vessels exposed to serum of women with preeclampsia28 or human brain endothelial cells exposed to plasma of women with preeclampsia29 reported that circulating factor(s) induce disruption of the BBB. Despite several candidates with the potential to harm the BBB present in the maternal circulation during preeclampsia, such as elevated levels of proinflammatory cytokines (i.e., tumor necrosis factor)18,28 or vascular regulators (i.e., vascular endothelial growth factor (VEGF))29,30,31, or oxidative molecules such as oxidized-lipoproteins (oxo-LDL)32,33, among others34, none of them establishes a direct connection between the placenta and the BBB. Recently, sEVs isolated from hypoxic placentas have shown the capacity to disrupt the BBB in nonpregnant female mice25. Since placental sEVs may carry most of the listed circulating factors with the capacity to disrupt the BBB, sEVs are considered suitable candidates to connect the injured placenta, be the carrier of harmful circulating factors, and disrupt the BBB in preeclampsia.
This protocol allows us to investigate whether sEVs isolated from placental explants cultured under hypoxic conditions can disrupt the BBB in nonpregnant female mice as a proxy for understanding the pathophysiology of cerebral complications during preeclampsia.

<table><tbody><tr><td>Adult mice brain slecer matrice 3D printed</td><td>Open access file</td><td>Adult mice</td><td>Adult mice brain slicer. Printed in PLA filament.</td></tr><tr><td>Anti &amp;beta;-Actin primary antibody</td><td>Sigma-Aldrich</td><td>Clon AC-74</td><td>Antibody for loading control (Western blot)</td></tr><tr><td>Anti-Claudin5 primary antibody</td><td>Santa cruz Biotechnology</td><td>sc-374221</td><td>Primary antibody for tight junction protein CLDN5 of mice BBB (Western blot)</td></tr><tr><td>BCA protein kit</td><td>Thermo Scientific</td><td>23225</td><td>Kit for measuring protein concentration</td></tr><tr><td>Culture media #200 500 mL</td><td>Thermo Fisher Scientific</td><td>m200500</td><td>Culture media for placental explants</td></tr><tr><td>D180 CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>RWD Life science</td><td>D180</td><td>Standard incubator to estabilize explants and culture sEVs-Nor</td></tr><tr><td>Evans blue dye&amp;nbsp; &gt; 75% 10 g</td><td>Sigma-Aldrich</td><td>E2129.10G</td><td>Dye to analize blood brain barrier disruption IN VIVO</td></tr><tr><td>Fetal bovine serum 500 mL</td><td>Thermo Fisher Scientific</td><td>16000044</td><td>Additive growth factor for culture media 200</td></tr><tr><td>Himac Ultracentrifuge CP100NX</td><td>Himac eppendorf group</td><td>5720410101</td><td>Ultracentrifuge for condicioned media &gt; 1,20,000 x g</td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Isoflurane x 100 mL</td><td>USP Baxter</td><td>212-094</td><td>Volatile inhalated anaesthesia agent for mice</td></tr><tr><td>Kit CellTiter 96 Non-radioactive&amp;nbsp;</td><td>Promega</td><td>0000105232</td><td>&lt;em&gt;In vitro&lt;/em&gt; assay for placental explants viability</td></tr><tr><td>Mouse IgG Secondary antibody</td><td>Thermo Fisher Scientific</td><td>MO 63103</td><td>Secondary antibody for CLDN5 (western blot)</td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical</td><td>90278090</td><td>Nanotracking analysis of particles from placental explants condicioned media</td></tr><tr><td>Paraformaldehide E 97% solution 500 mL</td><td>Thermo Fisher Scientific</td><td>A11313.22</td><td>Fixative solution for brain tissue slices and intracardial perfusion (once diluted)</td></tr><tr><td>PBS 1 X pH 7.4 500 mL</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>Wash solution for placenta explants</td></tr><tr><td>Peniciline-streptomicine 100x 20 mL</td><td>Thermo Fisher Scientific</td><td>10378016</td><td>Antiobiotics for placental explants culture media</td></tr><tr><td>ProOX C21 Cytocentric O&lt;sub&gt;2&lt;/sub&gt; and CO&lt;sub&gt;2&lt;/sub&gt; Subchamber Controller</td><td>BioSpherix</td><td>SCR_021131</td><td>CO&lt;sub&gt;2&lt;/sub&gt; regulator to induce Hypoxia in sealed chamber for sEVs-Hyp</td></tr><tr><td>Sodium Thiopental 1 g</td><td>Chemie</td><td>7061</td><td>humanitarian euthanasia agent</td></tr><tr><td>Somnosuite low flow anesthesia system</td><td>Kent Scientifics</td><td>SS-01</td><td>Isoflurane vaporizer for small rodents</td></tr><tr><td>Surgical Warming platform</td><td>Kent Scientifics</td><td>A41166</td><td>Warming platform for mainteinance anesthesia in mice</td></tr><tr><td>Syringe Filters, Polytetrafluoroethylene (PTFE), Hydrophobic, 0.22 &amp;micro;m, Sterile, 25 mm</td><td>Southern labware</td><td>10026</td><td>Filtration of condicioned media harvested from placental explants&amp;nbsp;</td></tr><tr><td>Tabletop High-Speed Micro Centrifuges&amp;nbsp;HITACHI himac CT15E/CT15RE</td><td>Hitachi medical systems</td><td>6020</td><td>Serial centrifugations of condicioned media &amp;lt; 1,20, 000 x g</td></tr><tr><td>Trinocular stereomicroscope transmided and reflective light 10x-160x&amp;nbsp;</td><td>Center Medical</td><td>2597</td><td>Stereomicroscope to register brain slices</td></tr></tbody></table>

disruption of the mouse blood-brain barrier by small extracellular vesicles from hypoxic human placentas

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated with preeclampsia. The underlying mechanisms of these cerebrovascular complications remain unclear. However, they are linked to placental dysfunction and blood-brain barrier (BBB) disruption. Nevertheless, the connection between these two distant organs is still being determined. Increasing evidence suggests that the placenta releases signaling molecules, including extracellular vesicles, into maternal circulation. Extracellular vesicles are categorized according to their size, with small extracellular vesicles (sEVs smaller than 200 nm in diameter) considered critical signaling particles in both physiological and pathological conditions. In preeclampsia, there is an increased number of circulating sEVs in maternal circulation, the signaling function of which is not well understood. Placental sEVs released in preeclampsia or from normal pregnancy placentas exposed to hypoxia induce brain endothelial dysfunction and disruption of the BBB. In this protocol, we assess whether sEVs isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the BBB in vivo.

This protocol evaluates the capacity of sEVs derived from placentas cultured in hypoxia to disrupt the BBB in nonpregnant mice. This method allows one to understand better the potential connection between the placenta and the brain in normal and pathological conditions. In particular, this method may constitute a proxy to analyze placental sEVs participation in the onset of cerebral complications in preeclampsia.
Contrary to mice injected with sEVs-Nor, mice injected with sEVs-Hyp show a progr...

Watch this Scientific Journal Video about Author Spotlight: Modeling an Aspect of Preeclampsia in Female Mice Using Hypoxic Human Placenta-Derived Small Extracellular Vesicles at JoVE.com

Author Spotlight: Modeling an Aspect of Preeclampsia in Female Mice Using Hypoxic Human Placenta-Derived Small Extracellular Vesicles

A protocol is presented to evaluate whether small EVs (sEVs) isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the blood-brain barrier in nonpregnant adult female mice.

Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated ...

disruption-mouse-blood-brain-barrier-small-extracellular-vesicles

Research

JoVE Journal

Medicine

753 Views.  Universidad del Bío-Bío, Chillan. Ons onderzoek richt zich op de communicatie tussen placenta en hersenen. We onderzoeken of kleine extracellulaire blaasjes de bloed-hersenbarrière kunnen verstoren, en of dat het mogelijke onderliggende mechanisme is dat geassocieerd wordt met cerebrovasculaire complicaties die worden waargenomen bij vrouwen met pre-eclampsie. We hebben eerder aangetoond dat plasma van vrouwen met pre-eclampsie de bloed-hersenbarrière kan aantasten.De aard van deze potentieel schadelijke factor die i...

Video: Auteur in de schijnwerpers: modellering van een aspect van pre-eclampsie bij vrouwelijke muizen met behulp van hypoxische menselijke placenta-afgeleide kleine extracellulaire blaasjes

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Developmental Biology

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Genetics

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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.

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 ...

Improving Ex Vivo Placental Explant Culture Using a Flow-Based Approach

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

Author Spotlight: Advancing Research Through Single Cell Sequencing and Spatial Histology in Placental Tissues 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

Immunology and Infection

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

Hyperglycemia-Enhanced Hemorrhagic Transformation in tMCAO-Induced Stroke

A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses significant limitations on the clinical application of recombinant tissue plasminogen activator (rt-PA). Unfortunately, there are currently no effective interventions available for HT in clinical practice. Therefore, there is an urgent need for stable and reliable experimental animal models to elucidate the pathogenesis of HT and develop effective intervention strategies. This study presented a protocol for establishing a mouse model of HT induced by acute hyperglycemia combined with transient focal ischemia (tMCAO). Male C57BL/6J mice were injected with 30% glucose to induce hyperglycemia and then subjected to 60 min of tMCAO with reperfusion. The infarct volume, integrity of the blood-brain barrier (BBB), and degree of intracranial hemorrhage were assessed at 24 h after MCAO. The results showed that glucose injection led to transient hyperglycemia (14.3-20.3 mmol/L), which significantly increased both the infarct volume and the incidence of HT. Hematoxylin-eosin (H&#38;E) staining indicated significant hemorrhagic lesions within the infarction zone in hyperglycemic mice. Additionally, hyperglycemic mice exhibited aggravated BBB disruption, as shown by more severe leakage of Evans blue (EB) and FITC-Dextran. In conclusion, acute hyperglycemia reliably and consistently resulted in macroscopic HT in a mouse model of tMCAO. This reproducible model offers a valuable tool for investigating the pathological mechanisms of HT and developing corresponding therapeutic interventions.

This study presents a protocol for establishing a highly reproducible animal model of hemorrhagic transformation (HT) using middle cerebral artery occlusion/reperfusion (MCAO/R) in C57BL/6 mice with acute hyperglycemia.

Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses ...

Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

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