isolation of normoxic and hypoxic human placenta-derived small extracellular vesicles for injection into mice

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

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.

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

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

Our research is focused on the placenta/brain communication. We are exploring whether small extracellular vesicles can disrupt the blood-brain barrier, and if that is the potential underlying mechanism associated with cerebrovascular complication observed in women with preeclampsia. We have previously shown that plasma of women with preeclampsia can impair the blood-brain barrier.However, the nature of this potential harmful factor present in the plasma is unknown. Results from our laboratory show that extracellular vesicles derived from the plasma of women with preeclampsia, or from placentas, cultured in hypoxia, impairs the blood-brain barrier. The underlying mechanism of how placental extracellular vesicles impair the blood-brain barrier needs further investigation.That includes analysis of the content of extracellular vesicles, such as specific microRNAs that control the expression of digestion proteins in brain endothelial cells.

Watch this Scientific Journal Video about Isolation of Normoxic and Hypoxic Human Placenta-Derived Small Extracellular Vesicles for Injection into Mice at JoVE.com

Isolation of Normoxic and Hypoxic Human Placenta-Derived Small Extracellular Vesicles for Injection into Mice

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JoVE Journal

Medicine

90 조회수.  Universidad del Bío-Bío, Chillan. 멸균된 겸자를 사용하여 인간 태반 샘플의 외엽을 추출하려면 태반의 기저판에서 혈전을 조심스럽게 제거합니다. 약 10g의 최종 조직 추출이 얻어질 때까지 작은 외엽을 추출합니다. 배양 배지에서 외식을 세척하고 재현탁시킨 후, 재현탁된 외식을 두 개의 100mm 배양 접시로 나눕니다.접시 중 하나를 산소 37%와 이산화탄소 8%가 있는 섭씨 5도의 표준 정상 산소 조건에 놓?...

비디오: 마우스에 주입하기 위한 Normoxic 및 Hypoxic Human Placenta-Derived Small Extracellular Vesicles의 분리

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.

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.

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