腦垂體pituitary巨噬細(xì)胞清除Macrophage depletion方案

垂體（pituitary）是下丘腦-垂體-腎上腺 （HPA） 軸的重要樞紐。垂體激素分泌細(xì)胞 （HPC） 釋放多種激素，以在正常和壓力條件下調(diào)節(jié)基本身體功能。垂體內(nèi)分泌腺通過釋放促腎上腺皮質(zhì)激素 （ACTH） 來調(diào)節(jié)免疫系統(tǒng)，以響應(yīng)下丘腦的神經(jīng)元激活。然而，目前尚不清楚全身炎癥如何調(diào)節(jié)垂體HPC的轉(zhuǎn)錄組學(xué)特征。在這里，我們對小鼠垂體進(jìn)行了單細(xì)胞RNA測序（scRNA-seq），發(fā)現(xiàn)在炎癥發(fā)生時，所有主要的垂體HPC都以細(xì)胞類型特異性方式產(chǎn)生強(qiáng)烈反應(yīng)，其中皮質(zhì)促物表現(xiàn)出強(qiáng)烈的反應(yīng)。全身炎癥還導(dǎo)致非典型生物活性分子的產(chǎn)生和釋放，包括皮質(zhì)激素的 Nptx2，以調(diào)節(jié)免疫穩(wěn)態(tài)。同時，HPCs上調(diào)了趨化因子的基因表達(dá)，促進(jìn)了HPCs與免疫細(xì)胞之間的通訊?？傊?，我們的研究揭示了垂體和免疫系統(tǒng)之間的廣泛相互作用，表明垂體在介導(dǎo)炎癥對身體生理學(xué)許多方面的影響方面發(fā)揮著多方面的作用。

Macrophage depletion and virus injection in the pituitary

Mice were anesthetized with pentobarbital (80 mg/kg, i.p.) before surgery and then placed in a mouse stereotaxic instrument. Injections were performed using a microsyringe pump and a Micro4 controller (World Precision Instruments). For macrophage depletion, liposome-PBS or liposome-Clodronate (Liposoma,CP-005-005) was stereotaxically microinjected into the anterior pituitary (2.5 mm posterior from Bregma, 0.4 mm lateral, 6 mm below pia). The liposomes were delivered to the target site at a rate of 60 nL/min for 500 nL per site. Mice received saline or 0.5 mg/kg LPS 18 h after liposome delivery and were killed 6 h after inflammation was established. For CCL2 expression and Nptx2-KO, AAV was delivered directly into the pituitary. The injection site, rate, and volume were the same as those used for the liposome injection. Subsequent experiments were performed at least 3 weeks after virus injection.

(C) Representative images showing Iba1 (the marker of macrophage) expression in the pituitary from mice that received liposome-PBS (left) or liposome-Clodronate (right) directly to the pituitary for 24 h. Scale bar, 100 μm.

(D) Serum concentrations of ACTH in saline- or LPS-treated (0.5 mg/kg LPS for 6 h) mice pretreated with liposome-PBS or liposome-Clodronate (n = 4–7 mice).

(E) Serum concentrations of corticosterone in LPS-treated (0.5 mg/kg LPS for 6 h) mice pretreated with liposome-PBS or liposome-Clodronate (n = 6–7 mice).