Dec 01, 2023
BBB permeability and NDP-MSH PK study
- 1Massachusetts General Hospital
Protocol Citation: Pranay Srivastava 2023. BBB permeability and NDP-MSH PK study. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22p43l1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91667
Keywords: ASAPCRN
Abstract
This protocol is to measure the integrity of BBB and assess NDP-MSH concentrations in plasma and brain
BBB permeability
BBB permeability
The integrity of BBB was measured through FITC-albumin (Millipore Sigma, Cat# A9771) leakage from vasculature into brain parenchyma as described previously (Chen et al. 2008). Mice were treated with MPTP+LPS and sacrificed after 6 h and 24 h after the last dose.
Mice were anaesthetized by isoflurane and perfused intracardially with heparin (100 units/kg) followed by 5 ml FITC albumin at a concentration of 5 mg/ml in PBS with a flow rate of 1.5 ml/min.
Subsequently, the brain was isolated and incubated in 4% paraformaldehyde overnight. The solution was changed to 30% sucrose in PBS. Coronal sections of striatum were mounted and analyzed under fluorescence microscope (Olympus BX51 microscope).
NDP-MSH PK study
NDP-MSH PK study
To assess NDP-MSH concentrations in plasma and brain, male mice were treated with
MPTP+LPS as described above and two concentrations of NDP-MSH (400 µg/kg and 1
mg/kg) and sacrificed after 5-, 30- and 90-min.
Blood samples were collected through cardiac puncture in 40 mM EDTA, and plasma was collected by centrifugation and stored at -80°C till further analysis.
Whole brain was dissected and homogenized in PBS.
Proteins in brain homogenate and plasma samples were crashed with 3 volumes of methanol containing internal standard (propranolol) and centrifuged.
Supernatants were analyzed by liquid chromatography/mass spectrometry (LC/MS).
NDP-MSH in plasma and brain samples was detected by LC/MS through a service contract with Cyprotex, LLC, MA, USA.