2.6. BSCB integrity analysis

For analysis of BSCB ultrastructure, microvessels in the cervical and lumbar spinal cords of cell-treated, media-treated, and controls were examined by an investigator blinded to the animal groups, using coded sections, and photographed with an AMT ActiveVu XR 16 digital camera, (Advanced Microscopy Techniques, Woburn, MA) attached to a FEI Morgagni transmission electron microscope (FEI, Inc., Hillsboro, OR), at 60kV.

Analyses of capillary morphologies in the spinal cords of cell-treated, media-treated, and controls were performed based on electron microscopic (EM) images. Total analyzed capillaries were: the cervical spinal cord – control (n=54), media (n=78), low dose (n=121), mid dose (n=99), and high dose (n=109); the lumbar spinal cord – control (n=55), media (n=72), low dose (n=127), mid dose (n=89), and high dose (n=81). Capillaries were considered of normal morphology if a) endothelial cells (ECs) were intact and the basement membrane was a single layer surrounded by astrocytes or oligodendrocytes, b) mitochondria had well preserved cristae in the cytoplasm of all cells including ECs, c) normal neuropil surrounded the capillaries, and d) no evidence of intra- or extracellular edema was displayed. Moderately impaired capillary morphology was determined by appearance of a) EC cytoplasm with some vacuoles and dilated endoplasmic reticulum, b) swollen mitochondria in ECs and in the neuropil, and/or c) minor extracellular edema between areas of neuropil and near capillaries. Severely compromised capillary morphology was determined by the presence of a) substantially vacuolated ECs, b) necrotic ECs with condensed cytoplasm, c) ECs detached from basement membrane, d) vacuolated mitochondria in the cytoplasm of ECs and neuropil with swelling and disruption of cristae, e) degenerated astrocyte end-feet surrounding the capillaries with free floating swollen mitochondria, and/or f) extensive protein-filled extracellular edema around the capillaries. Quantitative analysis for each capillary category was presented as a percentage of total capillary numbers per animal group for both cervical and lumbar spinal cords.