Semi-automated rapid quantification of brain vessel density utilizing fluorescent microscopy

• Published in: Journal of neuroscience methods Vol. 270; pp. 124 - 131
• Main Authors: Bohn, Kaci A., Adkins, Chris E., Mittapalli, Rajendar K., Terrell-Hall, Tori B., Mohammad, Afroz S., Shah, Neal, Dolan, Emma L., Nounou, Mohamed I., Lockman, Paul R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2016
• Subjects: Animals; Brain - anatomy & histology; Brain - blood supply; Brain - pathology; Brain Neoplasms - pathology; Cell Line, Tumor; Female; Fluorescent microscopy; Glioma - pathology; Humans; Image Processing, Computer-Assisted - methods; Immunohistochemistry; Indocyanine green; Male; Metastases; Mice, Transgenic; Microscopy, Fluorescence - methods; Neoplasm Metastasis - pathology; Pattern Recognition, Automated - methods; Rats, Inbred F344; Rats, Sprague-Dawley; TX Red; Vessel density; Immunohistochemistry; eGFP; BBB; kDa; MW; Indocyanine green; TX Red; ICG; F344; VD; Metastases; SD; Fluorescent microscopy; FITC; Vessel density; CD-1
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2016.06.012

[Display omitted] Vascular density varies significantly between brain regions and between healthy and diseased tissue. However, current methods are arduous and time consuming. Herein we present a rapid simple method to quantify vascular density in brain. Normal brain vasculature is seen with indocyanine green fluorescence within the image. •Semi-automated methodology utilizes fluorescence microscopy to calculate vascular density within brain and brain tumors.•Novel methodology produces data comparable to previously hand counting pathology methods.•Vascular density is decreased in experimental brain metastases compared to a preclinical glioma model. Measurement of vascular density has significant value in characterizing healthy and diseased tissue, particularly in brain where vascular density varies among regions. Further, an understanding of brain vessel size helps distinguish between capillaries and larger vessels like arterioles and venules. Unfortunately, few cutting edge methodologies are available to laboratories to rapidly quantify vessel density. We developed a rapid microscopic method, which quantifies the numbers and diameters of blood vessels in brain. Utilizing this method we characterized vascular density of five brain regions in both mice and rats, in two tumor models, using three tracers. We observed the number of sections/mm2 in various brain regions: genu of corpus callosum 161±7, hippocampus 266±18, superior colliculus 300±24, frontal cortex 391±55, and inferior colliculus 692±18 (n=5 animals). Regional brain data were not significantly different between species (p>0.05) or when using different tracers (70kDa and 2000kDa Texas Red; p>0.05). Vascular density decreased (62–79%) in preclinical brain metastases but increased (62%) a rat glioma model. Our values were similar (p>0.05) to published literature. We applied this method to brain-tumors and observed brain metastases of breast cancer to have a ∼2.5-fold reduction (p>0.05) in vessels/mm2 compared to normal cortical regions. In contrast, vascular density in a glioma model was significantly higher (sections/mm2 736±84; p<0.05). In summary, we present a vascular density counting method that is rapid, sensitive, and uses fluorescence microscopy without antibodies.