Enzymatic Depletion of Endothelial Glycocalyx

Seven-week-old male Sprague Dawley rats (approximately 250 g; Harlan, Bicester, UK) were euthanised by cervical dislocation and hearts quickly removed, mounted on a Langendorff apparatus and perfused in a non-recirculating mode with Krebs solution as described previously [30 (link)]. Contractile function was measured using a latex balloon in the left ventricle. Data acquisition and analysis used a PowerLab System (AD Instruments, Bella Vista, NSW, Australia). Measurement was initiated after hearts were stabilised by perfusion for 30 min. Hearts were then perfused for 40 min with the combination of hyaluronidase (14 μg/ml; Sigma Aldrich, UK) and chondroitinase (0.0022 u/ml; Sigma Aldrich) in Krebs solution to deplete eGlx or with Krebs solution alone as control. We have shown previously that this combination of enzymes reduces eGlx thickness and coverage in glomerular capillaries [21 (link)]. Some isolated hearts were also perfusion-fixed with glutaraldehyde and Alcian Blue for analysing eGlx by electron microscopy as above.

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Qiu Y., Buffonge S., Ramnath R., Jenner S., Fawaz S., Arkill K.P., Neal C., Verkade P., White S.J., Hezzell M., Salmon A.H., Suleiman M.S., Welsh G.I., Foster R.R., Madeddu P, & Satchell S.C. (2022). Endothelial glycocalyx is damaged in diabetic cardiomyopathy: angiopoietin 1 restores glycocalyx and improves diastolic function in mice. Diabetologia, 65(5), 879-894.

Publication 2022

PowerLab System hyaluronidase chondroitinase

Alcian blue Capillaries Cervical Chondroitinase Contractile Dislocation Electron microscopy Enzymes Glomerular Glutaraldehyde Hearts Krebs solution Latex Left ventricle Male Perfusion Sprague dawley rats

Corresponding Organization : University of Bristol

Other organizations : University of Nottingham, Manchester Metropolitan University, North Shore Hospital, Waitemata District Health Board

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Variable analysis

independent variables

Depletion of endothelial glycocalyx (eGlx) by perfusion with a combination of hyaluronidase (14 μg/ml) and chondroitinase (0.0022 u/ml) in Krebs solution

dependent variables

Contractile function measured using a latex balloon in the left ventricle

control variables

Seven-week-old male Sprague Dawley rats (approximately 250 g)
Hearts perfused with Krebs solution alone as control

controls

Positive control: Hearts perfused with the combination of hyaluronidase and chondroitinase to deplete eGlx
Negative control: Hearts perfused with Krebs solution alone

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