Analysis of Spinal Cord Blood Supply Combining Vascular Corrosion Casting and Fluorescence Microsphere Technique: A Feasibility Study in an Aortic Surgical Large Animal Model

Summary

This study combines fluorescence microsphere technique and vascular corrosion casting to simultaneously investigate spinal cord blood flow and visualize spinal cord feeding arteries in a large animal model. This model can be employed to investigate morphological vascular alterations and hemodynamic parameters in the same tissue.

Abstract

Spinal cord ischemia after cardiovascular interventions continues to be a devastating problem in modern surgery. The role of intraspinal vascular networks and anterior radiculomedullary arteries (ARMA) in preventing spinal cord ischemia is poorly understood. We are the first to combine the fluorescence microsphere technique and vascular corrosion cast to investigate spinal cord blood supply in a large animal model. Landrace pigs (n= 30, 35.1 ± 3.9 kg) underwent a lateral thoracotomy. Fluorescent microspheres were injected into the left atrium and a reference sample was aspirated from the descending aorta. Repeated measurements of spinal cord and renal cortical blood flow from the left and right kidneys with three different microsphere colors in 5 pigs were taken to validate reproducibility. Spinal cord blood flow to the upper thoracic (T1-T4), mid-thoracic (T5-T8), lower thoracic (T9-T13), and lumbar (L1-L3) levels were determined. After euthanasia, we carried out selective vascular corrosion cast and counted the left and right ARMAs from levels T1-T13. We observed no alterations in blood flow analysis and fluorescence background noise when using vascular corrosion casting in the same tissue. Repeated measurements of cortical renal blood flow and spinal cord blood flow were reproducible. Blood flow analysis of the left and right kidneys revealed a strong correlation (r = 0.94, p<0.001). We detected more left than right ARMAs, with the highest prevalence at T4 (p<0.05). The mean number of ARMAs was 8 ± 2. Their number in the upper thoracic region ranged from 2 to 7 (mean of 5 ± 1), while in the lower thoracic region they ranged from 0 to 5 (mean of 3 ± 1 (p<0.001)). This study shows that combining fluorescence microsphere technique and vascular corrosion cast is well suited for assessing the blood flow and visualizing the arteries at the same time.

Introduction

Microspheres

Since its introduction by Rudolph and Heymann in 1967, microsphere technique has undergone continuous development¹. Today it is the gold standard for measuring regional organ perfusion. Microspheres for blood flow studies are typically 15-µm-diameter particles labeled with colored, radioactive, or fluorescent substances. When injected into the left atrium, they mix into the central circulation and trigger microembolization in small capillaries ("trapping"). Blood flow is proportional to the number of microspheres in the region of interest. Following introduction of the reference sample method, ....

Protocol

This study was conducted at the University Medical Center Freiburg, Freiburg, Germany. Institutional Review Board (IRB) approval was obtained before beginning any experiment. The study animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals and in compliance with the guidelines established by the local German government (Protocol number G 14/39). An experienced veterinarian carried out anesthesia, pain control, perioperative monitoring, and euthanasia.

Discussion

We are the first to have combined the fluorescence microsphere technique and vascular corrosion cast in this feasibility study to visualize spinal cord vasculature and determine spinal cord blood flow at the same time.

The spinal cord's thoracic region has some important features that must be considered in cardiovascular surgery. The anterior spinal artery's supply of blood through the anterior radiculomedullary arteries leaves watershed areas with decreased blood flow next to the regi.......

Materials

Name	Company	Catalog Number	Comments
Fluospheres Green	Molecular Probes	F21010
Fluospheres Red	Molecular Probes	F8842
Fluospheres Yellow	Molecular Probes	F21011
Biofuge Primo R	Thermo Scientific
Water Bath GFL Type 1083	GFL
Weighting Scale	SCALTEC
Fluorescence spektrometer LS55	Perkin Elmer
PU4ii resin, hardener, pigment	vasQtec		blue pigment was used
15-ml-conicial-tubes	TPP	91015
Cellosolve acetate	Sigma-Aldrich	109967
potassium hydroxide >85%	Carl Roth	P747.1
Ethanol >99.5%	Carl Roth	5054.1
Triton X-100	Sigma-Aldrich	X100
Withdrawal pump 11	Harvard Apparatus
3-way stopcocks	B. Braun	16496
Perfusor lines PVC 150 cm/2.6 ml	B. Braun	8722960
4-0 prolene suture	Ethicon	EH7411H
Syringes	B. Braun	10ml, 20ml and 50ml
Vasofix Braun 18G	B.Braun	4268130B
Dormicum Midazolam 5mg/ml	Roche Pharma	3085793
Ketamin 10%	Medistar GmbH	3048734
Kaliumchlorid	B.Braun	3140598
tracheal tubus 6.5mm	Rüsch	112482
Fentanyl	Janssen-Cilag
Mepivacain Scandicain 2%	AstraZeneca	910860
Microtome blade S35	Feather	207500000
disposable blade #11, #10	B.Braun	8080168
French-14-catheter	B.Braun	4476142
French-3-catheter	Vygon	115,094