Download Evaluation of Acute Cardiac Damage in Swine Following

Evaluation of Acute Cardiac Damage in Swine Following Resuscitation –
A Microscopic and Genomic Pilot Study
Fiona Rogan1, Gerry Mahon2, Laura Davis3, Rebecca Di Maio3, Brian J Meenan1 and George A. Burke1
1Nanotechnology and Integrated Bioengineering Centre (NIBEC), School of Engineering, University of Ulster, N. Ireland.
2Centre for Experimental Medicine, Queen’s University Belfast, N. Ireland.
3HeartSine Technologies Ltd., Belfast, N. Ireland.
Email: [email protected]
Introduction
• Transthoracic defibrillation is performed to correct life threatening cardiac arrhythmias of the heart, such as ventricular fibrillation (VF) and
ventricular tachycardia (VT), which could result in cardiac arrest.
• These arrhythmias are treated by the delivery of an electrical shock from a defibrillator across the heart, of sufficient magnitude to depolarise
a critical mass of myocardium, helping restore normal electrical activity and a perfusing heart rhythm.
• In the event of sudden cardiac arrest, the 2010 American Heart Association (AHA) guidelines1 recommend defined cardiopulmonary
resuscitation (CPR) conditions and defibrillation energies to assist patient recovery.
• While it is accepted that resuscitation is required for life saving events, research is now investigating the potential of these techniques to
cause myocardial damage.
Figure 1 – HeartSine® samaritan® Public
Access Defibrillator (PAD) 350P
)
• This pilot study aimed to examine changes in cardiac tissue ultrastructure and gene expression following resuscitation.
Materials and Methods
OVERVIEW
HISTOLOGY METHODOLOGY
• Following the induction of VF in a porcine model, resuscitation was performed through CPR
a) and defibrillation as per the recommended guidelines.
• Upon protocol completion, animals were euthanised and cardiac tissues collected for
histopathological processing and scanning electron microscopy (SEM) examination with RNA
isolated for gene expression analysis.
• Samples were dehydrated in an increasing alcohol series,
cleared in xylene and embedded in paraffin wax for microtomy.
5µM section were cut, placed on glass slides and histologically
stained with haematoxylin and eosin.
• Representative sections were imaged using a Nikon 80i
upright microscope (Nikon Instruments, UK) and with 40, 100,
200 and 400 x magnification. Representative images are
illustrated below.
SEM PREPARATION
• Samples had 3 washes of DI water, post fixed in 1% osmium tetroxide prior to dehydration in an
increasing alcohol series.
• Then chemically dried in HDMS overnight, gold coated using a gold sputter target prior to
visualisation in a scanning electron microscope (FEI, UK). Representative samples are
illustrated below.
GENE EXPRESSION ANALYSIS
• Following Roche guidelines2, using Tripure Isolation Reagent
and SYBR Green, RNA was extracted, isolated and quantified.
Results and Discussion
SEM Analysis
Histological Analysis
A
3.13 ±0.55
3.05 ±0.36
B
C
3.30 ±0.42
Figure 2- Scanning Electron Micrograph of Cardiac tissue Left Ventricle A-C of cardiac tissue #1 at advancing magnification
Figure 3- Scanning Electron Micrograph of cardiac tissue Left Ventricle A-C of cardiac tissue #7 at advancing magnification
The above images indicate mild changes in the cardiac tissue, potentially
representing early degenerative processes within the tissue.
Figure 4 – Illustrates A- Abattoir ‘control’ at 40 x magnification, B- represents an image from cardiac tissue#1 at 100 x
magnification highlighting hypereosinophilic cytoplasm and C- shows a magnified image cardiac tissue#1 at 200 x
Figure 5 – Illustrates A- represents image from cardiac tissue #7 at 40 x magnification, B- magnified image from #7 at
100 x magnification highlighting haemorrhage and increase in neutrophilic numbers and C- cardiac tissue#7 at 200 x
H&E staining of the cardiac tissues indicated minor structural changes at a
cellular level such as cardiomyocytes exhibit a combination of sarcoplasmic
hypereosinophilia, haemorrhage and inflammation, these and others varied when
compared to the control tissue.
This process is used to amplify and simultaneously detect /
Real Time
quantify a targeted DNA molecule, this is detected as the reaction
- PCR
progresses, hence the ‘real time’. This detection occurs through
Study
the use of non-specific fluorescent dyes that intercalate with any
double stranded DNA and specific sequence DNA probes that
consist of oligonucleotides that are labelled with a fluorescent
label fluorophores permitting detection after hybridisation of the
probe with its complementary sequence (cDNA) to quantify RNA.
Connexin 43, Gata4, GJA1
Desmin, Troponin, HFABP3
The graphs adjacent illustrate 6 of the gene profiles examined on
the porcine cardiac tissue.
Conclusion
This pilot study aims to examine the acute structural and genomic changes that occur in a porcine model in the timepoint
immediately following implementing current resuscitation guidelines. While it is well recognized that defibrillation initiates
electroporative changes3 in the plasma membrane of cardiac myocytes, little is known about other structural and genomic
changes that occur downstream of the defibrillation process. The study examined changes in mRNA expression in key genes
associated with Cardiac muscle damage, Inflammatory response and Cardiac structural damage.
It is evident that additional research is required to fully investigate post resuscitation cardiac damage using a range of
conversion energies and CPR technique and duration. The work of this study hopes to investigate these potential changes in
the future.
Acknowledgements
This work was supported by HeartSine and the
Northern Ireland Department of Education and
Learning (DEL).
References
1.
2.
3.
Nanotechnology and Integrated Bioengineering Centre
AHA guidelines 2010;
http://www.heart.org/HEARTORG/CPRAndECC/Science/Guidelines/2010AHA-Guidelines-for-CPR-ECC_UCM_317311_SubHomePage.jsp
Roche Applied Science, Tripure Isolation Reagent Instruction Manual version
7, June 2013
Wilson, C.M., Allen, J.D., Bridges, J.B., and Adgey, A.A. Death and damage
caused by multiple direct current shocks: studies in an animal model. Eur
Heart J 9, 1257, 1988.