Research Article: The Unusual Circulation of the Newt Heart after Ventricular Injury and Its Implications for Regeneration

Date Published: September 15, 2011

Publisher: Hindawi Publishing Corporation

Author(s): Yukihisa Miyachi.


Why do newts survive after needle puncture of the heart despite significant hemorrhage into the thoracic cavity? The answer involves the unique anatomical changes in the circulation that occur after ventricular injury. If the ventricle ruptures, newts quickly develop valve hyperplasia at the location of both the ventricular inflow and outflow tracts so as to redirect blood flow away from the injured ventricle. In addition, there is collateral flow between the left anterior caval vein and the conus arteriosus (a part of the aorta) after ventricular injury that supplements the systemic circulation and helps maintain vital organ perfusion. During this time period, the damaged ventricle can regenerate.

Partial Text

Many studies of newts support the notion that regeneration is largely achieved via a dedifferentiation process [1, 2]. For example, the transplantation of fluorescent-labeled cardiomyocytes into regenerating limb blastema results in the downregulation of cardiac marker genes and the upregulation of undifferentiated genes, which indicate that these cells can dedifferentiate [3]. I reported that lens regeneration in the newt requires the destruction and engulfment of damaged tissue by immune cells. I hypothesize that VEGFR expression by antigen-presenting cells is essential for angiogenesis and the subsequent regeneration [4].

Adult newts, Cynops pyrrhogaster, were reared in polycarbonate cages in an environmentally controlled room (water temperature: 22 ± 1°C), with a standard 12-hour light/dark cycle. The hearts from 600 newts were pricked twice with a needle (18 G syringe) through the skin to induce injury. Chest portions were fixed for 24 hours in a 10% formalin solution that was buffered with 0.15 M sodium-phosphate at a pH of 7.3. They were then washed in several changes of the same buffer and embedded in paraffin. Serial sections of 2 μm were prepared and stained with Mayer’s hematoxylin and eosin, and the degree of heart damage and subsequent remodeling were evaluated. Masson’s trichrome stain (acid fuchsin-orange G and aniline blue stain) was used for the characterization of collagen fibrogenesis, and alcian blue or toluidine blue was used to stain chondrocytes. The immunohistochemistry was used to detect CD31, similar to that described by Cursiefen et al. [5]. Briefly, the chest portion of each newt was frozen on dry ice in Tissuetek and sectioned into 8-μm thick slices. The sections were collected onto microscope slides, dried at room temperature, fixed in acetone, rinsed in PBS, blocked in 2% BSA, and stained with horseradish-peroxidase- (HRP-) conjugated CD31 antibody overnight. All staining procedures were performed at room temperature. Staining with secondary antibody alone, or with an isotype control instead of with CD31 primary antibody, was negative. Cell death was indicated by the expression of LC3 protein, which is a protein associated with autophagocytosis. All experiments were approved by the animal ethics committee of the National Cerebral and Cardiovascular Center.

How do newts survive ventricular puncture that causes hemorrhage into the thoracic cavity? The answer lies in the unique adaptive changes in the circulatory system that develop rapidly after ventricular injury. When I punctured the ventricle with a needle, there was immediate collateral blood flow between the left anterior cava vein and the conus arteriosus. This duct allowed blood to be diverted away from the injured ventricle and flow directly into the ascending aorta. In addition, the valve between the ventricle and the conus arteriosus and between the left atrium and ventricle underwent rapid hyperplasia so as to redirect blood flow away from the injured ventricle. During the time when collateral flow and valve hyperplasia prevented additional hemorrhage into the thoracic cavity, the injured ventricle regenerated.




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