An in situ perfusion protocol of rat epididymal adipose tissue useful in metabolic studies

Abstract

Experimental approaches involving the perfusion of tissues and organs offer the advantage of improved physiological relevance over the use of isolated tissues or cells while at the same time being much more controlled and tissue-specific than studies in vivo. Nevertheless, there have been few metabolic studies performed in perfused white adipose tissue, largely because of the difficulty of the surgical technique involved. Although some methods have been described, they are difficult to use as perfusion protocols and their reproducibility is poor. We have modified a rat perfusion method, based on a modification of the Ho and Meng technique, for use with epididymal white adipose tissue (eWAT), and we present it here as a protocol to be reproduced. We also offer surgical solutions for the most common variants of vessel distributions in rats. Using the protocol described here, the perfused adipose tissue is viable and metabolically active, as indicated by the maintenance of tissue ATP levels and adiponectin secretion and by endogenous lipolysis regulation. Moreover, there is a high level of lipoprotein lipase activity in the endothelium of the tissue, which is heparin-releasable. Thus, this method is a useful and reproducible tool that allows the perfusion of eWAT for use in metabolic studies. Perfusion is a technique that faithfully reproduces the physiological environment in an isolated tissue. It allows the study of metabolic processes in physiological conditions because it involves the whole organ, maintaining its structure and cellular diversity. Moreover, substrates, hormones, drugs, etc., reach cells through physiological channels, the arteries and veins.In the early 20th century, the development of perfusion methodology in laboratory animals began in organs such as the heart and liver. Later, some studies were performed in perfused white fat tissue. However, this technique is more complicated than in other tissues. In 1963, Robert and Scow (1) published the first perfusion of rat parametrial white fat tissue, used in later studies (1-5). In 1964, Ho and Meng (6) described a perfusion technique for isolated rat epididymal white adipose tissue (eWAT), used as a reference for subsequent epididymal tissue studies (6-10). In all cases, the protocols described are very difficult to reproduce and use in metabolic studies.Alternatively, in humans, the use of microdialysis and arteriovenous techniques has been proposed to study white adipose tissue (WAT) metabolism (11). Although these methods are applicable to humans, they have several disadvantages, such as minimal control over the studied tissue and over the different factors released from the rest of the body.Based on the modification by Gubser, Di Francesco, and Bickel (7) of the Ho and Meng technique (6), we developed a detailed in situ perfusion method with the aim of providing an easily reproducible protocol useful for other researchers. This method is appropriate for use in metabolic studies involving the perfusion of different substances and collec...

Nitric Oxide and the Release of Lipoprotein Lipase from White Adipose Tissue

“…Briefly, the method entails perfusion of the right epididymal fat pad, from the aorta and spermatic artery to the spermatic vein and cava. LPL activity measured in the perfused right fat pad is similar to that measured in the non-perfused left fat pad, and the perfused tissue is viable and metabolically active [25].…”

“…The dose of the NO donor used in the perfusion does not interfere with the assay of LPL activity in WAT homogenates (N. Jané, unpublished data). Furthermore, the activity found in the perfusate after NO donor perfusion was lower than that found after heparin perfusion [25], probably because heparin has a greater capacity to displace LPL from its anchorage and it also stabilizes the dimer.…”

“…The technique used is described in our previous study [25]. Briefly, the method entails perfusion of the right epididymal fat pad, from the aorta and spermatic artery to the spermatic vein and cava.…”

Application of Proteomic Tools To Detect the Nonspecificity of a Polyclonal Antibody against Lipoprotein Lipase

“…A classic procedure for obtaining antibodies against LPL was immunization of hens with LPL purified from bovine milk. – Several recent studies have used polyclonal antibodies obtained following this strategy to immunodetect LPL by Western blot or ELISA. ,,– …”

“…In our laboratory, for years, we used P66, a polyclonal antibody obtained following this protocol, for LPL immunodetection by Western blot. , The obtaining of P66 was described in a previous study, in which this antibody was used for coating in a sandwich ELISA combined with the widely used monoclonal antibody (mAb) 5D2 for detection.…”