A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

Summary

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4^th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Abstract

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4^th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Introduction

For over three decades, researchers have been modifying and improving rat lung transplantation models so that the data generated are more consistent and more reflective of the actual clinical condition. In our laboratory's time performing this model, we have determined four areas of improvement: cuffing techniques for anastomoses, identification of the recipient's 4^th intercostal space, lung inflation and wound closure during the recipient's procedure, and the harvesting of samples for analysis.

Cuffing technique modifications for anastomoses can improve the entire transplantation procedure by shortening handling time....

Protocol

Male Sprague-Dawley rats (180-270 g body weight) were purchased commercially (e.g., Envigo) and were housed under pathogen-free conditions at The Ohio State University Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol # 2012A00000135-R2).

1. Initial setup

1. Set up surgical devices.
   NOTE: This is a non-survival surgery. If survival surgery is to be performed, sterile instruments and barrier....

Results

In order to measure pulmonary edema, the wet-to-dry weight ratio was calculated. The donor's native lobe, the transplanted lobe, and the recipient's native lobe were collected as described in the protocol and weighed immediately for wet weight, dried at 60 °C for 48 h, and then weighed again for the dry weight. An increased wet-to-dry weight ratio would be indicative of pulmonary edema. Our results indicate that the transplanted lobe did have a significant increase in wet-to-dry .......

Discussion

In this report, we have intervened at several critical steps in a rat lung transplantation protocol to optimize the procedure. While various cuffing techniques for rat lung transplantation has been reported^{1,2,3,4,5,6,7,8,9.......}

Materials

Name	Company	Catalog Number	Comments
12 Gauge angio-catheter	BD	382277
14 Gauge angio-catheter	B. Braun	4251717-02
16 Gauge angio-catheter	B. Braun	4252586-02
18 Gauge angio-catheter	B. Braun	4251679-02
20 Gauge angio-catheter	B. Braun	4252527-02
4-0 silk suture	Surgical Specialties Corp.	SP116
6-0 nylon suture	AD Surgical	S-N618R13
7-0 nylon suture	AD Surgical	S-N718SP13
8-0 nylon suture	AD Surgical	XXS-N807T6
Betadine Spray	Avrio Health L.P	UPC 367618160039
Clippers	VWR	MSPP-023326
Castroviejo micro dissecting spring scissors	Roboz Surgical Instrument Co	RS-5668
Dumont #5 - Fine Forceps	Fine Science Tools	11254-20
Electrocautery	Macan	MV-7A
Endotracheal intubation kit	Kent Scientific	ETI-MSE
Forceps	Fine Science Tools	11027-12
Halsted-mosquito hemostat	Roboz Surgical Instrument Co	RS-7112
Heparin	Fresnius Medical Care	C504701
Insulin syringe	Life Technologies	B328446
Isoflurane	Piramal Critical Care	NDC 66794-017-25
Isopropyl Alcohol Swabs	BD	326895
Ketamine	Hikma Pharmaceuticals PLC	NDC 0413-9505-10
Dieffenbach Bulldog Clamp	World Precision Instruments	WPI14117
Needle holder/Forceps, Curved	Micrins	MI1542
Needle holder/Forceps, Straight	Micrins	MI1540
Perfadex Plus (Organ Preservation Solution)	XVIVO Perfusion AB	REF# 19950
PhysioSuite	Kent Scientific	PS-MSTAT-RT	Used to check SpO2 and heartbeat
Retractor	Roboz Surgical Instrument Co	RS-6560
Saline	PP Pharmaceuticals LLC	NDC 63323-186-10
Scissors	Fine Science Tools	14090-11
SomnoSuite Small Animal Anesthesia System	Kent Scientific	SS-MVG-Module
Sterile  Cotton Gauze Pad	Fisherbrand	22-415-469
Surgical Microscope	Leica	M500-N w/ OHS
Syringe 5mL	BD	309646
Vannas-Tubingen Spring Scissors	Fine Science Tools	15008-08
Xylazine	Korn Pharmaceuticals Corp	NDC 59399-110-20
Yasargil Clamp	Aesculap, Inc	FT351T	Used to clamp bronchus
Yasargil Clamp	Aesculap, Inc	FT261T	Used to clamp hilum
Yasargil Clamp Applicator	Aesculap, Inc	FT484T