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Results: For diameters greater than 1.5 mm, the mean anastomosis time decreased from. 17.9 ± 0.9 ..... fication to facilitate end-to-side portacaval anastomosis in.
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Clinics and Research in Hepatology and Gastroenterology (2012) xxx, xxx—xxx

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ORIGINAL ARTICLE

Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Franck Marie P. Leclère a,∗,b, Gregory A. Lewbart c, Robert Rieben a, Esther Vögelin a a

Department of Plastic and Hand Surgery, University of Bern, Bern, Switzerland Inserm, U 703 (French National Institute of Health and Medical Research), University Lille Nord de France, Lille University Hospital, CHRU, Lille, France c North Carolina State University, College of Veterinary Medicine, Raleigh, NC, USA b

Summary Background: Experiments using animal models are the most common way to learn microsurgery. This expertise is necessary for liver research, microsurgical reconstruction of the esophagus by free jejunum or reconstruction of the hepatic artery during reimplantation from living donors. The goal of this prospective study is to assess the reliability of an invertebrate model for microsurgical training. Methods: Between November 2011 and January 2012, 150 microsurgical trainings simulating vascular end-to-end microanastomoses were performed on 134 earthworms. The trainings were divided into 10 periods of 1 week each that included 15 simulations of end-to-end vascular microanastomoses: larger than 1.5 mm (n = 5), ranging in size from 1.0 to 1.5 mm (n = 5), and size less than 1.0 mm (n = 5). The technique is presented and documented. Results: For diameters greater than 1.5 mm, the mean anastomosis time decreased from 17.9 ± 0.9 min to 9.9 ± 0.2 min between the first and last week of training. For training with smaller diameters, the results showed a decrease in execution time of 41.8% (diameters between 1.0 and 1.5 mm) and 38.6% (diameters < 1.0 mm) between the first and last periods. The study underlines an improvement in the dexterity and speed of nodes’ execution. Conclusion: The earthworm appears to be a reliable experimental model for microsurgical training. It is more ethical than using rats, less expensive than vertebrate animal models, and allows the operator to gain in both confidence and time of execution. © 2012 Published by Elsevier Masson SAS.



Corresponding author. Department of Plastic and Hand Surgery, University of Bern, Inselspital Bern, Freiburgstrasse, Bern, Switzerland. E-mail addresses: [email protected], [email protected], [email protected] (F.M.P. Leclère).

2210-7401/$ – see front matter © 2012 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.clinre.2012.04.008

Please cite this article in press as: Leclère FMP, et al. Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Clin Res Hepatol Gastroenterol (2012), http://dx.doi.org/10.1016/j.clinre.2012.04.008

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F.M.P. Leclère et al.

Introduction Microsurgery has been progressing steadily over the last five decades. This progress stems from technical improvements in microsurgical devices, optical instrumentation, and improvements in microsurgeon training. Today, in the domains of hepatic and gastroenterologic surgery, it serves many applications including reconstruction of the esophagus by free jejunum, reconstruction of the hepatic artery during re-implantation from living donors, and liver research. Animal models have improved the training of microsurgeons via technical exercises of increasing difficulty [1—4] and have significantly contributed to the development of liver research [5—8]. The limitations of animal models are numerous and include:

Table 1 Specific requirements for use of Lumbricidae in experimental studies. Animal

Family: Lumbricidae Order: Haplotaxida Phylum: Annelida Weight: 1 to 10—15 g

Preparation of the animal for microsurgical training

Mean duration: 5 min

Kill the earthworm by immersion in 5% ethanol [12,13] Section the body of the animal proximally and distally Wash the animal with a thin wire of steel Cut the earthworm in the transverse plane at the level of the desired training Place a double microvascular clip Perform the microsurgical training simulating a vascular end-to-end microanastomosis with the biangulation technique

• the strict ethical rules governing the use of vertebrate animals and; • the time required for the training (as pointed out by Dumont et al. [9], the installation of the animal and the exposure are lengthy procedures that must be repeated for each operation); • anesthesia of the animal requires competence and adds to the duration of training [10]; • only one or two exercises are conducted on an animal that is then sacrificed. Since 2010, we have been working towards developing an efficient model for basic microsurgical training. Based on the literature [11] and our previous observations, the earthworm is a reliable model for microsurgical training because of the following: • the technique does not require anesthesia; • the texture of the earthworm is comparable to the arterial wall texture; • the wall tends to collapse perfectly mimicking the behaviour of mammalian blood vessels during anastomoses; • breeding earthworms is very easy and does not require expensive equipment; • it is possible to choose the size of the earthworm for each level of difficulty desired; • no assistance is needed which facilitates regular training.

Main principles of earthworm breeding in the laboratory

As well as dead organic matter, the earthworm also ingests any other soil particles that are small enough No food supplement Temperature of the room Change the soil every week Space required for each: 15 cm2

the laboratory (Fig. 1). The earthworms weighed between 1 g and 8 g. The mean size of the animals was 15 cm (5 to 32 cm). Their main characteristics are summarized in Table 1. The trainings were divided into 10 periods of 1 week each that included 15 simulations of end-to-end vascular

In this article, we present a prospective series of microsurgical training with earthworms and address the further development of this invertebrate model for microsurgical training.

Material and methods Animals Between November 2011 and January 2012, 150 microsurgical training exercises were performed in 134 earthworms (Lumbricus terrestris) belonging to the order Haplotaxida of the phylum Annelida (Table 1). Among the five families belonging to this order, the Lumbricidae were chosen for their ubiquity in European soils and ease of storage in

Figure 1 Lumbricidae belonging to the order Haplotaxida of the phylum Annelida.

Please cite this article in press as: Leclère FMP, et al. Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Clin Res Hepatol Gastroenterol (2012), http://dx.doi.org/10.1016/j.clinre.2012.04.008

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Lumbricus terrestris for microsurgical training ?

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Figure 2 Microsurgical training using the biangulation technique. A. Clamping the Lumbricus. B. Dilatation of the extremities. C. Placement of the two cardinal microsutures. D. Microanastomosis of the anterior wall.

microanastomoses: larger than 1.5 mm (n = 5), ranging in size from 1.0 to 1.5 mm (n = 5), and size less than 1.0 mm (n = 5).

Surgical technique After killing the earthworm with hot water the body of the animal was sectioned proximal and distal. Euthanasia with 5% ethanol was used in subsequent experiments and is a preferred method ((Table 1) [12,13]). The animal was washed by passing a thin wire of steel. The earthworm was then cut in the transverse plane at the level of the desired training. A double microvascular clip was placed. The microsurgical vascular training was then performed using the biangulation technique (Fig. 2). Each exercise was performed by the same operator (FML).

Objective parameters The external diameter of the animal was measured with an eyepiece reticule at ×40 magnification prior to division. The time necessary for the microanastomoses was recorded in every case. An O’Brian patency test was not possible and was replaced by a permeability test conducted by injecting water into the earthworm using a syringe (Fig. 3).

Subjective parameters A subjective evaluation was completed at the end of the training by the surgeon. A qualifier between excellent, very

Figure 3 Result after microsurgical training (diameter 2.1 mm, magnification ×40) and permeability test conducted by injecting water into the earthworm using a syringe.

good, good, and bad was given for the following parameters: dexterity, speed of nodes execution, improvement in the precision of movement and improvement in the quality of microanastomoses.

Statistical analysis The prospective study includes 150 microsurgical training exercises in 134 earthworms, spread over 10 weeks of practice, each with three sub-groups depending on the size of the animals. Statistical analysis was performed using XL Stat program (Addinsoft). Data are presented as mean ± standard error of the mean (SEM). Student’s test (two samples) is used to calculate the P-values and P < 0.05 is considered to be statistically significant.

Please cite this article in press as: Leclère FMP, et al. Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Clin Res Hepatol Gastroenterol (2012), http://dx.doi.org/10.1016/j.clinre.2012.04.008

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F.M.P. Leclère et al. Table 2

Average time necessary for the training in the different groups and periods of training. Time for training (Ø > 1.5 mm) (Mean ± STD [min])

1 2 3 4 5 6 7 8 9 10

17.9 17.0 15.9 13.8 12.7 12.1 11.5 10.2 10.9 9.9

± ± ± ± ± ± ± ± ± ±

0.9 0.7 1.2 0.9 0.6 1.1 1.3 0.3 0.8 0.2

Time for training (Ø = 1—1.5 mm) (Mean ± STD [min]) 18.2 17.4 17.0 14.8 14.2 13.4 10.7 11.3 10.8 10.6

± ± ± ± ± ± ± ± ± ±

0.7 0.3 0.7 1.6 1.0 1.0 0.6 0.7 0.7 1.0

Results Microsurgical training exercises (150 occurrences) were performed on 134 earthworms (Fig. 2). All the microanastomoses, controlled with our permeability test were patent. The results are presented in Tables 2 and 3.

Anastomosis time As shown in Table 2, for microanastomoses of diameter greater than 1.5 mm, the mean anastomosis time decreased from 17.9 ± 0.9 min to 9.9 ± 0.2 min (P < 0.01) between the first and last week of training. For training with smaller diameter, the results showed a decrease in execution time of 41.8% (P < 0.01) (diameter between 1.0 mm and 1.5 mm) and 38.6% (P < 0.01) (diameter < 1.0 mm) between the first and last periods.

Subjective aspect The subjective evaluation of training is presented in Table 3. The study noted a subjective improvement for all the subjective items, especially for the groups in which size was less than 1 mm.

Discussion In this prospective study, 150 microsurgical training exercises were performed on 134 earthworms. The results underline an improvement in speed of execution and microsurgical dexterity for all sizes of animals. Table 3

Time for training (Ø < 1.0 mm) (Mean ± STD [min]) 20.7 19.5 18.5 17.2 15.8 15.6 14.2 13.2 13.1 12.7

± ± ± ± ± ± ± ± ± ±

0.9 0.3 0.6 0.9 0.9 0.4 1.09 0.7 0.6 0.7

Since the early work of Jacobson and Suarez [14], the exponential development of microsurgery has been achieved through increasing attention to learning and regular training [15]. Numerous microsurgical training models are available that can impart skill and provide an appropriate level of rehearsal. Models range from basic devices to live animals. In the former, different sorts of materials, including silastic tubing or rubber gloves [16], have been used to reproduce a model for first step training. Recently Dumont et al. [9] introduced the double clock technique in order to improve the dexterity of future microsurgeons. However, the texture of these inert models is completely different from the clinical situation. Their rigidity does not reproduce the flexibility and collapse of the vascular walls observed in vivo. Animal models used in the laboratory of microsurgery are numerous and include pigs, rats, rabbits, chickens and fish [10]. The disadvantages of these models were highlighted in the introduction and include: ethical rules, cost, time consuming and expensive anaesthesia, and surgical approach to vessels before performing the microsurgical training.Our series of 150 trainings objectively confirmed the numerous advantages associated with the annelid model for microsurgical training: • the storage of this animal model is simple and does not require expensive care; • due to the symmetry of the animals, it is possible to section only the required part of the animal in order to reuse the remainder for future training; • it is possible to perform several trainings with only a few cm of the models; • no anaesthesia is required because the animal is killed before operation. In this context it is important to note

Subjective evaluation. Excellent

Dexterity Speed of nodes execution Improvement in the precision of movement Improvement in the quality of microanastomoses

Very good

X X X

X

Good

Bad

Remarks For all groups For all groups Especially for the groups in which size was less than 1 mm This model does not assess any intimal injuries

Please cite this article in press as: Leclère FMP, et al. Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Clin Res Hepatol Gastroenterol (2012), http://dx.doi.org/10.1016/j.clinre.2012.04.008

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Lumbricus terrestris for microsurgical training ? that despite its death, the texture of the animal remains similar to the texture of the arterial wall; • the training is immediately possible since no lengthy preparation is required; • this training can also be performed with magnifying glasses at home if the surgeon wishes to continue the training over the weekend or during holidays. This in vivo model can be used for the following: basic first step microsurgical training, regular training to improve speed of nodes and dexterity, training to reduce physiologic tremor, and training to become familiar with the alternative techniques of conventional vascular microanastomoses [17—19]. Despite the numerous advantages, this model has some shortcomings both in technique and methodology. First, it is not possible to perform a conventional patency test. However, this difficulty was somewhat overcome by the evaluation via an injection of water through the animal. Second, with this technique, it is not possible to mimic an adventicectomy. This step is essential for obtaining patent microanastomosis. Third, this model does not assess any intimal injuries, since the models will not be followed over time. Fourth, this model does not incorporate possible postoperative bleeding after dropping the clamps found in other in vivo models and clinical procedures. Additionally, the texture of the wall which is close to vertebrate arterial texture, is significantly different from that of veins. Finally, this article presents only trainings for end-to-end microanastomoses. Historically, the first microsurgical training was done by Buncke [20] in his garage. After a short time, microsurgery has become omnipresent in all fields of surgery. In this context, this specialty is essential and must be learned and practiced regularly. The proposed technique allows a regular, low-cost, and easily accessible training. This model will also help maintain the use of the rat for exercises of greater difficulties. We propose the hypothesis that this training will grow and develop significantly in the near future.

Conclusion The earthworm appears to be a reliable experimental model for microsurgical training. It is more ethical than using rats, less expensive, and allows the operator to gain in both confidence and speed of execution.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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Please cite this article in press as: Leclère FMP, et al. Microsurgery and liver research: Lumbricus terrestris, a reliable animal model for training? Clin Res Hepatol Gastroenterol (2012), http://dx.doi.org/10.1016/j.clinre.2012.04.008