Mouse Skull 3d Model

[Francisca Noggles]

Copyright2021 Bainier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Stereotaxic brain surgery is a common procedure routinely performed to this end in neuroscience research [1]. However, stereotaxic surgery remains a challenging technique requiring high technical and manual skills for which surgeons need extensive training.

Proper surgical techniques are fundamental for animal welfare and for high-quality science [2]. From viral injections to long-term implantations, improper surgery techniques could lead to tissue damage, infections, and animal distress. Moreover, for injections and implantations, an inaccurate targeting or placement will lead to inadequate data collection thus making the animal not usable.

Microsurgery skills improve with frequent practice and experience requiring extensive and lengthy training before safely performing stereotaxic surgeries on live animals. The learning process includes a few techniques that can be overwhelming such as anesthesia, sterile work, craniotomies, suturing, etc.

At present, surgeries are typically learned and practiced on dead animals. Besides questions relating to limited animal numbers and availability, the use of animals for this training raises ethical concerns; should animal euthanasia for training individuals be allowed? Unfortunately, to the best of our knowledge there is no current animal-free option to train on rodent stereotaxic surgeries.

While 3D printing technologies have been used extensively in human healthcare for a wide variety of applications: surgery training [3], simulation with patient-specific anatomy reproduction [4], patient-tailored implant/prosthetic production [5], etc., to the best of our knowledge, this technology has never been translated to animal research. In humans, realistic 3D printed models have been successfully used in surgical training for trainees to practice and master relevant procedures [6]. Furthermore, the skills acquired by simulation-based training have been shown to transfer to the operative setting [7].

In the spirit of the 3Rs (Replacement, Reduction and Refinement) we wanted to provide a new method enabling hands-on training of surgeons while improving animal welfare. For that, we developed and validated a new animal-free neurosurgical training model using 3D printing technologies. These 3D printed real-size skin-skull-brain models are meant to enable training on all different types of brain surgeries for both rats and mice.

Rat [8] and Mouse [9] 3D skull models made from microCT pictures were used in stl format (Fig 1). The process used, from anatomical imaging data to 3D models, has been described step by step by Bernd M Pohl and his colleagues [10].

Both models have been optimized (surface optimization, quality check) using Insight 3D printing software (Stratasys Ltd, USA). Vertebras were removed from the rat 3D model and landmarks made more visible using a dot at the intersections. Size of 3D models can be adapted to the size of the animals used.

Rat skulls were printed using Fortus 380mc FDM (Fused Deposition Modeling) Printer nozzle T10 (Stratasys Ltd, Eden Prairie, MN, USA) with 0.1270mm slice height. The choice of material was oriented to the PC-ABS, which provides better rendering and better haptic feedback, thanks to its flexibility. Black PC-ABS was used to get a better visualization of white dental/bone cement finishes used for long term implantation.

Mice skulls were printed using Formlabs Form3 LFS (Low Force Stereolithography) printer (Formlabs Inc., Somerville, MA, USA) due to the extremely small details of the model. The skulls were printed out of Durable Resin (Formlabs Inc., Somerville, MA, USA) with a 0.05mm layer thickness. After printing, 3D printed skulls were washed with isopropyl alcohol for 10 minutes using a Form Wash machine (Formlabs Inc., Somerville, MA, USA) and cured for 60 minutes at 60C using a Form Cure machine (Formlabs Inc., Somerville, MA, USA).

The brain was made out of Polyurethane expanding foam (PU 500, Fischer Deutschland Vertriebs GmbH, Germany). The white color of the material makes it possible to see dyes to train on viral injections. Foam was projected from the foramen magnum and was gently filling the whole plastic skull model (Fig 2A). Once fully dry, the foam excess was removed using forceps (Fig 2B).

We invited ten staff neurosurgeons from Roche to evaluate and test various surgical procedures on the 3D printed skin-skull-brain models. Experience level in stereotaxic surgery varied between participants, from students to experienced scientists.

The skin-skull-brain model was fixed in the stereotaxic frame using teeth and ear bars, as usually done during a live animal procedure. A wide range of neurosurgery techniques, listed below, were successfully tested using our 3D printed models.

These constitute the first step of every brain surgery. They involve the fixation of the skull into the stereotaxic frame, the identification of skull landmarks (bregma, lambda), the positioning of the drill on the target area and the careful drilling and removal of bone tissue. Detailed rodent craniotomies protocol have been described previously [11].

A proper positioning of screws in the craniotomy area is essential to achieve good screw stability, in turn allowing screws to work as anchors for potential implants. It is important to set these screws at a reproducible depth, to ensure good data acquisition, e.g. for EEG screws [12].

A wide range of probes can be implanted in rodents for different functions (brain stimulation, electrophysiology recordings, etc). For acute and long-term implantation, implants need to be firmly secured using dental cement on the skull couple to anchoring screws. Several implantation methods are available, from optic fiber [13] to chronic neural electrode implantations [14, 15].

Successful brain injections require a very precise positioning of the needle to target specific brain regions. Identification of brain landmarks and identification of the right coordinates are crucial to ensure that the area of interest is appropriately targeted [16].

Each 3D printed skull required around 8 hours of non-supervised printing time and a cost of 21 CHF for rat against 2 hours and 5 CHF for mouse. Brain assembly takes a few minutes while the skin addition process (not including setting time) requires less than 20 minutes per skull.

The feedback from ten experienced neurosurgeons revealed high satisfaction (Fig 4) with very good anatomical accuracy (4.5). Both models show a very detailed real-size (5) replica of actual rodent skulls anatomy (Fig 5). Bone sutures forming Bregma and Lambda landmarks are identifiable (4.2). The 3D printed rodent models were perceived as very realistic, and even if the model did not perfectly replicate the feeling of real-animal brain surgery, tactile fidelity was positively rated (4.4).

Our models were reported to be appropriate and very useful for surgical training applications (4.9) as well as good alternatives to cadaveric skulls (4.9). They were stated to help trainees develop their confidence before working on live animals (4.7). All surgeons responded that they would actively use the model to train someone or to be trained on new techniques and to test the feasibility of new surgeries (5) (Fig 7).

Q5: Are these models appropriate and useful for surgery training? Q6: Are these models good alternatives to cadaveric skulls for training purposes? Q7: Would these models help trainees to develop their confidence? Q8: Would you use these models to train someone or to be trained on new techniques? Q9: Would you use these models to test the feasibility of new surgeries? Data presented with mean and SD.

Within the last decade, 3D printing technologies have penetrated and improved several fields of human healthcare from ophthalmology [18] to cardiovascular diseases [19] and neurosurgery [20], amongst others. 3D printing applications are now widely used for multiple purposes: pre-operative planning, teaching and training of nave surgeons, counselling of patients before a surgery, and creating patient-specific implantable devices [21, 22].

3D printing use in neurosurgery is becoming more widespread with the creation of patient-specific anatomical models (e.g. 3D printed hollow models of cerebral aneurysms [23]). Such models provide an excellent tool for practice and rehearsal of the surgery by reproducing the patient exact anatomy and pathology.

Since the emergence of 3D printing technologies, cutting-edge research efforts are ongoing to develop and share 3D structures of vertebrates for education and research. Indeed, rat [8] and mouse [9] skull designs used here were shared by Bernd M. Pohl, Timothy Rowe and their colleagues in this spirit. To the best of our knowledge, 3D technology has never been translated to animal research in neuroscience for surgery training purposes.

The 3D printed skin-skull-brain models described here are high-fidelity and real-size replicas of rat and mice skulls. These 3D printed models are rapidly made (8 hours of unsupervised printing for the rat model and 2 hours for the mouse model) and low-cost (the raw materials cost 21 and 5 CHF, respectively).

Though 3D-printing materials are known for their inability to properly mimic soft biological tissues such as skin tissues, the recent development of a new technique called freeform 3D printing has revolutionized the field by allowing soft matters such as hydrogels and silicone elastomers to be printed [28]. The use of such techniques could improve our skin model; they would likely make the skin addition on our models more time-efficient, and by opening the door to other soft materials, they could provide an improved haptic representation of living animal skin, particularly useful for sutures.

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