Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Otol Neurotol. 2021 Oct 1;42(9):e1197–e1202. doi: 10.1097/MAO.0000000000003255

A Novel 3D-Printed Head Holder for Guinea Pig Ear Surgery

Chris Valentini 1, Young Jae Ryu 2, Betsy Szeto 1, Michelle Yu 1, Anil K Lalwani 1,2,*, Jeffrey Kysar 1,2,*
PMCID: PMC8448908  NIHMSID: NIHMS1708685  PMID: 34172663

Abstract

Background:

Otologic surgery in guinea pig requires head immobilization for microscopic manipulation. Existing commercially available stereotaxic frames are expensive and impede access to the ear as they rely on ear bars or mouthpieces to secure the head.

Method:

Prototype head holders were designed using the Solidworks 2019 software and 3D-printed using Formlabs Form 2 Printers with photopolymer resin. The head holder consists of a C-shaped brace with adjustable radial inserts of 1⁄4–20 UNC standard screws with cone point tips providing head fixation for animals of various sizes. The C-shaped brace is attached to a rod that can be secured to a commercially available micromanipulator. The head holder design was tested during in vivo guinea pig experiments where their head motion with (n=22) and without the head holder (n=2) was evaluated visually through a stereotaxic microscope at 24x magnification during surgery.

Results:

The head holder design was easy to use and allowed for both nose cone administration of anesthesia and access to the ear for intraoperative auditory testing and manipulation. Functionally, the head holder successfully minimized head movement. Furthermore, harvested round window membranes evaluated at 72 hours following surgery showed precise perforations with the use of head holder.

Conclusion:

The novel 3D-printed head holder enables simultaneous access for nose cone administration of anesthesia and surgical manipulation of the ear and brain. Moreover, it provides a modular, intuitive, and economical alternative to commercial stereotaxic devices for minimizing head motion during small animal surgery.

Keywords: Guinea pig, head holder, head brace, stereotaxic instruments, otologic surgery, neurosurgery

1. INTRODUCTION

Guinea pigs and other small laboratory animals often require immobilization and precise positioning of the head during otologic and neurological surgeries while under anesthesia. Existing commercial head holders typically secure the head through the use of ear bars, nose clamps, or mouthpieces [1–5]. Ear bars prevent surgical access to the external acoustic meatus, thereby preventing audiological testing and surgical access to the ear. Nose clamps and mouth pieces are affixed to the nasal cavity and the hard palate and teeth respectively, which impede the use of nose cone anesthesia [2–5]. Furthermore, these commercial frames are expensive and cumbersome within most experimental setups.

Our laboratory became acutely aware of the need for a head immobilization apparatus that does not impede access to the ear while conducting experiments to assess a novel microneedle design to aspirate perilymph from the inner ear via the round window membrane (RWM) [6]. Previously, we had successfully created perforations in the RWM without tearing or causing substantial damage to the membrane [7–9]; however, these perforations were introduced within one breath cycle, thus absolute head immobilization was not crucial. In contrast, the use of the microneedle to aspirate 1 μl perilymph from the inner ear—which required the needle to stay in place approximately 45 seconds, the timespan of multiple respiration cycles—was associated with tearing of RWM [10]. Thus, for procedures that exceed the length of one breath cycle, head stability becomes critical to prevent RWM tearing. However, there is currently no commercially available head holder that simultaneously allows for the use of sustained nose cone anesthesia, prevents disruptive movement from respiration without injuring the animal, and permits surgical access to the ear and postauricular space.

To overcome this limitation, we have designed a novel 3D-printed head holder that provides a solution for the aforementioned problems surrounding in vivo otologic experiments. The inspiration for the design comes from the pin-type head frames commonly used in human neurosurgical surgeries, such as the MAYFIELD®skull clamp (Integra Life Sciences Corporation, Cincinnati, OH, USA). These pin-type frames encircle the skull to provide rigid fixation for the head and neck by applying force from opposite directions at two or three points. They protect the patient from injury and accommodate most equipment necessary during surgery because of their modular and slim design. By using multiple pins, instead of a headband with a larger surface area, surgeons avoid cutting off circulation that could lead to skin breakdown and injury [11]. The three-pin skull clamp is the gold standard and generally considered safe for prolonged patient immobilization [11]. The proposed head holder implements a similar design to current neurosurgical pin-type head clamps to provide stabilization and unimpeded access to allow aspiration of perilymph from the inner ear through the RWM. Additionally, the novel head holder design is easy to share among researchers and is inexpensive to produce.

2. METHODS

2.1. Design of the head holder

The head holder was designed to use commercially available material stock and fasteners. It is modular and can be assembled for use with as few as two parts and up to four parts (Figure 1). It consists of a C-shaped brace with a 0.45-inch diameter hole at the extrema and a smaller threaded hole on its perpendicular surface (Figure 2). In order to ensure the structural integrity of the brace, four ribs were placed along the curve arms of the head holder. A fitted rod can be inserted into the 0.45-inch diameter hole and can rotate freely, forming a cylindrical joint (Figure 1, part number 2). The joint is fixed by tightening a ¼−20 screw (Threads per inch: 20, Major diameter: 6.35mm, Tap drill diameter: 5.35mm) inserted through the smaller threaded hole. Eight threaded holes along the length of the C-brace, four on either side of the cylindrical joint, accommodate adjustable radial inserts of ¼−20 UNC standard screws.

Figure 1:

Figure 1:

Open in a new tab

Guinea pig head holder. Part 1: The brace of the head holder with peripherally variable insertable screw slots and rod insert slot. Part 2: Ball joint with rod attachment to the head brace. Part 3: Ball socket mid-joint as an intermediary attachment for flexible brace adjustment. Part 4: Socket joint to attach to a clamping mechanism or micromanipulator.

Figure 2:

Figure 2:

Open in a new tab

The design of the brace of the head holder. It allows for sharp screw inserts at various positions to achieve optimal head orientation and stabilization.

The modular design of the head holder allows for assembly in multiple ways. Parts 1 and 2 (Figure 1) are essential for a functional head holder, where part 2 secures to a commercially available micromanipulator (MM3 Marzhauser Wetzlar, Germany). For additional degrees of freedom in brace adjustment, two other attachments were created (Figure 1, parts number 3 and 4) to allow for up to two ball-and-socket joints, which can also be fixed using ¼−20 screws. Each ball-and-socket joint provides an additional three degrees of freedom; adding two of these ball-and-socket joints therefore provides six degrees of freedom, allowing for any desired orientation—rotation or translation—of the brace. In addition, the researcher can choose the number and placement of screws, which can be tightened or loosened to provide head fixation to guinea pigs between 150 and 300 grams. The length of the screws must be long enough to be inserted through the threaded holes and come in contact with the head of the animal.

For the purposes of our study, two 2-inch long socket head screws were inserted into the threaded holes furthest from the cylindrical joint on either side in opposing positions. The tips of the screws were tapered to a cone shape at a 40-degree tip angle using a Fryer Easy Turn-14” Tool Room Lathe (Fryer Machine Systems Inc., Patterson, NY, USA). Our head holder was assembled with parts 1, 2, and 3 (Figure 1), using only one ball and socket joint, which permitted the necessary flexibility for our surgical setup; part 4 was not required.

2.2. 3D Printing

Prototypes and the final design of the head holder were drafted using SolidWorks 2019 (Dassault Systems SolidWorks Corporation, Concord, NH, USA) computer-aided design (CAD) software to generate stereolithography Standard Triangle Language (STL) files. The STL files were printed using Form 2 SLA Desktop 3D Printer (FormLabs Inc., Somerville, MA, USA) with Grey Pro Resin (FormLabs Inc.). The Grey Pro Resin material has a tensile strength of 61.0 MPa (megapascal) and a modulus of 2.2 GPa (gigapascal). The STL files were first compiled on the Preform software (Formlabs Inc.). The user-controlled parameters were adjusted accordingly to the Form 2 SLA Desktop 3D Printer (Formlabs Inc.). The layer height was set to 100 microns, the positioning was autogenerated, and the supports were autogenerated with 0.3 mm contact points. Once printed, the parts were placed in an ultrasonic bath with 99% isopropyl solution for 20 minutes and then under a UV light to cure remaining resin residues for 30 minutes. All sharp edges were sanded down using a 400-grit sandpaper. The brace was submerged in the ultrasonic isopropyl bath to remove any further residue and dried at room temperature.

2.3. Animal surgery

The head holder design was tested during in vivo guinea pig experiments involving insertion of a hollow microneedle into the RWM for at least 45 seconds while the guinea pig was under isoflurane anesthesia administered via nose cone [10]. All surgical experimental procedures were in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC). Twenty-two juvenile guinea pigs (Hartley strain) between 150 and 300 grams were acquired from a commercial vendor (Charles River, Inc., Wilmington, MA, USA). Twenty guinea pig surgeries were performed with the head holder and two were performed without.

The guinea pig was placed in a left lateral recumbent position, with the right side of the body facing up. The surgeries were survival experiments and therefore 0.5 mg/kg meloxicam and 1.0mg/kg buprenorphine sustained release formula were administered subcutaneously as additional analgesics. The snout of the anesthetized guinea pig was placed in the nose cone and the guinea pigs were then anesthetized with isoflurane gas (3.0% for induction and 1.0–3.0% for maintenance). Lidocaine was injected for topical anesthesia subcutaneously at the desired incision site and at the two positions where the screws of the head holder were to be placed, after the guinea pigs were on isoflurane maintenance. The head holder was secured to the skull approximately 10 minutes after induction of anesthesia by placing the screws anterior to the external auditory meatus and posterior to the orbit without piercing the skin (Figures 3 and 4). The screws are tightened to exert no more than the minimal required amount of pressure that limits animal head motion from breaths. The nose cone is stabilized with a clamp attached to a micromanipulator and acts as a third point of contact, which prevents flexion or extension of the head that may be created by the two-point fixation from the head holder. During the surgery, the isoflurane was adjusted to 1.5–2% to achieve an appropriate depth of anesthesia, confirmed by negative toe pinches.

Figure 3:

Figure 3:

Open in a new tab

Approximate site of the screw placement on the guinea pig skull; posterior to the orbit and anterior to the external auditory meatus.

Figure 4:

Figure 4:

Open in a new tab

Surgical photograph of the head holder device in use. The head holder is making contact with the guinea pig’s preauricular skin bilaterally. The right ear is surgically open, exposing the bulla with a drill hole. The retractor is out of the way and allowing for an unobstructed view of the round window through the drill hole.

A postauricular surgical approach was used to expose the bulla and view the RWM. With the head holder secure, a syringe with a hollow microneedle affixed to the tip was mounted onto a UMP3 UltraMicroPump (World Precise Instruments, Sarasota, FL, USA), and the pump was fixed to a micromanipulator. The microneedle was then gradually advanced toward the RWM. The RWM was perforated and 1 μl of perilymph was aspirated from the cochlea over the course of 45 seconds. Head motion was evaluated visually through stereotaxic microscope at 24x magnification. Following surgery, the guinea pigs were assessed for complications through once daily monitoring for signs of pain or distress, abnormal circling behavior indicating dizziness, and general activity. The surgical site and sites of head holder application were assessed daily as being clean, dry, without erythema, edema, purulence, or signs of dehiscence. No animals showed any signs of infection.

72 hours after aspiration, the animals were sacrificed with pentobarbital overdose.

2.4. Confocal microscopy of perforated membranes

Temporal bones were harvested immediately after euthanasia using blunt dissection and were fixed for 1 hour in 10% formalin and stored in phosphate-buffered saline (PBS). Temporal bones were drilled open using Stryker S2 πDrive drill (Stryker, Kalamazoo, MI, USA) to expose the RWM. Fixed RWMs were soaked in 1 mM rhodamine B, a fluorescent stain selective for elastic tissue, for 5 minutes and then rinsed with PBS and soaked in PBS for 15 minutes. Confocal imaging was performed with a Nikon A1R scanning confocal microscope (Nikon Instruments, Melville, NY, USA). Images were projected in the stacking direction with maximum intensity to view a flattened membrane and examined for perforation closure. An excitation wavelength of 561 nm was chosen for the laser, and emitted light from 570 to 620 nm was allowed to pass to the detector. A stack of images was generated at several focal heights spaced 5 mm and 1 mm apart for the x10 objective. At x10 magnification, the pinhole was set at 0.8 Airy unit (AU) to create an optical Z section of 6.35 mm. A qualitative visual assessment was performed to assess the degree of RWM tearing from microneedle insertion as evaluated by confocal microscopy imaging served as an additional measure of head holder efficacy (Figure 5).

Figure 5:

Figure 5:

Open in a new tab

Confocal images of guinea pig RWM 72 hours following surgery. Left image shows a tear of the membrane caused by head movement during surgery without use of the head holder. Right image shows a well healed perforation with use of head holder.

3. RESULTS

The guinea pigs on whom the head holder was applied in this investigation were part of a study evaluating the effectiveness of hollow microneedles in aspirating perilymph from the inner ear without anatomic or functional injury. The head holder design efficaciously accommodated various adolescent guinea pig head sizes and enabled simultaneous access to nose cone anesthesia and audiologic testing. The absence of ear bars allowed for intraoperative auditory testing using distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) [10]. The two-screw design of the head holder created enough space, while maintaining immobilization, for microphone insertion into the external auditory meatus and probe placement on the cochlea through the postauricular surgical opening for these auditory tests both before and after perforating the RWM [10]. The modularity of the device allowed it to be used effectively in a crowded surgical set up and the device was easy to adjust on the spot to provide desired positioning.

In the two guinea pigs undergoing RWM procedure without the head holder, there was significant head movement associated with larger uncontrolled tears in RWM. Confocal microscopy of these two RWM samples from surgeries performed without the head holder demonstrated large tears with granulation tissue (Figure 5). Furthermore, and consistent with the large tears to the RWM observed in the two guinea pigs in which the head holder was not used, there was damage to hearing. The head holder was implemented immediately after the failure of these two experiments to create precise RWM perforations because hearing preservation was paramount to the perilymph aspiration experiments. Visual inspection of the guinea pig heads under stereotaxic microscope at 24x confirmed virtually no head motion from breath movement with the head holder compared to obvious head motion without the head holder. The resultant perforations viewed under confocal microscopy were precise, small, and well-healed (n=22) when compared to the perforations made without the head holder (Figure 5). Harvested RWMs evaluated at 72 hours following initial surgery demonstrated no tearing and no visible granulation tissue around perforations in experiments that used the head holder (n=22). Immobilization of the head with the head holder allowed the microneedle tip to remain in the RWM for multiple breath cycles without tearing the RWM.

4. DISCUSSION

The head holder described herein is a cost-effective, innovative, and modular solution for small animal ear and brain surgery that requires head immobilization and nose cone anesthesia. The slim C-shaped design with two fixation points on the skull gives the researcher tremendous flexibility in choosing a surgical setup while providing reliable immobilization that is safe for survival surgeries. It provides stability without injuring the animal as it contacts the skin above the skull anterosuperior to the external acoustic meatus without piercing it or damaging soft tissue (Figure 3). Possible injuries and complications from use of the head holder include injury to the animal’s eye, soft tissue damage, infection, and skull fractures, but if used appropriately these risks are small, as demonstrated by the uncomplicated and safe usage described in these experiments. The current design overcomes limitations of many commercially available stereotaxic head holders for small animal surgery by permitting simultaneous access of the ear and the snout. Other head holder designs rely on fixation using the hard palate, incisors, masticators, or the back of the nose, which all block the nose and mouth from receiving inhaled anesthesia via nose cone [1–5]. Fried et al. used a U-shaped head holder design with 4 pairs of bolts applied to side of mouse skull, without ear bars or nose clamp, to allow for rostral and unimpeded ear access; however, their design used a mouth bar preventing simultaneous nose cone anesthesia [1]. Thus, the current design, by avoiding mouth and nose clamps, ear bars, and bolt fixation to the skull to secure the head, permits delivery of nose cone anesthesia, excellent head immobilization, and surgical access to the ear and cranium.

A significant limitation of the study is the use of two control animals. The head holder was developed in response to an issue faced as part of a guinea pig perilymph aspiration study [10]. In the first two experiments in this study, during perforation and perilymph withdrawal, head movement associated with the guinea pig’s respiration led to large tears in the RWM and hearing loss. The head holder was designed to in response to minimize head movement to facilitate these experiments. Therefore, only two controls were used because once the head holder was implemented into the perilymph aspiration surgeries, there were no longer tears to the RWM. The two control animals’ weights were 186.2 g and 258.0 g, within range of the animals on which the head holder was used (mean weight: 235.6 g; standard deviation: 34.9 g).

The head holder was designed using SolidWorks 2019 software and printed on a 3D-printer, which are resources that have become increasingly available to all researchers. The ubiquitous availability of CAD software and 3D-printers allows researchers to quickly and inexpensively print this device for immediate use in their lab. The full cost to print and configure our head holder with all materials and screws was less than $50 USD. The design has the flexibility to accommodate different head sizes of guinea pigs; the guinea pigs used in this study ranged between 150 and 310 grams. For smaller or larger animals such as mice, rats, or cats, the design is versatile and could be scaled, although it is not currently parametric and other animal species were not tested in this study. This device showed no signs of wear throughout or after the 22 surgeries, but it should be noted that because it is plastic, it potentially has less longevity than a traditional metal variant. Further testing with a single device would enable longer-term observation for signs of wear, such as loosening of grip from repeated insertion of screws, and allow for a better understanding of longevity.

As 3D-printing technology is increasingly leveraged within medical research, it will more frequently provide greater customization of surgical tools. The current head holder is a case study of how a customized 3D printed surgical device designed for specific experimental requirement can more effectively drive valuable research, effect care, and impact outcomes. In our case, we were able to overcome the tearing of the RWM by designing and implementing a head holder that effectively immobilized the head and provided access to the ear. Looking forward, it is possible to imagine similar 3D-printed devices or iterations of this device solving numerous specific issues in small animal surgeries. The files for the head holder are available upon request by emailing the corresponding author.

Acknowledgements

The authors gratefully acknowledge support by the National Institutes of Health (NIH) National Institute on Deafness and Other Communication Disorders (NIDCD) with award number R01DC014547. The authors would also like to thank Prof. Elizabeth Olson, Robert Stark, Bill Miller and Albert Tai for use of their facilities and materials.

Footnotes

Conflicts of Interest

Dr. Anil K. Lalwani serves on the Medical Advisory Board for Advanced Bionics and on the Surgical Advisory Board for MED-EL. For the remaining authors, no conflicts of interest were declared.

REFERENCES

  • 1.Fried H-U, Linnig H-D, and Korsching SI, An inexpensive mouse headholder suitable for optical recordings. Physiology & Behavior, 2001. 74(3): p. 253–255. [DOI] [PubMed] [Google Scholar]
  • 2.Gray DS and Cheung BS, An inexpensive surgical headholder for the rat. Physiology & Behavior, 1982. 28(1): p. 181–182. [DOI] [PubMed] [Google Scholar]
  • 3.Haidarliu S, An anatomically adapted, injury-free headholder for Guinea pigs. Physiology & Behavior, 1996. 60(1): p. 111–114. [DOI] [PubMed] [Google Scholar]
  • 4.Haidarliu S, Ahissar E, and Saraf-Sinik I, A self-adjusting head holder without ear-bars for guinea pigs. The Journal of Physiological Sciences, 2018. 68(6): p. 875–880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Szlávik L and Tóth S, New headholder for sensory stimulation and stereotaxic operations in small animals. Physiology & Behavior, 1974. 13(6): p. 849–853. [DOI] [PubMed] [Google Scholar]
  • 6.Szeto B, Chiang H, Valentini C, Yu M, Kysar JW, Lalwani AK. Inner ear delivery: Challenges and opportunities. Laryngoscope Investigative Otolaryngology. 2019; 1–10. 10.1002/lio2.336 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wazen JM, et al. , Silver/silver chloride microneedles can detect penetration through the round window membrane. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2017. 105(2): p. 307–311. [DOI] [PubMed] [Google Scholar]
  • 8.Aksit A, et al. , In-vitro perforation of the round window membrane via direct 3-D printed microneedles. Biomedical microdevices, 2018. 20(2): p. 47–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yu M, et al. , Anatomical and Functional Consequences of Microneedle Perforation of Round Window Membrane. Otology & Neurotology, 2020. 41(2): p. e280–e287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Szeto B, Aksit A, Valentini C, Yu M, Werth EG, Goeta S, Tang C, Brown LM, Olson ES, Kysar JW, Lalwani AK. Novel 3D-printed hollow microneedles facilitate safe, reliable, and informative sampling of perilymph from guinea pigs. Hearing Research, 2021. doi: 10.1016/j.heares.2020.108141.Epub 2020 Dec 2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Matouk CC, et al. , Skull fracture secondary to application of a Mayfield skull clamp in an adult patient: Case report and review of the literature. Clinical Neurology and Neurosurgery, 2012. 114(6): p. 776–778. [DOI] [PubMed] [Google Scholar]

RESOURCES