The study assessed the histological effects of perineural injections of propylene glycol and three medical gels in a mouse model utilizing ultrasound guided microinjection techniques.
The study was approved by the University of Queensland Animal Ethics Committee (Anatomical Biosciences) according to the principles of the National Health and Medical Research Council’s Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th Edition 2004) and the Animal Care and Protection Act (2001).
Perineural injection model
Micro-ultrasound guided perineural injection was performed in six to eight week old female CD1 wild-type mice.
The mice were anesthetized with 5% isoflurane in oxygen in an anesthetizing chamber and maintained with 1.5% isoflurane by nose cone for the duration of the procedure. The fur over the hind limb was clipped with an animal clipper. The mouse was turned to the lateral position and the hind limb was passed through an orifice in the bottom of a Petri dish sealed with a silicone membrane. The skin was prepared with 70% ethanol and allowed to dry. The Petri dish was filled with sterile phosphate buffered saline (PBS) to act as an ultrasound conduction medium for imaging, thus avoiding the use of ultrasound gel.
A Vevo 770 (VisualSonics, Canada) micro-ultrasound machine with a 25 MHz probe was used to image a cross section of the sciatic nerve in the proximal hind limb. The leg was scanned proximally and distally to ensure consistent identification of the sciatic nerve. The sciatic nerve can be identified as a circular structure running in the fascial layer deep to the biceps femoris muscle at the mid-thigh (Figure 1).
A 30 G needle was inserted into the thigh using an in-plane technique and the tip was positioned 0.5 mm away from the nerve, deep to the biceps femoris muscle (Figure 2). 0.1 ml of the test substance was injected at a single point. The injected fluid spread over the nerve (Figure 3). The preparation was mixed with 12.5 mcl/ml of 2 micrometre Fluoresbrite® Yellow Green Microspheres (Polysciences Inc, Warrington PA, USA). These inert, non-toxic polystyrene and latex microspheres are readily visible under ultraviolet light. They served as a visual marker of injection location during the dissection stage [16, 17]. The investigators performing the injections were not blinded to the injected fluid.
The animals were kept for three days until euthanasia was performed. Three days was chosen because Schwann cell changes and macrophage migration begin to be visible two days after peripheral nerve injury [18]. After the initial myelin extrusion, Schwann cell division is maximal at three days [18]. The sciatic nerve was dissected with care to prevent crushing. The location of the injection around the sciatic nerve was confirmed by examination under ultraviolet light demonstrating fluorescence of the microspheres around the sciatic nerve. This area of nerve was sampled for histological assessment. The nerve was fixed and processed for histology. Semi-thin Toluidine blue transverse plastic sections were examined for nerve injury by two blinded neuropathologists. The nerves were assessed for axonal degeneration, myelin degeneration, intraneural and perineural inflammation on a qualitative scale (present, possible or absent).
Propylene glycol concentration-neurotoxicity relationship
The first stage assessed the relationship between the concentration of PG injected and histological evidence of neurotoxicity in 42 animals.
Propylene glycol (Sigma Aldrich, Australia) was diluted with saline to make five solutions of 2.5%, 10%, 35%, or 70% (v/v) in addition to a saline control. For each concentration, eight animals had 0.1 ml of solution injected around one sciatic nerve. One additional experiment was performed with a saline control and 70% propylene glycol (n=9).
Neurotoxicity assessment of gels
The second stage of the project was to perform perineural injection of three sterile gels: Aquasonic® 100 Ultrasound Transmission Gel (Parker Laboratories, USA); K-Y® Lubricating Jelly (Johnson & Johnson, USA); and PDI® Lubricating Jelly (Professional Disposables International, USA).
Each gel was tested in eight animals (n=24). Each animal had bilateral perineural injections with saline on one side and gel on the other. Sample size calculations were not performed because no data was available to provide an estimate of effect size. Descriptive statistics were used.