Analyzed specimens represent narrow-ridged finless porpoises (N.asiaeorientalis) that were killed incidentally in fishing nets or were found washed ashore. They were successively collected in Dalian with the permission from Chinese Authorities for Animal Protection. The study of these carcasses was approved by the Ethics Committee of Dalian Medical University. All of the collected dead bodies were conducted arterial perfusion through the aorta with 10% formalin solution. All methods were carried out in accordance with relevant guidelines and regulations.
Methods
CT three-dimensional reconstruction
Two specimens’ heads and necks were continuously scanned by GE 128-row VCT, and dual phase serial computer tomography (CT) images were obtained, the slice thickness and pitch were set to 0.6mm. The images were analyzed for modeling and reconstruction in MIMICS software (MIMICS 18.0.0.525, Materialise, Leuven, Belgium).
Dissection of postoccipital region
Four specimens were dissected layer-by-layer at the posterior occipital region to expose the atlanto-occipital interspace. A dorsal midline incision was made at the neck, the skin, subcutaneous fascia, and superficial neck muscles were gradually removed to expose deep post-occipital musculature. Subsequently, the rectus capitis dorsalis (RCD) was detached from its cranial attachment carefully to reveal the other muscle lying deep. The musculature and other structures in the atlanto-occipital interspace along with a part of cervical spinal dura mater were isolated as tissue block by an electric handsaw. The tissue blocks were preserved for histology and scanning electron microscope. The photographic documentations were carried out by Canon 7D and 450D camera.
P45 sheet plastination
One specimen of N.asiaeorientalis was sliced in sagittal section for p45 sheet plastination. The P45 sections are semi-transparent, durable slices with a clear delineation of the tissue morphology including the connective tissues [28]. Anatomical structures in posterior occipital region and the connections between the postoccipital muscles and cervical spinal dura mater were observed. The experimental procedure of the technique is described as follow [29]:
Slicing.
The embalmed specimens of the head and neck were frozen at -70℃ for two weeks and then embedded in polyurethane foam and frozen at -70℃ again for two days. After freezing, 3 mm sagittal slices were made from side to side with a high-speed band saw.
Bleaching.
All the slices were rinsed overnight in cold running water, and afterwards, the slices were immersed in 5% dioxogen overnight.
Dehydration. After bleaching, the slices were dehydrated with 100% acetone by the freeze substitution method.
Casting and forced impregnation.
After dehydration, the casting mold was prepared. The slices were lifted from the acetone bath and placed between two glass plates. The molds were then filled with polyester (Hoffen polyester P45, Dalian Hoffen Bio-Technique Co. Ltd., Dalian, P. R. China.).
The filled mold was placed upright into a vacuum chamber at room temperature for impregnation. The absolute pressure was slowly decreased to 20, 10, 5, and 0 mm Hg, according to the rate of bubble releasing. The pressure was maintained at 0 mm Hg until bubbling ceased. The impregnation time lasted for more than eight hours.
Curing.
After the vacuum was released, the air bubbles within the sheets were checked and removed. The top of the mold was clamped with large fold back clamps, and the sheet was then ready for curing. The sheets were cured using a heated water bath and were placed upright in the water bath at 40℃ for 3 days. After curing, the sheets were removed from the bath and cooled to room temperature in a rack. The slices were then removed from the flat chamber and covered appropriately with adhesive plastic wrap for protection. The sheets were then observed and photographed.
Histological study
Two tissue blocks were prepared containing the postoccipital musculature, the periosteum of the adjacent cervical vertebrae and the occiput, adjoining spinal dura mater, and spinal cord. After washed in running water overnight, these tissue blocks were dehydrated with ethanol in increasing grades, passed through xylene, infiltrated and then embedded in paraffin wax. A rotary microtome was used to cut 10-µm-thick sections. Sections were mounted on the glass microscope slides, then rehydrated for Van Gieson (VG, picric acid and acid fuchsin) staining. The staining sections were analyzed and photographed under a Nikon Eclipse 80i light microscope, with the support of Nikon NIS image software.
Scanning electron microscopic study
Through layer-by-layer dissection, two tissue blocks were used for scanning electron microscope (SEM) study. After washing in running water overnight, the specimens were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer at PH 7.3 for more than 2h. Then, the specimens were repeatedly washed in the buffer solution. They were subsequently dehydrated through a graded alcohol series, vacuum dried with 100% tert-butyl alcohol, and sputter-coated with platinum by ION SPUTTER JFC-1100 ion sputtering equipment. Tissues of the specimens were observed under a scanning electron microscope (model FEI QUATA 200, voltage:20KV, manufacture: the Netherlands FEI company). Fibers connections were photographed, digitized and analyzed.