Fig 1.
Dorsal approach of the left frontal sinus for olfactory mucosa harvesting and later spinal cord transplantation, in a dog with chronic paralysis.
The nasal and frontal bones are cut above the nasal cavity and frontal sinus to form a bone flap. The periosteal layer (black arrows) is preserved to be sutured at the end of the procedure. The rostral aspect of the bone flap is kept intact (white arrow). A hole (visible on the cranial aspect of the bone flap) has been made using surgical drill to suture the bone flap back into position at the end of the procedure.
Fig 2.
Surgical approaches to the frontal sinus.
(A) frontal sinus region of a dog prepared for surgery; the frontal sinus can be approached caudo-laterally through an incision above the left eye (red curved line) and then through the temporal bone, or more classically along the nose midline through the nasal bone (yellow shape and dashed red-line–see Fig 1 as well); (B) lateral view of a dog cadaver showing the incised left temporal muscle detached and elevated from the temporal bone to expose the cranial part of the temporal bone that forms the caudal wall of the frontal sinus (location suggested by the green shapes); a circular bony window (black arrow) was drilled in the caudal wall of the frontal sinus to access it; the window allowed the insertion of biopsy instruments (alligator or long curved forceps); the blue shapes represent the nasal cavities; (C) lateral radiograph showing the anatomical relationship between the frontal sinus (red dotted line), the nasal cavities (green arrows) and the cribriform plate (yellow dotted curved line) just cranial to the brain; (D) caudal view of a dog cadaver with the left frontal sinus open, showing the difference between the pink pale respiratory mucosa (*) and the brown olfactory mucosa (white arrow) deeper in the cranial aspect of the frontal sinus and communicating through the ostium with the nasal cavity; (E) 5-day post-operative view of a dorso-lateral incision above the left eye made to collect olfactory mucosa from the frontal sinus in a paraplegic dog, weighing > 10 kg.
Fig 3.
Endoscopic approach to the olfactory mucosa via the natural nasal passage.
(A) dogs were positioned in sternal recumbency and one side of the nasal passages was washed with sterile saline containing gentamicin; (B) silicon tube attached to the end of the endoscope. The space inside the tube kept the vision clear and allowed clean biopsy of olfactory mucosa; (C) pale-coloured respiratory mucosa could initially be seen; (D) brown-coloured olfactory mucosa could be observed more caudally in the nasal cavity; (E)(F) harvesting olfactory mucosa using endoscopic biopsy forceps without damaging other structures; (G) bleeding after biopsy; (H) fragments of olfactory mucosa biopsied and placed in L15 medium.
Table 1.
Dog breeds included in the three different methods.
Fig 4.
Immunocytochemical and morphological characteristics of olfactory mucosa cell cultures obtained from three different methods.
All images are from in vitro cell cultures except image I obtained from an olfactory mucosa biopsy. Upper layer (A)(B)(C): cells obtained via rhinotomy; middle layer (D)(E): cells obtained via keyhole approach; and right middle and bottom layer (F)(G)(H): cells obtained via endoscopy; (I) immunohistochemistry of olfactory mucosa. Immunocytochemical characteristics of olfactory mucosa cell cultures obtained by rhinotomy in three dogs, showing variation in p75 positive cell (red) purity (A)(B)(C); images show a purity of ~25% (A), of ~50% (B) and of ~75% (C). Morphological characteristics of olfactory mucosa cells obtained by keyhole approach in bright light (D): note the spindle-shaped bipolar cells that form the majority of cells. Olfactory mucosa cell cultures obtained by the keyhole approach which are double stained with p75 and fibronectin (E): spindle-shaped bipolar cells expressed p75. The p75-positive cells obtained by endoscopy were morphologically and immunocytochemically identical to the p75-positive cells obtained using the other two methods (F)(G)(H). Most of the p75 positive cells were also positive for GFAP (G)(H). In rare instances, other cells with a bipolar and elongated morphology were solely GFAP-positive (white arrows in G). Immunofluorescence of the olfactory mucosa showed p75-positive area (cells) in the olfactory epithelium (OE) layer (I). LP: lamina propria. Bar = 50μm.
Fig 5.
Proportion and population of each phenotype of cells obtained by three different methods.
Upper histogram: number of cells (black). Lower histogram: corresponding proportion (%) of p75-positive cells (in red), fibronectin positive cells (in green) and unidentified cells (in blue) for each dog in different method.
Fig 6.
Comparison of proportion (left) and population (right) of p75 positive cells in between different harvesting methods. There were significant differences in p75-positive cell proportion and population in between cultures obtained by rhinotomy and endoscopy (P<0.01, **; P<0.05, *).
Fig 7.
Correlation between olfactory ensheathing cell yield (population and proportion) and age, or olfactory ensheathing cell yield (population and proportion) and body weight.
No correlation was observed.
Table 2.
Summary of Cell Proportion and Phenotypic Characteristics in Cultures Obtained by Rhinotomy (n = 27), Keyhole approach (n = 7) and Rhinoscopy (Cadaver n = 12, Living dog n = 7) at 21 Days In Vitro.
Table 3.
Summary of Estimated Cell Population and Phenotypic Characteristics in Cultures Obtained by Rhinotomy (n = 27), Keyhole approach (n = 7) and Rhinoscopy (Cadaver n = 12, Living dog n = 7) at 21 Days In Vitro.