Challenges of transsphenoidal pituitary surgery in severe brachycephalic dogs.

Introduction: Transsphenoidal hypophysectomy is the standard surgical technique for the excision of pituitary neoplasms. Anatomy may be more obscured in brachycephalic skull types due to the crowding of soft tissue and osseous structures. We describe the unique challenges to approach the sphenoid bone and localize the correct burr hole site in severe brachycephalic dogs. Materials and methods: A single institution retrospective case series of brachycephalic dogs with pituitary-dependent hypercortisolism (PDH). Preoperative computed tomography enabled 3D-, and cross-sectional reconstruction to plan and dry-practice the position of the ideal burr hole in relation to the sella turcica, pterygoid hamular processes, and hard palate. Rostral burring of the caudal hard palate obscuring the direct sphenoid approach necessitated adaptations to the original transsphenoidal hypophysectomy procedure. Postoperative outcomes and complications with respect to those seen in mesocephalic dogs are described. Results: Ten brachycephalic dogs including French Bulldogs (n = 9) and a single Dogue de Bordeaux were included. All dogs were diagnosed with PDH and had preoperative advanced imaging performed on the skull. All but one dog had an enlarged pituitary gland, with a median pituitary/brain value of 0.5 (range 0.21-0.9). A total of 11 transsphenoidal hypophysectomy procedures were performed in these 10 dogs. Rostral extension of the soft palate incision into the hard palate was performed to access the burr hole site on the sphenoid bone. Major complications included aspiration pneumonia (n = 1), severe gastroesophageal reflux (n = 1), and central nervous signs (=1). All dogs survived until discharge, with a median time to follow-up of 618 days (range 79-1,669 days). Seven dogs experienced long-term remission of PDH. Conclusion: Brachycephalic dogs undergoing transsphenoid al hypophysectomy benefit from meticulous presurgical planning and extension of the approach into the caudal hard palate. Advanced surgical skills can render a good outcome in a technically challenging environment.

Comparison of Single versus Double Lateral Plating in Treatment of Feline Ilial Fractures Using Veterinary Cuttable Plates.

OBJECTIVE: The aim of this study was to compare the frequency of implant failure and the extent of pelvic canal narrowing associated with the fixation of ilial fractures in cats with a single veterinary cuttable plate (SLP) or double veterinary cuttable plates (DLP) applied to the lateral surface of the ilium. STUDY DESIGN: Radiographic evaluation of feline ilial fractures plated laterally using SLP or DLP. Pelvic canal narrowing directly postoperatively and at 6 weeks follow-up was objectively measured using the sacral index (SI). Radiographs were evaluated for implant failure and fracture healing. RESULTS: Seventy-seven cats satisfied the inclusion criteria. Twenty-nine fractures were treated with a SLP and 48 with DLP. Implant failure occurred significantly more (p = 0.001) in the SLP group (14/29) compared with the DLP group (6/48). Follow-up SI was significantly different between the two groups (p = 0.048, SLP median: 1.0 range: 0.83-2.4, DLP median: 0.98; range: 0.76-1.45). Median change in SI was -0.04 (range: -1.4 to 0.05) in the SLP group and 0.0 (range: -0.23 to 0.23) in the DLP group. This difference was significantly different (p = 0.031). CONCLUSION: DLP leads to significantly less implant failure and significantly less pelvic canal narrowing compared with SLP. This difference in pelvic canal narrowing was small and the clinical relevance remains unclear.

Expression and clinical relevance of paired box protein 7 and sex determining region Y-box 2 in canine corticotroph pituitary adenomas.

Pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, caused by an adrenocorticotrophic hormone secreting pituitary tumour of the anterior or intermediate lobe. The prognosis of intermediate lobe adenomas is worse than that of anterior lobe adenomas, indicating the possible usefulness of melanotropic markers as prognosticators. Another possible origin of pituitary adenomas is found in cancer stem cells. The aim of the present study was to investigate the expression of melanotroph specific transcription factor paired box protein 7 (Pax7) and stem cell marker and reprogramming factor sex determining region Y-box 2 (Sox2) and to relate their expression to clinical parameters. The mean ± SD of labelling index (LI) for Pax7 was 8.6% ± 21.7% in the adenomas; 1/6 controls had positive staining (LI, 15.2%). For Sox2, the LI in the adenomas was 16.9% ± 15.2% and 19.5% ± 11.6% in the controls. Pax7 expression was significantly higher in enlarged pituitaries, compared to non-enlarged pituitaries (P = 0.05), but Pax7 or Sox2 immunopositivity did not correlate to other clinical parameters such as histological diagnosis, survival time or disease-free interval. Gene expression of Pax7 target genes, such as proconvertase 2 (PC2), pro-opiomelanocortin (POMC), and dopamine D2 receptor (DRD2), was significantly lower in the adenoma samples compared to normal tissue, indicating that Pax7 signalling was not activated in adenomas. It was suggested that Pax7 and Sox2 remain interesting targets for molecular investigations into their role in pituitary tumorigenesis, but were unsuitable as clinical prognosticators in dogs.

Expression stability of reference genes for quantitative RT-PCR of healthy and diseased pituitary tissue samples varies between humans, mice, and dogs.

Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.

Stem cells in the canine pituitary gland and in pituitary adenomas.

Cushing's disease (CD) or pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, with an estimated prevalence of 1 or 2 in 1000 dogs per year. It is caused by an adrenocorticotropic hormone secreting adenoma in the pars distalis or pars intermedia of the pituitary gland. The pituitary gland is a small endocrine gland located in the pituitary fossa. In the postnatal individual, the hypothalamus-pituitary axis plays a central role in maintaining homeostatic functions, like control of metabolism, reproduction, and growth. Stem cells are suggested to play a role in the homeostatic adaptations of the adult pituitary gland, such as the rapid specific cell-type expansion in response to pregnancy or lactation. Several cell populations have been suggested as pituitary stem cells, such as Side Population cells and cells expressing Sox2 or Nestin. These cell populations are discussed in this review. Also, stem and progenitor cells are thought to play a role in pituitary tumorigenesis, such as the development of pituitary adenomas in dogs. There are limited reports on the role of stem cells in pituitary adenomas, especially in dogs. Further studies are needed to identify and characterize this cell population and to develop specific cell targeting therapeutic strategies as a new way of treating canine CD.

Complex structure of engineered modular domains defining molecular interaction between ICAM-1 and integrin LFA-1.

Intermolecular contacts between integrin LFA-1 (α(L)ß(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the ß-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.

Identification and characterisation of side population cells in the canine pituitary gland.

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.