Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Diagnostic immunohistochemistry of primary and secondary central nervous system neoplasms of dogs and cats.

The diagnosis of primary and secondary CNS neoplasms of dogs and cats relies on histologic examination of autopsy or biopsy samples. In addition, many neoplasms must be further characterized by immunohistochemistry (IHC) for a more refined diagnosis in specific cases. Given the many investigations assessing the diagnostic and prognostic IHC profile of CNS neoplasms in the veterinary literature, it may be difficult for the diagnostic pathologist or pathology trainee to narrow the list of reliable diagnostic IHCs when facing a challenging case. Here we compile a comprehensive list of the most diagnostically relevant immunomarkers that should be utilized for the diagnostic support or confirmation of the most common primary and secondary CNS neoplasms of dogs and cats.

Case report: The first description of a Dieulafoy's lesion in the gastric mucosa of a dog.

An approximately 12-year-old, 31 kg, male neutered Labrador Retriever was presented to the referring hospital with an acute onset (less than 1 day) of hematemesis and melena. The dog was treated supportively for a presumptive gastric ulcer for 4 days with intravenous fluids, gastro protectants, such as pantoprazole, misoprostol, sucralfate, and barium, as well as an anti-emetic (maropitant) and analgesics (fentanyl, gabapentin, and tramadol). Throughout medical management, the dog continued to require blood transfusions approximately every 24 h. Given the poor medical response, the patient was subjected to an exploratory laparotomy. During surgery, a grossly raised, blister-like lesion on the mucosal surface of the stomach was appreciated on the lesser curvature of the stomach. A partial gastrectomy was performed, and the segment was submitted for histological evaluation. Histologically, there were multiple, tortuous, medium-caliber muscular arteries (>1.0 mm in diameter) in the submucosa. A single large-caliber artery (>0.75 mm in diameter) containing a partially occlusive thrombus extruded through the mucosa and projected on the ulcerated surface. The patient's signs were similar clinically and histopathologically to Dieulafoy's lesion in people. A Dieulafoy's lesion is a potentially life-threatening disorder that causes gastrointestinal (GI) hemorrhage. This lesion is characterized by a dilated, large-caliber, aberrant submucosal artery that erodes through the epithelium and ruptures, resulting in massive and potentially fatal hemorrhage. This lesion has never been documented previously in a dog.

Safety and interim survival data after intracranial administration of M032, a genetically engineered oncolytic HSV-1 expressing IL-12, in pet dogs with sporadic gliomas.

OBJECTIVE: The diagnosis of glioma remains disheartening in the clinical realm. While a multitude of studies and trials have shown promise, improvements in overall survival have been disappointing. Modeling these tumors in the laboratory setting has become increasingly challenging, given their complex in situ behavior and interactions for therapeutic evasion. Dogs, particularly brachycephalic breeds, are known to spontaneously develop gliomas that resemble human gliomas both clinically and pathophysiologically, making canines with sporadic tumors promising candidates for study. Typically, survival among these dogs is approximately 2 months with palliation alone. METHODS: The authors have completed the first stage of a unique phase I dose-escalating canine clinical trial in which the safety and tolerability of M032, a nonneurovirulent oncolytic herpes simplex virus-1 vector genetically engineered to express interleukin-12, are being studied in pet dogs with gliomas undergoing maximum safe tumor resection and inoculation of the cavity with the viral infusate. RESULTS: Twenty-five canine patients were enrolled between January 2018 and August 2020. One patient was electively withdrawn from the trial by its owner, and 3 did not receive the virus. For the 21 dogs that remained, 13 had high-grade gliomas, 5 had low-grade gliomas, and 3 were undetermined. According to histopathological analysis, 62% of the tumors were oligodendrogliomas. At the time of this report, the median overall survival from the date of treatment was 151 days (± 78 days). No significant adverse events attributable to M032 or dose-limiting toxicities have been observed to date. CONCLUSIONS: In this largest study of oncolytic viral therapy for canine brain tumors to date, treatment with M032 did not cause harm and the combination of surgery and oncolytic viral therapy may have contributed to prolonged survival in pet dogs with spontaneous gliomas. Forthcoming in-depth radiographic, immunohistochemical, and genetic analyses will afford a more advanced understanding of how this treatment impacts these tumors and the immune system. Our goal is to utilize these findings bitranslationally to inform human studies and refine therapies that will improve outcomes in both humans and pet dogs with gliomas.

Generating primary murine vaginal fibroblast cell lines.

Primary human vulvovaginal fibroblast cell lines are useful for studying biological mechanisms underlying genital pain, pelvic organ prolapse, and the spread of sexually transmitted infections. However, the vaginal biopsies necessary for establishing these cell lines are invasive and relatively difficult to obtain. Primary mouse fibroblast cell lines derived from pre-clinical animal models of these conditions can be used for better controlled experiments that can help us dissect disease mechanisms. To our knowledge, there are no published protocols for establishing primary murine vaginal fibroblast cell lines to date. Here, we describe a protocol for the establishment of murine vaginal fibroblast cell lines via enzymatic digestion of vaginal canal tissue. Cell lines generated using this method can be used for in vitro studies of these important structural cells in a variety of pre-clinical mouse models; such studies are required to identify and characterize relevant regulatory and therapeutic targets in a wide array of diseases of interest. As shown in our representative data, this protocol yields viable cell lines from ND4 Swiss outbred mice. These cells bear surface markers characteristic of fibroblasts and are capable of producing inflammatory cytokines in response to treatment with bacterial and yeast antigens in vitro.

Volume and Infusion Rate Dynamics of Intraparenchymal Central Nervous System Infusion in a Large Animal Model.

Thalamic infusion of adeno-associated viral (AAV) vectors has been shown to have therapeutic effects in neuronopathic lysosomal storage diseases. Preclinical studies in sheep model of Tay-Sachs disease demonstrated that bilateral thalamic injections of AAV gene therapy are required for maximal benefit. Translation of thalamic injection to patients carries risks in that (1) it has never been done in humans, and (2) dosing scale-up based on brain weight from animals to humans requires injection of larger volumes. To increase the safety margin of this infusion, a flexible cannula was selected to enable simultaneous bilateral thalamic infusion in infants while monitoring by imaging and/or to enable awake infusions for injection of large volumes at low infusion rates. In this study, we tested various infusion volumes (200-800 µL) and rates (0.5-5 µL/min) to determine the maximum tolerated combination of injection parameters. Animals were followed for ∼1 month postinjection with magnetic resonance imaging (MRI) performed at 14 and 28 days. T1-weighted MRI was used to quantify thalamic damage followed by histopathological assessment of the brain. Trends in data show that infusion volumes of 800 µL (2 × the volume required in sheep based on thalamic size) resulted in larger lesions than lower volumes, where the long infusion times (between 13 and 26 h) could have contributed to the generation of larger lesions. The target volume (400 µL, projected to be sufficient to cover most of the sheep thalamus) created the smallest lesion size. Cannula placement alone did result in damage, but this is likely associated with an inherent limitation of its use in a small brain due to the length of the distal rigid portion and lack of stable fixation. An injection rate of 5 µL/min at a volume ∼1/3 of the thalamus (400-600 µL) appears to be well tolerated in sheep both clinically and histopathologically.

Differential Expression of miRNAs in Hypoxia ("HypoxamiRs") in Three Canine High-Grade Glioma Cell Lines.

Dogs with spontaneous high-grade gliomas increasingly are being proposed as useful large animal pre-clinical models for the human disease. Hypoxia is a critical microenvironmental condition that is common in both canine and human high-grade gliomas and drives increased angiogenesis, chemo- and radioresistance, and acquisition of a stem-like phenotype. Some of this effect is mediated by the hypoxia-induced expression of microRNAs, small (~22 nucleotides long), non-coding RNAs that can modulate gene expression through interference with mRNA translation. Using an in vitro model with three canine high-grade glioma cell lines (J3T, SDT3G, and G06A) exposed to 72 h of 1.5% oxygen vs. standard 20% oxygen, we examined the global "hypoxamiR" profile using small RNA-Seq and performed pathway analysis for targeted genes using both Panther and NetworkAnalyst. Important pathways include many that are well-established as being important in glioma biology, general cancer biology, hypoxia, angiogenesis, immunology, and stem-ness, among others. This work provides the first examination of the effect of hypoxia on miRNA expression in the context of canine glioma, and highlights important similarities with the human disease.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Canine Ependymoma: Diagnostic Criteria and Common Pitfalls.

Reports of canine ependymoma are generally restricted to single case reports with tumor incidence estimated at 2% to 3% of primary central nervous system (CNS) tumors. While most commonly reported in the lateral ventricle, tumors can occur anywhere in the ventricular system and in extraventricular locations. Rosettes and pseudorosettes are a common histologic feature; however, these features can be mimicked by other CNS neoplasms. Thirty-seven potential ependymoma cases were identified in a retrospective database search of 8 institutions, and a histologic review of all cases was conducted. Of 37 cases, 22 candidate cases were further subjected to a consensus histologic and immunohistochemical review, and only 5 of 37 (13.5%) were conclusively identified as ependymoma. The neuroanatomic locations were the lateral ventricle (3/5), third ventricle (1/5), and mesencephalic aqueduct (1/5). Subtypes were papillary (4/5) and tanycytic (1/5). Histologic features included rosettes (5/5), pseudorosettes (5/5), ependymal canals (2/5), tanycytic differentiation (1/5), blepharoplasts (1/5), ciliated cells (1/5), and high nuclear to cytoplasmic ratio (5/5). Immunolabeling for GFAP (4/4) and CKAE1/3 (3/4) was found in pseudorosettes, rosettes, and scattered individual neoplastic cells. Diffuse but variably intense cytoplasmic S100 immunolabeling was detected in 3 of 4 cases. Olig2 intranuclear immunolabeling was observed in less than 1% of the neoplastic cells (3/3). Tumors that had pseudorosettes and mimicked ependymoma included oligodendroglioma, choroid plexus tumor, pituitary corticotroph adenoma, papillary meningioma, and suprasellar germ cell tumor. These findings indicate that canine ependymoma is an extremely rare neoplasm with histomorphologic features that overlap with other primary CNS neoplasms.

A Revised Diagnostic Classification of Canine Glioma: Towards Validation of the Canine Glioma Patient as a Naturally Occurring Preclinical Model for Human Glioma.

The National Cancer Institute-led multidisciplinary Comparative Brain Tumor Consortium (CBTC) convened a glioma pathology board, comprising both veterinarian and physician neuropathologists, and conducted a comprehensive review of 193 cases of canine glioma. The immediate goal was to improve existing glioma classification methods through creation of a histologic atlas of features, thus yielding greater harmonization of phenotypic characterization. The long-term goal was to support future incorporation of clinical outcomes and genomic data into proposed simplified diagnostic schema, so as to further bridge the worlds of veterinary and physician neuropathology and strengthen validity of the dog as a naturally occurring, translationally relevant animal model of human glioma. All cases were morphologically reclassified according to a new schema devised by the entire board, yielding a majority opinion diagnosis of astrocytoma (43, 22.3%), 19 of which were low-grade and 24 high-grade, and oligodendroglioma (134, 69.4%), 35 of which were low-grade and 99 were high-grade. Sixteen cases (8.3%) could not be classified as oligodendroglioma or astrocytoma based on morphology alone and were designated as undefined gliomas. The simplified classification scheme proposed herein provides a tractable means for future addition of molecular data, and also serves to highlight histologic similarities and differences between human and canine glioma.

A case of basal cell carcinoma of the nictitating membrane in a dog.

A case of a basal cell carcinoma (BCC) of the nictitating membrane (NM) in a 9-year-old female spayed dachshund is reported. Computed tomography and resection of the NM followed by cryosurgery was performed. Although uncommon, BCC should be considered as a differential diagnosis for tumors of the NM.

Use of a Barbed Knotless Suture for Laparoscopic Ablation of the Nephrosplenic Space in 8 Horses.

OBJECTIVE: To evaluate use of a knotless suture for laparoscopic closure of the equine nephrosplenic space. STUDY DESIGN: Experimental in vivo study. ANIMALS: Normal horses without previous history of abdominal surgery (n=8). METHODS: The nephrosplenic space was closed under laparoscopic visualization using a unidirectional, barbed 0 metric absorbable suture (copolymer of glycolic acid and trimethylene carbonate). Intracorporeal suturing of the nephrosplenic space was performed in a cranial-to-caudal direction in a simple continuous fashion. Repeat evaluation was performed laparoscopically in 2 horses and by necropsy in 6 horses. The length of closure was measured and nature of the healed tissue was evaluate grossly. RESULTS: Total surgery time was 65-167 minutes (mean ± SD, 89.6 ± 22.6). Suturing time was 30-65 minutes (40.4 ± 16.3). Second laparoscopy in 2 horses was performed at days 198 and 227. Necropsy was performed at day 69-229 postoperatively (132.7 ± 63.0) in 6 horses. The closure measured 12-14 cm in length (13 ± 1) and consisted of mature fibrous tissue bridging the splenic capsule and the nephrosplenic ligament. No residual suture material was identified grossly in any horses. The procedure was easily performed; extracorporeal suture management to hold it taut was unnecessary since the barbs had excellent purchase in the apposed tissues, and intracorporeal knot tying was not required. CONCLUSION: The barbed knotless suture appears to be a valid alternative to facilitate laparoscopic closure of the nephrosplenic space in normal horses; however, further work is necessary to investigate its suitability in clinically affected horses.

Creation of an NCI comparative brain tumor consortium: informing the translation of new knowledge from canine to human brain tumor patients.

On September 14-15, 2015, a meeting of clinicians and investigators in the fields of veterinary and human neuro-oncology, clinical trials, neuropathology, and drug development was convened at the National Institutes of Health campus in Bethesda, Maryland. This meeting served as the inaugural event launching a new consortium focused on improving the knowledge, development of, and access to naturally occurring canine brain cancer, specifically glioma, as a model for human disease. Within the meeting, a SWOT (strengths, weaknesses, opportunities, and threats) assessment was undertaken to critically evaluate the role that naturally occurring canine brain tumors could have in advancing this aspect of comparative oncology aimed at improving outcomes for dogs and human beings. A summary of this meeting and subsequent discussion are provided to inform the scientific and clinical community of the potential for this initiative. Canine and human comparisons represent an unprecedented opportunity to complement conventional brain tumor research paradigms, addressing a devastating disease for which innovative diagnostic and treatment strategies are clearly needed.

Evaluation of the genes encoding CD39/NTPDase-1 and CD39L1/NTPDase-2 in horses with and without abnormal hemorrhage and in horses with pathologic evidence of exercise-induced pulmonary hemorrhage.

BACKGROUND: Exercise-induced pulmonary hemorrhage (EIPH) is a common disorder of equine athletes. The role of polymorphisms in genes encoding hemostasis-regulatory proteins in horses with abnormal hemorrhage is unknown. OBJECTIVES: The goal of this study was to evaluate the genes encoding 2 ectonucleotidases, CD39/NTPDase-1 and CD39L1/NTPDase-2, and one ecto-5' nucleotidase, CD73, in horses with abnormal hemorrhage or pathologic changes consistent with EIPH. METHODS: Twenty-three horses with histories of abnormal hemorrhage, 8 horses with gastrointestinal signs, and 45 healthy horses were evaluated using polymerase chain reaction-based techniques. Formalin-fixed tissues from 21 horses with pathologic changes consistent with EIPH were also evaluated. RESULTS: Three single nucleotide polymorphisms (SNPs) were identified in the gene encoding CD39 and one SNP was identified in the gene encoding CD39L1. No SNPs were identified in the gene encoding CD73. CD39 SNPs were identified in 19 of 20 (95%) horses with unexplained hemorrhage and 20 of 21 (95%) horses with pathologic features consistent with EIPH. CD39L1 SNPs were identified in 6 of 20 (30%) horses with unexplained hemorrhage and 8 of 21 (38%) horses with pathologic features consistent with EIPH. CD39 and CD39L1 SNPs were identified in 5 of 8 (62.5%) and one of 8 (12.5%) horses, respectively, presenting with colic or weight loss. CD39 and CD39L1 SNPs were identified in 28 of 45 (62%) and 13 of 45 (28.8%) healthy horses, respectively. CONCLUSIONS: CD39 and CD39L1 are critically important in maintaining normal hemostasis and limiting inflammation. Further studies are needed to evaluate their role in the pathogenesis of equine EIPH.

Subependymal glioneuronal hamartoma in the mesencephalic aqueduct of a giraffe.

A 1-day-old male giraffe calf (Giraffa camelopardalis) was submitted for necropsy examination after sustaining postnatal head trauma from the cow. In addition to the expected findings of severe cerebral edema and epidural and subarachnoid hemorrhage, there also was present an incidental finding of a subependymal glioneuronal aqueductal hamartoma. Reports of this type of congenital lesion are rare in the human literature, and the lesion has not, to the authors' knowledge, previously been reported in this or any other veterinary species.