Hilde De Rooster

INVITED REVIEW—Morphologic and Functional Features of the Canine Cruciate Ligaments

Objective— To review the gross, microscopic, and functional anatomy of the cranial cruciate ligament (CCL) in dogs. Study Design— Literature review. Methods— Reports of the anatomy and function of the cruciate ligaments in dogs were retrieved by search of the 1975–2005 PubMed database. Results— The CCL has an important biomechanical function resisting cranial drawer, hyperextension, and internal rotation and acts to fine tune and guide the stifle through its rolling and sliding motion. It has a complex architecture, and distinct geographic regions within the ligament have different functional roles depending on the angle and loading conditions. Collagen type I is the main component of the extracellular matrix; the fibrils have a crimped structure. The cruciate ligaments are almost completely covered by synovium, protecting them from synovial fluid. Cruciate blood supply is mainly of soft tissue origin. The intraligamentous network is relatively limited whereas the core of the middle third of the CCL is even less well vascularized. Neurohistologic studies are very limited in the dog. Various mechanoreceptors and proprioceptive receptors have been identified within the substance of the cruciate ligaments. Conclusions— CCL structural characteristics play an important part in its complex behaviour with the crimped pattern of the collagen fibrils being an important determinant of its biomechanical properties. In contrast to reports of managing CCL rupture, there are few reports describing the microanatomy and neurovascular morphology of the cruciate ligaments. Clinical Relevance— Cruciate disease is likely multi-factorial. Improved understanding of CCL degradation leading to CCL rupture is critical to development of new diagnostic tests for cruciate disease in dogs. Appropriate intervention during the early stages of disease process might preserve CCL structural properties by preventing further collagen degradation. Accurate knowledge of functional and fiber bundle anatomy is imperative for reconstruction and restoration of normal stifle joint physiology. Reconstructive goals should alleviate existing instability and mimic normal kinematics. Knowledge of the exact function of the CCL in the neuromuscular control around the stifle joint could possibly explain osteoarthritis progression after CCL damage.

Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

Penetrating injuries in dogs and cats. A study of 16 cases.

The objective of this retrospective study was to assess radiographical and surgical findings, surgical management and outcome of penetrating injuries in dogs and cats by evaluating patient records. Sixteen patients were identified (15 dogs and one cat), four with gunshot wounds, and 12 with fight wounds (11 with bite wounds, one struck by a claw). The thoracic cavity was affected in six patients, the abdominal cavity in three cases. Both cavities were affected in five dogs and the trachea in two cases. All of the patients with fight wounds were small breed dogs. Multiple injuries to internal organs that required intervention were found surgically after gunshot wounds and a high amount of soft tissue trauma requiring reconstruction was present after fight wounds. Radiography diagnosed body wall disruption in two cases. All of the affected thoracic body walls in the fight group had intercostal muscle disruptions which was diagnosed surgically. Fourteen patients survived until discharge and had a good outcome. In conclusion, penetrating injuries should be explored as they are usually accompanied by severe damage to either the internal organs or to the body wall. A high level of awareness is required to properly determine the degree of trauma of intercostal muscle disruption in thoracic fight wounds.

Various ways to improve whole cancer cell vaccines

Immunotherapy based on whole cancer cell vaccines is regarded as a promising avenue for cancer treatment. However, limited efficacy in the first human clinical trials calls for more optimized whole cancer cell vaccines and better patient selection. It is suggested that whole cancer cell vaccines consist preferably of immunogenically killed autologous cancer stem cells associated with dendritic cells. Adjuvants should stimulate both immune effector cells and memory cells, which could be achieved through their correct dosage and timing of administration. There are indications that whole cancer cell vaccination is less effective in patients who are immunocompromised, who have specific genetic defects in their immune or cancer cells, as well as in patients in an advanced cancer stage. However, such patients form the bulk of enrolled patients in clinical trials, prohibiting an objective evaluation of the true potential of whole cancer cell immunotherapy. Each key point will be discussed.

Outcome from status epilepticus after portosystemic shunt attenuation in 3 dogs treated with propofol and phenobarbital

Objective – To describe outcome of treatment with propofol and phenobarbital for status epilepticus (SE) after portosystemic shunt (PSS) attenuation. Case or Series Summary – Three dogs without preceding seizure activity, were diagnosed with a single extrahepatic PSS. Following standard preoperative medical therapy, an ameroid constrictor was placed surgically. Recovery was uneventful until spontaneous SE developed 46–96 hours after surgery. After unsuccessful seizure control with benzodiazepines, dogs were treated with a bolus of propofol followed by a propofol constant rate infusion. Phenobarbital was concurrently administered and supportive care was optimized. All dogs recovered uneventfully over the next 7–9 days. Over the following months phenobarbital was slowly tapered. All dogs have been free from antiepileptic drugs for several months, without recurrence of neurologic signs. New or Unique Information Provided – In this case series, we describe the treatment of 3 dogs with propofol and phenobarbital for refractory SE following attenuation of a single congenital PSS. After weaning of the propofol constant rate infusion, and tapering and discontinuation of phenobarbital over the following months, all dogs experienced a complete recovery. This study provides evidence that use of propofol in combination with phenobarbital may be efficacious for management of SE in dogs after PSS surgery.OBJECTIVE To describe outcome of treatment with propofol and phenobarbital for status epilepticus (SE) after portosystemic shunt (PSS) attenuation. CASE OR SERIES SUMMARY Three dogs without preceding seizure activity, were diagnosed with a single extrahepatic PSS. Following standard preoperative medical therapy, an ameroid constrictor was placed surgically. Recovery was uneventful until spontaneous SE developed 46-96 hours after surgery. After unsuccessful seizure control with benzodiazepines, dogs were treated with a bolus of propofol followed by a propofol constant rate infusion. Phenobarbital was concurrently administered and supportive care was optimized. All dogs recovered uneventfully over the next 7-9 days. Over the following months phenobarbital was slowly tapered. All dogs have been free from antiepileptic drugs for several months, without recurrence of neurologic signs. NEW OR UNIQUE INFORMATION PROVIDED In this case series, we describe the treatment of 3 dogs with propofol and phenobarbital for refractory SE following attenuation of a single congenital PSS. After weaning of the propofol constant rate infusion, and tapering and discontinuation of phenobarbital over the following months, all dogs experienced a complete recovery. This study provides evidence that use of propofol in combination with phenobarbital may be efficacious for management of SE in dogs after PSS surgery.

Prepubertal gonadectomy in cats: different surgical techniques and comparison with gonadectomy at traditional age

Feasibility, surgical time and complications of different surgical techniques for prepubertal gonadectomy (PPG; 8–12 weeks of age) in cats were studied and compared to gonadectomy at traditional age (TAG; 6–8 months of age). Kittens were randomly assigned to PPG or TAG. Ovarian pedicle haemostasis for PPG was achieved by ligatures (n=47), vascular clips (n=50), bipolar electrocoagulation (n=50), or pedicle tie (n=50); for TAG (n=34) ligatures were used. In male cats, PPG consisted of closed castration by spermatic cord knot (n=92) or ligature (n=91) while TAG (n=34) was an open castration by spermatic cord knot. A linear (surgical time) and a logistic regression (complications) model were designed. Significance was set at 0.05. For female PPG, clips and coagulation were the fastest procedures; placement of ligatures was most time-consuming. In male PPG, knot placement was significantly faster than ligation. In both sexes, very few intraoperative or wound complications were observed, irrespective of the surgical technique used. Surgical times in females (ligatures) as well as in males (knot) were significantly shorter for PPG than for TAG. PPG was as safe as TAG, yet took less time to perform and did not result in a greater rate of postoperative complications.

Prepubertal gonadectomy in cats: different injectable anaesthetic combinations and comparison with gonadectomy at traditional age

Anaesthetic and analgesic effects of three different injectable anaesthetic combinations for prepubertal gonadectomy (PPG) in cats were studied. One anaesthetic protocol was compared with a similar one for gonadectomy at traditional age (TAG). Kittens were randomly assigned to PPG or TAG. For PPG, three different protocols were compared: (1) intramuscular (IM) administration of 60 μg/kg dexmedetomidine plus 20 μg/kg buprenorphine followed by an IM injection of the anaesthetic agent (20 mg/kg ketamine) (DB-IM protocol); (2) oral transmucosal (OTM) administration of 80 μg/kg dexmedetomidine plus 20 μg/kg buprenorphine followed by an IM injection of 20 mg/kg ketamine combined with 20 µg/kg dexmedetomidine (DB-OTM protocol); (3) IM injection of a 40 μg/kg medetomidine–20 μg/kg buprenorphine–20 mg/kg ketamine combination (MBK-IM protocol). For TAG, a DB-IM protocol was used, but with different doses for dexmedetomidine (40 μg/kg) and ketamine (5 mg/kg). All cats (PPG and TAG) received a non-steroidal anti-inflammatory before surgery. Anaesthetic and analgesic effects were assessed pre- and postoperatively (until 6 h). Cumulative logit, linear and logistic regression models were used for statistical analysis. Compared with the DB-OTM protocol, the DB-IM and MBK-IM protocols provided better anaesthesia with fewer adverse effects in PPG cats. Postoperative pain was not significantly different between anaesthetic protocols. PPG and TAG cats anaesthetised with the two DB-IM protocols differed significantly only for sedation and pain scores, but sedation and pain scores were generally low. Although there were no anaesthesia-related mortalities in the present study and all anaesthetic protocols for PPG in cats provided a surgical plane of anaesthesia and analgesia up to 6 h postoperatively, our findings were in favour of the intramuscular (DB-IM and MBK-IM) protocols.

Regional cerebral blood flow assessed by single photon emission computed tomography (SPECT) in dogs with congenital portosystemic shunt and hepatic encephalopathy

Regional cerebral blood flow (rCBF) in eight dogs with congenital portosystemic shunt (PSS) and hepatic encephalopathy (HE) was compared with rCBF in eight healthy control dogs using single photon emission computed tomography (SPECT) with a 99mtechnetium-hexamethylpropylene amine oxime (99mTc-HMPAO) tracer. SPECT scans were abnormal in all PSS dogs. Compared to the control group, rCBF in PSS dogs was significantly decreased in the temporal lobes and increased in the subcortical (thalamic and striatal) area. Brain perfusion imaging alterations observed in the dogs with PSS and HE are similar to those in human patients with HE. These findings suggest that dogs with HE and PSS have altered perfusion of mainly the subcortical and the temporal regions of the brain.

Immunological, anti-angiogenic and clinical effects of intratumoral interleukin 12 electrogene therapy combined with metronomic cyclophosphamide in dogs with spontaneous cancer: A pilot study.

The immunological, anti-angiogenic and clinical effects of metronomic cyclophosphamide and 3 consecutive intratumoral interleukin (IL)-12 gene therapy (electrogene therapy (EGT)) treatments were evaluated in 6 dogs with spontaneous cancer. In all dogs, a decrease in peripheral leukocytes 2 days after IL-12 EGT coincided with erythema and swelling of the tumor. In the tumor, a transient increase in IL-12 levels was measured, whereas a continuous increase in interferon γ (IFNγ) and thrombospondin 1 (TSP-1) were determined in contrast to a continuous decrease in vascular endothelial growth factor (VEGF). In the serum, a transient increase in IL-12 and IL-10 levels were noted in contrast to a transient decrease in VEGF and TSP-1. The treatment resulted in a significant anti-angiogenic effect. Although all primary tumors continued to progress in time, this progression was slower than before treatment according to the contrast-enhanced ultrasound data. Besides the encouraging immunostimulatory and anti-angiogenic effects observed in all dogs we also noticed in 4 out of 6 dogs clinically relevant improvements in quality of life and weight. These results hold great promise for combinatorial strategies of IL-12 EGT and metronomic chemotherapy with conventional antitumor (immuno)therapies.

Can dendritic cells improve whole cancer cell vaccines based on immunogenically killed cancer cells

Immunogenic cell death (ICD) offers interesting opportunities in cancer cell (CC) vaccine manufacture, as it increases the immunogenicity of the dead CC. Furthermore, fusion of CCs with dendritic cells (DCs) is considered a superior method for generating whole CC vaccines. Therefore, in this work, we determined in naive mice whether immunogenically killed CCs per se (CC vaccine) elicit an antitumoral immune response different from the response observed when immunogenically killed CCs are associated with DCs through fusion (fusion vaccine) or through co-incubation (co-incubation vaccine). After tumor inoculation, the type of immune response in the prophylactically vaccinated mice differed between the groups. In more detail, fusion vaccines elicited a humoral anticancer response, whereas the co-incubation and CC vaccine mainly induced a cellular response. Despite these differences, all three approaches offered a prophylactic protection against tumor development in the murine mammary carcinoma model. In summary, it can be concluded that whole CC vaccines based on immunogenically killed CCs may not necessarily require association with DCs to elicit a protective anticancer immune response. If this finding can be endorsed in other cancer models, the manufacture of CC vaccines would greatly benefit from this new insight, as production of DC-based vaccines is laborious, time-consuming and expensive.