Guillaume Chanoit

Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3β

The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta (GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3beta inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by zinc.

Molecular mechanism underlying Akt activation in zinc-induced cardioprotection

Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3beta (GSK-3beta). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with ZnCl(2) (10 microM) in the presence of the zinc ionophore pyrithione (4 microM) for 20 min enhanced Akt phosphorylation (Ser(473)), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation.

Static hydraulic urethral sphincter for treatment of urethral sphincter mechanism incompetence in 11 dogs

OBJECTIVE To review the postoperative results and complications associated with urethral sphincter mechanism incompetence managed with a static hydraulic urethral sphincter. METHODS Case records and a telephone owner questionnaire were retrospectively used to assess postoperative urinary continence scores (1 - dripping constantly to 10 - completely dry) and presence and frequency of complications. RESULTS Eleven spayed females were included. Median continence score/10 (range) awarded preoperatively was 3 (2 to 6), and scores at two weeks, three and six months were 8 (4 to 10), 9 (4 to 10) and 8 (4 to 10), respectively. At the last survey, the median continence score of 9 (5 to 10) was significantly better (P=0·004) than before surgery. Complete continence was achieved in 36·4% of dogs. The median (range) follow-up time was 412 (118 to 749) days. Complications occurred in 9 of 11 dogs and included dysuria (n=7), bacterial cystitis (n=7), longer urination time (n=8), urinary retention (n=3), haematuria (n=1), pain (n=3) and incisional seroma (n=3). CLINICAL SIGNIFICANCE Static hydraulic urethral sphincter was frequently associated with minor complications but no major complications (i.e. those requiring further surgery). Continence scores were significantly improved compared with those before surgery, with the possibility of further improvement following inflation of the sphincter.

Heterogeneity of T-Tubules in Pig Hearts

Background T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue. Methods Cardiac tissue was obtained from young female Landrace White pigs (45–75 kg, 5–6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT). Results & Conclusion The spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 μm, n = 17) than in either ventricular cells (1.33±0.05 μm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 μm, n = 5, P<0.05). These data demonstrate considerable heterogeneity between pig cardiomyocytes in the extent of t-tubule network, which correlated with cell size.

Outcomes and Complications Associated With Epicardial Pacemakers in 28 Dogs and 5 Cats

OBJECTIVE To report signalment, history, indications, complications and outcome for 28 dogs and 5 cats in which 34 permanent epicardial pacing leads were surgically placed by transdiaphragmatic approach (32) or intercostal thoracotomy (2). METHODS Medical records (2005-2010) were reviewed. Signalment, age, species, gender, clinical signs, presence of structural heart disease and/or congestive heart failure, ECG diagnosis, body weight (<10 or >10 kg), and overall survival rate were recorded. Statistical correlations were made between these variables and major and minor complications rates. RESULTS Except for body weight, no statistical differences were identified in prevalence of major (life threatening or requiring replacement of the pacemaker system) or minor (self-limiting) complications; dogs weighing >10 kg had significantly more major complications (P = .03). There was a trend (P = .051) for lower survival in animals that had major complications. CONCLUSIONS Larger dogs (>10 kg) may be predisposed to more major complications with epicardial pacemaker (EP) implantation. Major complication rate and survival time are similar to those reported for transvenous pacing and therefore implantation of EPs remains a suitable alternative.

Surgical treatment of a pulmonary artery vascular hamartoma in a dog

CASE DESCRIPTION A 6-year-old Siberian Husky-mix dog was examined for episodes of collapse. CLINICAL FINDINGS Physical examination, echocardiography, abdominal ultrasonography, ECG, and thoracic computed tomography with contrast were performed and revealed a 2.5 × 2.3 × 2.0-cm mass over the pulmonic valve leaflets, resulting in moderate pulmonic stenosis. Other abnormal findings included systemic hypertension, right bundle branch block, proteinuria, and a urinary bladder mass. TREATMENT AND OUTCOME Pulmonary arteriotomy was performed under inflow occlusion, and the mass was resected with transesophageal echocardiographic guidance and direct visualization. Results of histologic examination of the mass revealed a vascular hamartoma. Sequential follow-up examinations and telephone contacts (at 0.5, 5, and 15 months after surgery) revealed that the patient had been free from episodes of collapse since surgery. No regrowth of the mass was noted on follow-up echocardiograms, and the pulmonic stenosis had resolved, although mild to moderate pulmonary insufficiency later developed. The bladder mass was excised 15 months after the first surgery when hematuria developed, and results of histologic examination of this mass revealed a vascular hamartoma. The dog was eventually euthanized 31 months after the initial surgery for reasons that could not be directly linked to any recurrence of the pulmonary artery mass. CLINICAL RELEVANCE Hamartomas are benign tumors that can be located in various tissues, including large arteries. Computed tomography was helpful in predicting the resectability of the intracardiac mass in this dog. Treatment with arteriotomy under inflow occlusion and mild hypothermia resulted in a favorable outcome.

Pacemaker malfunction due to mechanical failure of the lead–header interface

An 8 year old female spayed Boxer dog, diagnosed with concurrent vasovagal syncope and arrhythmogenic right ventricular cardiomyopathy, presented for routine evaluation approximately three months following epicardial pacemaker implantation. Routine device interrogation revealed intermittent loss of ventricular capture and intermittent failure to appropriately sense. Following evaluation of chronic impedance data, failure of the pacemaker lead-header interface or lead conductor fracture was suspected. Radiographic and pacemaker interrogator findings suggested incomplete lead insertion into the device header with intermittent loss of ventricular capture and variable pacemaker sensing. We hypothesize that either the presence of a loose or cross-threaded set screw or operator error at the time of device implantation may have caused this complication. This report details the diagnosis of mechanical failure of the lead-header interface, a complication not previously reported in a veterinary patient.

Complications after thoracic surgery: don't (necessarily) blame it on the approach

In this issue of JSAP, Meakin et al. (2013) report the results of a retrospective study looking at risk factors associated with intrathoracic infection (pyothorax) following thoracic surgery. One of the major findings of their study is that the risk of postoperative pyothorax is significantly increased in dogs treated for chylothorax. The fact that the presence of pleural effusion, the presence of chest drains, and the history of previous (multiple) thoracocentesis were all potential risk factors for the development of pyothorax can obviously explain this result. However, interestingly, the surgical approach in all the dogs with chylothorax in this study was via a median sternotomy. Although the authors have emphasized in their discussion that the occurrence of pyothorax following thoracic surgery is likely multifactorial and does not incriminate only the surgical approach, I am fairly convinced that after reading this study, a few surgeons (including myself probably) will think twice before approaching the chest via a median sternotomy when treating a dog with chylothorax. Rightly or wrongly, median sternotomy is already an unpopular chest approach even though sternums have been cut longitudinally (or even transversally) since the time experimental dogs were paying a large contribution to advances in thoracic procedures (Andreasen & Watson 1953). It is interesting to note that median sternotomy was critically evaluated in clinical peer-reviewed studies (Bright et al. 1983, Burton & White 1996), much earlier than lateral thoracotomy (Moores et al. 2007). Clearly, in the veterinary surgeon’s mind, there has always been a large difference between these two techniques. However, when comparing results, one needs to remember that these studies are not contemporaneous and many aspects of veterinary surgery and postoperative care have positively evolved between the early 1980s and the late 2000s. Evaluating the complications and morbidity (let alone mortality) associated with different approaches to the thoracic cavity is a very complicated task unless one can evaluate the same condition (e.g. lung tumours, coronary by-pass) and compare the approaches. Similar studies in humans show that median sternotomy is not associated with an increase in wound infection and probably not associated with an increased level of postoperative pain compared to a lateral thoracotomy (Asaph et al. 2000, Rogers et al. 2012). The conformation of the chest in dogs is different than in humans and we should probably not directly compare results. Yet it is clear from the human literature that sternotomy is no longer considered as daunting an approach compared to 20 or 30 years ago when large sample studies and meta-analysis were not available to increase the level of evidence. Some veterinary studies have incriminated the thoracic approach to explain the outcome of one particular condition (e.g. pyothorax) suggesting that the high complication rate following surgical treatment of pyothorax is due, in part, to the approach taken (namely the median sternotomy) rather than simply the results of the underlying pathology. This is probably partially true but the chances are that if you are going to surgically treat a pyothorax, you will need to enter the chest via a median sternotomy (until we can prove that we can obtain the same results via keyhole approaches) as you probably gain better exposure and are more efficacious than by using a lateral approach. Thus it may be hazardous to neglect the right to a sternotomy based on presumptions. Tattersall & Welsh (2006) rightly stated that “...prospective studies are required to fully evaluate the true effect of the thoracotomy approach on postoperative wound complications...”. Such studies are still lacking in the veterinary literature. So how can we minimize postoperative complications after thoracic approaches, especially if we assume that the complication rate varies greatly between techniques? As infection is a potential complication after sternotomy, would a change in closure techniques be a reasonable measure to evaluate critically? The common belief is that we should use wire to close a sternotomy (Pelsue et al. 2002). However, monofilament suturing of the sternum is common in human surgery (Luciani et al. 2006) and appears biomechanically equivalent to wiring (Gines et al. 2011). There is clearly a need to look prospectively at these methods in veterinary patients. The effect of chest approach on postoperative pain (one other major complication after thoracic surgery) should also be a topic of critical appraisal. There is no veterinary study comparing the degree of pain following sternotomy and thoracotomy even if we know that some methods of chest closure can potentially lead to a lower level of postoperative pain (Rooney et al. 2004). To minimize pain it would seem logical to continue the trend towards keyhole approaches. Thoracoscopy has become the preferred method to evaluate the chest fully, to take biopsies (Schmiedt 2009) and for some procedures such as pericardiectomy (Dupre et al. 2001). Certainly, some studies tend to report a favourable outcome of thoracic procedures done via minimal access approaches in terms of pain control. Yet reported measurable outcomes, such as reduced hospital stay, at the moment do not necessarily completely support the superiority of this method over the standard method (Lansdowne et al. 2005). Let’s not forget that the evidence that key-hole thoracic surgery in small animals results in less pain than the open approach remains weak. The only study that has critically compared the level of pain following these two different approaches was done on healthy dogs and used cortisol and blood glucose concentrations to assess the degree of postoperative pain (Walsh et al. 1999); these outcome measures are not considered very robust to evaluate acute pain in animals (Lascelles 2012). In human prospective case-matched studies, results have shown no difference in terms of chronic pain post thoracoscopy versus thoracotomy (Furrer et al. 1997).

Electrosurgery reduces blood loss and immediate postoperative inflammation compared to cold instruments for midline celiotomy in dogs: A randomized controlled trial

Objectives: To compare the use of an electrosurgical device with traditional cold instruments (scalpel and scissors) for midline celiotomy incision. Study design: Prospective randomized controlled clinical trial. Sample population: One hundred and twenty client‐owned dogs undergoing abdominal surgery. Methods: Dogs were prospectively recruited and randomized to receive electroincision or cold instrument incision. For cold incision, surgeons used basic surgical instruments including scalpel and scissors. For electroincision, surgeons only used the electrosurgical device in cutting mode. Time for the approach, blood loss, and the incision length were recorded. A blinded observer assessed pain and incision redness, swelling, and discharge at 24 and 48 hours postoperative (graded 0‐3). Owner assessment of incision healing was recorded by telephone interview. Results: Blood loss during surgery was significantly lower for electroincision (mean 0.7, SD 1.7 mL) than cold incision (mean 3.0, SD 4.3 mL, P < .0001) with no significant difference in incision length or time for approach. Electroincision was associated with significantly less incision redness (cold median 1, range 0‐3; electroincision median 0, range 0‐2, P = .02) and less incision discharge (cold median 0.5 range 0‐3; electroincision median 0, range 0‐1, P = .006) at 24 hours postoperative. There was no significant difference in pain scores or incision healing in dogs receiving the two techniques. No incisional hernias were reported. A surgical site infection occurred in 1 dog (cold incision). Conclusions: Electroincision for a celiotomy approach in the dog reduces blood loss, and incision redness and discharge in the immediate postoperative period without affecting the occurrence of wound complications such as infection and dehiscence (including linea alba).

Long-term outcome of female dogs treated with static hydraulic urethral sphincter for urethral sphincter mechanism incompetence

The purpose of the study was to report the postoperative outcome, complications, and long-term follow-up of the use of a static hydraulic urethral sphincter for the management of urethral sphincter mechanism incompetence in female dogs. Medical records were reviewed to extract information on long-term (>365 days) outcome data. Telephone owner questionnaire was performed to assess postoperative urinary continence scores (scale 1-10, where 10 is complete continence) and the presence and frequency of complications. Twenty female dogs were included. Mean (±standard deviation) time to follow-up was 1,205.1 (±627.4) days. Median continence score/10 (range) was 3.5 (2-6) preoperatively, and 9.0 (7-10) at the last follow-up. Median continence score was significantly higher at all time points postoperatively compared with before surgery (P < .001). Complete continence was achieved in 90% of bitches. Minor complications occurred in 13 bitches and included dysuria (8), bacterial cystitis (8), longer urination time (10), incisional seroma (5), urinary retention (3), hematuria (2), and pain when urinating (2). Major complications occurred in one dog (static hydraulic urethral sphincter removed 28 mo after placement). Continence scores were sustainably improved in the long-term. Complications were mostly minor. Urinary tract infections were the most common but resolved with conventional antibiotic treatment.