Canine Model Research Articles

Comparative analysis of canine and human HtrA2 to delineate its role in apoptosis and cancer.

Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system. Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared with laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines.

Allograft to bone-tunnel integration in a canine anterior cruciate ligament reconstruction model: a comparison study of allograft preparation methods

The procedure of anterior cruciate ligament (ACL) allograft preparation can be divided into fresh-frozen method (FF-allograft) or freeze-dried method (FD-allograft). This study aims to biomechanically and histologically compare the graft to bone tunnel integration between the two allografts. In-vitro results indicated that FF-allograft and FD-allograft showed excellent biocompatibility and biomechanics, while FD-allograft showed a denser collagen fiber arrangement than FF-allograft and autograft. Then, in-vivo preformation of the FF-allograft, FD-allograft, and autograft on bone tunnel integration was evaluated via a canine ACL reconstruction model. In-vivo results indicated that no signs of infection or osteoarthritis were shown in the femur-graft-tibia complexes, but more vascularity and synovitis formed around the implanted FF-allograft. Micro-computed tomography showed that peri-graft bone in the FF-allograft group was significantly increased and remodeled compared with the FD-allograft group; Histologically, the FF-allograft group exhibited similar graft-bone tunnel healing to the FD-allograft group. Tartrate-resistant acid phosphatase (TRAP) staining showed significantly more osteoclasts presented in the FD-allograft group compared to the FF-allograft group. Meanwhile, a significantly higher failure load was shown in the FF-allograft group when compared with the FD-allograft group (P < 0.05). In conclusion, the FF-allograft integrated more firmly into the bone tunnel than the FD-allograft when used in ACL reconstruction.

First, do no harm: the role of preclinical animal models in predicting adverse events in gene therapy clinical trials for Duchenne muscular dystrophy and X-Linked myotubular myopathy

The occurrence of severe adverse events (SAEs) in patients with Duchenne muscular dystrophy (DMD), X-linked myotubular myopathy (XLMTM), and other neuromuscular diseases treated with adeno-associated virus (AAV) constructs has prompted studies to improve the safety and efficacy of gene therapy. Physicians have weighed the medical tenet of “first, do no harm” against the perspective of patients with progressive life-threatening conditions who may accept greater risk. Regarding SAE pathogenesis, discussion has focused on total AAV exposure and patient mutations more likely to induce immunity, while stressing the limitations of animal models in predicting adverse events. Therapeutic strategies for reducing side effects have employed more myotropic AAV serotypes and efficient transgenes. Other recommendations include excluding certain DMD gene mutations associated with SAEs and substituting less immunogenic transgenes such as utrophin (DMD) and myotubularin-related protein (XLMTM). For the sake of preclinical studies, emphasis has been placed on outbred rodents and larger animals that better predict immunity. Here, the absence of side effects in canine DMD and XLMTM models might be explained partly by phenotypic differences between affected humans and dogs. Specifically, dystrophin- and myotubularin-deficient dogs exhibit milder lesions, including less muscle fat deposition and the absence of hepatopathy, respectively, which could lead to reduced immune responses to AAV constructs. To better predict future problems, thought should be given to tracking early subclinical markers of the innate immune response, especially complement activation. Regardless of steps taken to improve the predictive value of animal models for SAEs, some questions will only be answered through human clinical trials after carefully considering the risk-benefit ratio.

Vasodilatory shock: a review of pathophysiology and vasopressor therapy

Vasodilatory shock is characterised by a global loss of vasomotor tone, leading to maldistribution of blood volume, low systemic arterial pressure and hypoperfusion. This syndrome can be caused by sepsis, anaphylaxis and a wide range of other aetiologies. This review article explores the pathophysiology of vasodilatory shock, including well-understood mechanisms and emerging avenues of future investigation. Options for vasopressor therapy are reviewed, including evidence from preclinical canine models, small animal clinical research, large human clinical trials and the Surviving Sepsis Campaign. Recommendations for rational vasopressor choice are extrapolated from this evidence. Future directions include the development of novel vasoactive agents, clinical data comparing the safety and effectiveness of vasopressors in small animals and the development of a veterinary-specific consensus statement guiding best practices for the treatment of vasodilatory shock.

Point-of-care viscoelastic coagulation monitoring device shows promise for informing resuscitation strategies in a canine hemorrhagic shock model.

To use a point-of-care viscoelastic coagulation monitoring device (VCM-Vet; Entegrion) and traditional tests to assess hemostatic changes during hemorrhagic shock in dogs. 8 healthy, purpose-bred Beagles were enrolled in a hemorrhagic shock model conducted from September through December 2021. Dogs were anesthetized, had baseline hemostatic variables measured after blood pressure was stabilized at 70 to 80 mm Hg for 10 minutes (T1), had blood withdrawn from a jugular vein to achieve a mean blood pressure of 40 ± 5 mm Hg for 10 minutes (T2), were resuscitated with 100% shed blood, and then had hemostatic variables evaluated 10 minutes later (T3). At each time point, hemostatic variables were measured with traditional tests (Hct, WBC count, platelet count, mean platelet volume, prothrombin time, activated partial thromboplastin time [aPTT], and concentrations of fibrinogen, antithrombin, and D-dimer) and the VCM-Vet device (clot time, clot formation time, α-angle, maximum clot formation, amplitude at 10 minutes [A10], amplitude at 20 minutes [A20], clot lysis index at 30 minutes [LI30], and clot lysis index at 45 minutes [LI45]). All dogs survived without complication. At T2 and T3 (vs T1), samples had significantly higher coagulability (increased median α-angle, A10, and A20) and fibrinolysis (decreased median LI30 and LI45) and significantly longer aPTT; however, all values remained within reference limits. The use of VCM-Vet helped identify complex hemostatic disturbances in dogs with hemorrhagic shock. The use of VCM-Vet shows promise in aiding veterinarians in optimizing resuscitation strategies based on real-time clotting data.

Iron Chelators Augment Large Osteochondral Allograft Osseointegration in a Preclinical Canine Model.

Osteochondral allograft transplantation (OCAT) can be a successful joint restoration treatment option for large post-traumatic articular defects but is still associated with significant revision and failure rates. Despite recent advances that have improved OCAT success, insufficient osteochondral allograft (OCA) osseointegration remains a major cause of failure. Deferoxamine (DFO) is an effective angiogenic and osteo-anabolic iron chelator that consistently promotes bone neovascularization and regeneration. This study was designed to investigate local delivery of DFO for augmenting OCA osseointegration using a preclinical canine model for OCAT in the knee and hip as commonly affected joints. On Institutional Animal Care and Use Committee (IACUC) approval, 12 purpose-bred dogs underwent OCAT of the femoral head or femoral condyles with DFO or DFO-free (controls) microspheres in recipient sites. OCA revascularization, cellular repopulation, and integration were evaluated based on functional, diagnostic imaging, microcomputed tomography, histology, and immunohistochemistry outcome measures. Local delivery of DFO into OCAT recipient sites was associated with maintained or improved joint function, superior radiographic appearance, significantly greater trabecular thickness, higher bone volume, and new bone ingrowth compared with DFO-free controls. OCA osseointegration is dependent on cellular repopulation and neovascularization, resulting in new bone ingrowth through creeping substitution, and insufficient osseointegration with resorption and subsidence of the OCA remains a major cause of failure after transplantation. The results of this study suggest that local delivery of DFO using a controlled microsphere release system may reduce resorption and improve revascularization and cellular repopulation to increase new bone ingrowth, potentially expediting OCA osseointegration after transplantation.

Evaluation of Injectate Distribution of the Middle Mental Nerve Block Within the Mandibular Canal in a Cadaveric Canine Model.

Awareness among veterinarians has increased regarding the need for comprehensive pain relief, but many companion animal veterinarians do not administer regional analgesia pre-emptively during dental procedures. The middle mental nerve (MMN) block desensitizes the ipsilateral mandibular incisor and canine teeth as well as soft tissues rostral to the delivery site. There is little published information on the efficacy of the MMN block in dogs. The objective of this study was to determine injectate distribution within the mandibular canal using a radiopaque contrast media/methylene blue solution. Half a milliliter of solution was injected within the opening of the middle mental foramen using a standard hypodermic syringe and a 25G x 25 mm needle. The course of the injectate was traced both via computed tomography (CT) and, in some cadavers, gross dissection. Post-treatment CT revealed that in 90% of the cases, the contrast diffused at least as far caudally as the mesial root of the third premolar tooth. The injectate was not identified within the canal of 5% of cadaveric mandibles examined. Although the solution used diffused caudally within the mandibular canal when injected using recommended clinical techniques, this may not completely represent the extent of clinical effects experienced in live patients. This technique also confirmed that the needle does not need to be advanced into the mandibular canal to achieve adequate diffusion to at least the mesial root of the third premolar tooth.

Comparison of three approaches for accessory lung lobectomy in the canine cadaveric model: Intercostal thoracotomy, median sternotomy, and a transdiaphragmatic approach combined with caudal median sternotomy.

To describe a combined transdiaphragmatic and caudal median sternotomy (TDCM) approach to the accessory lung lobe and to compare its accessibility with intercostal thoracotomy (ICT) and median sternotomy (MS). Cadaveric study. Twelve canine cadavers. Cadavers underwent an accessory lung lobectomy using an articulating EndoGIA stapler via randomly assigned approach: ICT (n = 4), MS (n = 4) or TDCM (n = 4). The percentage of accessory lung tissue removed was measured in surface area and weight. Exposure was measured as area of cavitary or bicavitary exposure at maximal retraction, by tracing a line around the circumference of the exposed cavity using an imaging software. Staple line leak pressures were evaluated to 40 cmH2O. The average area of exposure was larger in the TDCM approach (TDCM = 193.5 cm2, MS = 106.5 cm2, ICT = 73.5 cm2); (p = .01). Two of four ICT staple lines leaked at 40 cmH2O or lower, and 1/4 MS resulted in iatrogenic damage to an adjacent lobe. There was no difference in the percentage of the lobe excised by weight or surface area between groups. The transdiaphragmatic and caudal median sternotomy approach provided greater exposure, although the percentage of the lobe excised and the surgical time did not differ between approaches. All three approaches allowed for adequate excision of the accessory lung lobe (ALL) with similar surgical times; however, the TDCM approach provided a greater area of exposure, which could increase accessibility to the ALL.

Combined PVI and LAAC is feasible using RFA or PFA in a canine model despite acute tissue edema in PFA versus RFA

Abstract Background Combined procedure with pulmonary vein isolation (PVI) using thermal energy followed by left atrial appendage closure (LAAC) has been used in select patient populations with positive results. Pulsed field ablation (PFA) is a novel non-thermal ablation modality that ablates cardiac tissue by irreversible electroporation. The effect of PFA on cardiac tissue and its interaction with a LAAC device in a single procedure has not been studied. Purpose To evaluate using transesophageal echo (TEE) imaging the acute tissue response following ablation by PFA compared to radiofrequency ablation (RFA) and its interaction with the WATCHMAN FLX Pro LAAC device when used in a combined procedure in a healthy canine model. Methods Nine mongrel canines underwent PVI using either circumferential PFA (FARAPULSE) energy application (n=6) or RFA energy (n=3). TEE imaging was obtained at the time of the procedure before and immediately after ablation energy application. The general appearance and echogenicity of the left lateral ridge was assessed and the os of the left atrial appendage (LAA) was measured in the four standard TEE views (0, 45, 90, and 135 degrees). Decisions about device sizing were made accordingly. PVI was evaluated by entry and exit block in all veins except the right inferior pulmonary vein (RIPV), which was not ablated. Following ablation, the LAAC device was implanted using TEE and fluoroscopic guidance in all animals and the percentage of compression measured. PASS criteria were assessed before the release of each device. All animals received dual antiplatelet therapy post implant. Results PVI was confirmed in all animals (except RIPV which was not targeted for ablation) using PFA and RFA. LAA dimensions (at device landing site) did not change substantially after RF ablation. With PFA, there was evidence of more diffuse edema and decrease in LAA diameter (Table). The LAAC device was implanted successfully after meeting PASS criteria in all animals within 30 minutes post ablation. The device size was decreased following PFA in two (2/6) animals, whereas there was no deviation from baseline measurements in device size for any of the RFA animals. Average LAAC device compression was higher for PFA versus RFA animals (Table). TEE post implant confirmed absence of any peri-device leaks in all animals. Conclusion Concomitant PVI using PFA (FARAPULSE) and LAAC (WATCHMAN FLX Pro) is feasible with 100% success and no residual acute peri-device leak. In the canine model, PFA is associated with more diffuse edema compared to RFA, which might affect device sizing. Long term consequences of this acute edema need to be evaluated in serial imaging and future larger clinical trials.Table

Efficacy comparison of transcatheter aorticorenal ganglion ablation and renal denervation in a hypertensive canine model

Abstract Background Transcatheter renal sympathetic denervation (RDN) has been proposed to be an adjunctive treatment in uncontrolled hypertension. However, non-responders to RDN were not rare. Considering the lack of a procedure endpoint and the limitation of anatomic-based ablation strategy, incomplete or futile denervation may contribute to non-responsiveness. Aorticorenal ganglion (ARG) is a purely sympathetic ganglion that projects to the proximal and distal renal artery, forming the peri-arterial nerve network. Ablation of this ganglion results in significant renal denervation. However, it remains unknown whether ARG ablation has a superior effect in lowering blood pressure (BP) compared to RDN. Purpose The aim of the study was to determine whether ARG ablation improved the BP-lowering effect compared to RDN in a hypertensive canine model. Methods A total of 21 male Chinese Kunming dogs were included and randomized into three groups: ARG ablation group (n=7), RDN group (n=7) and control group (n=7). In the ARG ablation group, anatomically based high-frequency stimulation (HFS) was performed in the aorta and inferior vena cava (IVC) under the guidance of 3D anatomical construction (Figure 1A). ARG ablation sites were identified by HFS-induced BP elevation response (&amp;gt;10mmHg) and renal artery vasoconstriction (Figure 1B). In the RDN group, HFS-guided ablation was conducted at the positive site (BP elevation response &amp;gt;5mmHg) from the distal to proximal renal artery. Endpoint of the procedure was defined as the elimination or attenuation of BP elevation induced by HFS. BP was assessed at baseline and 4 weeks after the procedure. Results The left ARG ablations were performed at the inferior aspect of superior mesenteric artery (4/7), the abdominal aorta (2/7) and the proximal renal artery (1/7), while the right ARG ablations were performed at the posterior aspect of the IVC (6/7) and the ostium of the left renal vein (1/7). The gross anatomy of bilateral ARGs was shown in Figure 1C. The hemodynamic response to ARG stimulation was significantly greater than that of renal artery stimulation (40.0±13.4 vs 13.3±3.7 mmHg, Figure 2A, B); meanwhile, ARG ablation is more effective in blunting the BP elevation response compared to RDN (30.7±11.7 vs 9.6±2.6, P=0.001, Figure 2A, C). With a 4-week follow-up, ARG ablation demonstrated a greater systolic BP-lowering effect than RDN (-29.6±12.0 vs -15.3±6.9 mmHg, P=0.018, Figure 2D). No complications were found during the procedure or follow-up. Conclusion Our study suggests that ARG ablation could lead to a more efficient BP-lowering effect compared to RDN, which provides a novel paradigm for autonomic modulation in the treatment of hypertension.Figure 1Figure 2

Machine Learning Models for Tracking Blood Loss and Resuscitation in a Hemorrhagic Shock Swine Injury Model

Hemorrhage leading to life-threatening shock is a common and critical problem in both civilian and military medicine. Due to complex physiological compensatory mechanisms, traditional vital signs may fail to detect patients’ impending hemorrhagic shock in a timely manner when life-saving interventions are still viable. To address this shortcoming of traditional vital signs in detecting hemorrhagic shock, we have attempted to identify metrics that can predict blood loss. We have previously combined feature extraction and machine learning methodologies applied to arterial waveform analysis to develop advanced metrics that have enabled the early and accurate detection of impending shock in a canine model of hemorrhage, including metrics that estimate blood loss such as the Blood Loss Volume Metric, the Percent Estimated Blood Loss metric, and the Hemorrhage Area metric. Importantly, these metrics were able to identify impending shock well before traditional vital signs, such as blood pressure, were altered enough to identify shock. Here, we apply these advanced metrics developed using data from a canine model to data collected from a swine model of controlled hemorrhage as an interim step towards showing their relevance to human medicine. Based on the performance of these advanced metrics, we conclude that the framework for developing these metrics in the previous canine model remains applicable when applied to a swine model and results in accurate performance in these advanced metrics. The success of these advanced metrics in swine, which share physiological similarities to humans, shows promise in developing advanced blood loss metrics for humans, which would result in increased positive casualty outcomes due to hemorrhage in civilian and military medicine.

Staphylococcus felis C4 exhibits invitro antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius in a novel canine skin explant model.

Canine superficial pyoderma is a common bacterial skin infection of dogs, generally caused by Staphylococcus pseudintermedius. The C4 strain of Staphylococcus felis was recently discovered to have strong antimicrobial activity against S. pseudintermedius in mice. We aimed to evaluate invitro if this antimicrobial activity was maintained using a novel canine skin explant model. Punch biopsies (8 mm) of skin from recently euthanised dogs were collected and placed into six-well plates on top of an agarose pedestal. Histological examination of the skin explants showed an intact dermal-epidermal organisation and a stratum corneum that was successfully colonised by S. pseudintermedius after topical application. The number of colony forming units of S. pseudintermedius showed a 2 log increase after 24 h colonisation, indicating that the explant supported bacterial growth. By contrast, co-treatment with S. felis C4 live bacteria and its sterile protein product significantly reduced the growth of a methicillin-susceptible (ST540, p = 0.0357) and a methicillin-resistant (MR) strain (ST71, p = 0.0143) of S. pseudintermedius. No detectable bacteria were recovered from or visualised on skin 24 h posttreatment with the S. felis C4 sterile protein product. Using a novel canine explant model, we demonstrate that the S. felis C4 strain inhibits the growth of S. pseudintermedius and that it is a promising candidate for a new probiotic therapy to treat cutaneous infections caused by S. pseudintermedius, including MR strains.

Bovine pulp extracellular matrix hydrogel for regenerative endodontic applications: in vitro characterization and in vivo analysis in a necrotic tooth model

BackgroundRegenerative endodontic procedures (REPs) offer the promise of restoring vitality and function to a previously necrotic and infected tooth. However, the nature of regenerated tissues following REPs remains unpredictable and uncontrollable. Decellularized extracellular matrix scaffolds have gained recent attention as scaffolds for regenerative endodontics.ObjectivesPreparation and characterization of a bovine dental pulp-derived extracellular matrix (P-ECM) hydrogel for regenerative endodontic applications. Biocompatibility and regenerative capacity of the prepared scaffold were evaluated in vivo in a canine animal model.MethodsFifteen freshly extracted bovine molar teeth were used to prepare P-ECM hydrogels following approval of the institutional review board of the faculty of dentistry, Alexandria University. Decellularization and lyophilization of the extracted pulp tissues, DNA quantification and histological examination of decellularized P-ECM were done. P-ECM hydrogel was prepared by digestion of decellularized pulps. Prepared scaffolds were evaluated for protein content and release as well as release of VEGF, bFGF, TGF-β1 and BMP2 using ELISA. Rabbit dental pulp stem cells’ (rDPSCs) viability in response to P-ECM hydrogels was performed. Finally, proof-of-concept of the regenerative capacity of P-ECM scaffolds was assessed in an infected mature canine tooth model following REPs versus blood clot (BC), injectable platelet-rich fibrin (i-PRF) or hyaluronic acid (HA). Statistical analysis was done using independent t test, the Friedman test and chi-square tests (p value ≤ 0.05).ResultsDNA was found to be below the cut-off point (50 ng/mg tissue). Histological evaluation revealed absence of nuclei, retention of glycosaminoglycans (GAGs) and collagen content, respectively. P-ECM hydrogel had a total protein content of (493.12 µg/µl) and protein release was detected up to 14 days. P-ECM hydrogel also retained VEGF, bFGF, TGF-β1 and BMP2. P-ECM hydrogel maintained the viability of rDPSCs as compared to cells cultured under control conditions. P-ECM hydrogel triggered more organized tissues compared to BC, i-PRF and HA when used in REPs for necrotic mature teeth in dogs. Periapical inflammation was significantly less in HA and P-ECM groups compared to blood-derived scaffolds.ConclusionBovine dental pulp-derived extracellular matrix (P-ECM) hydrogel scaffold retained its bioactive properties and demonstrated a promising potential in regenerative endodontic procedures compared to conventional blood-derived scaffolds.

In Vivo Evaluation of Demineralized Bone Matrix with Cancellous Bone Putty Formed Using Hydroxyethyl Cellulose as an Allograft Material in a Canine Tibial Defect Model.

Demineralized bone matrix (DBM) is a widely used allograft material for bone repair, but its handling properties and retention at defect sites can be challenging. Hydroxyethyl cellulose (HEC) has shown promise as a biocompatible carrier for bone graft materials. This study aimed to evaluate the efficacy of DBM combined with cancellous bone putty formed using HEC as an allograft material for bone regeneration in a canine tibial defect model. Experiments were conducted using dogs with proximal tibial defects. Four groups were compared: empty (control group), DBM + HEC (DH), DBM + cancellous bone + HEC (DCH), and DBM + cancellous bone + calcium phosphate + HEC (DCCH). Radiographic, micro-computed tomography (CT), and histomorphometric evaluations were performed 4 and 8 weeks postoperatively to assess bone regeneration. The Empty group consistently exhibited the lowest levels of bone regeneration throughout the study period, indicating that DBM and cancellous bone with HEC significantly enhanced bone regeneration. At week 4, the DCCH group showed the fastest bone regeneration on radiography and micro-computed tomography. By week 8, the DCH group showed the highest area ratio of new bone among all experimental areas, followed by the DH and DCCH groups. This study demonstrated that HEC significantly enhances the handling, mechanical properties, and osteogenic potential of DBM and cancellous bone grafts, making it a promising carrier for clinical applications in canine allograft models. When mixed with allograft cancellous bone, which has high porosity and mechanical strength, it becomes a promising material offering a more effective and reliable option for bone repair and regeneration.

Characterization of the Angiogenic and Proteomic Features of Circulating Exosomes in a Canine Mandibular Model of Distraction Osteogenesis.

Distraction osteogenesis (DO) represents a highly effective method for addressing significant bone defects; however, it necessitates a long treatment period. Exosomes are key mediators of intercellular communication. To investigate their role in the angiogenesis and osteogenesis of DO, we established a canine mandibular DO model with a bone defect (BD) group as the control. Higher levels of angiogenesis were observed in the regenerating tissue from the DO group compared to those from the BD group, accompanied by earlier osteogenesis. Proteomic analysis was performed on circulating exosomes at different phases of the DO using a data-independent acquisition method. Data are available via ProteomeXchange with the identifier PXD050531. The results indicated specific alterations in circulating exosome proteins at different phases of DO, reflecting the regenerative activities in the corresponding tissues. Notably, fibronectin 1 (FN1), thrombospondin 1 (THBS1), and transferrin receptor (TFRC) emerged as potential candidate proteins related to the angiogenic response in DO. Further cellular experiments validated the potential of DO-associated circulating exosomes to promote angiogenesis in endothelial cells. Collectively, these data reveal previously unknown mechanisms that may underlie the efficacy of DO and suggest that exosome-derived proteins may be useful as therapeutic targets for strategies designed to improve DO-related angiogenesis and bone regeneration.

Icariin-loaded chitosan/β-glycerophosphate thermosensitive hydrogel enhanced infection control and bone regeneration in canine with infectious bone defects.

Faced with infectious bone defects, the development of a thermosensitive hydrogel containing icariin (ICA) represents a promising therapeutic strategy targeting infection control and bone regeneration. In this study, we prepared and evaluated the physicochemical properties, in vitro and in vivo drug release, antimicrobial activity, anti-inflammatory properties, and bone repair effects of ICA/Chitosan/β-Glycerophosphate (ICA/CTS/β-GP) thermosensitive hydrogel. Our findings demonstrate that the ICA/CTS/β-GP thermosensitive hydrogel undergoes a liquid-to-gel transition at body temperature, which is crucial for maintaining local drug release at the defect site. Additionally, the hydrogel exhibited sustained release of ICA over 28days, showing high antimicrobial activity against Staphylococcus aureus and good biocompatibility in blood compatibility tests. In a canine model of infectious bone defects, the ICA/CTS/β-GP thermosensitive hydrogel showed effective infection control and modulated inflammation, vascular formation, and bone factor expression, while also activating the Wnt/β-catenin signaling pathway. In conclusion, the ICA/CTS/β-GP thermosensitive hydrogel could control infection and repair bone tissue. Its antimicrobial and osteogenic properties provide hope for its clinical application.

Transcriptional changes of genes encoding sarcoplasmic reticulum calcium binding and up-taking proteins in normal and Duchenne muscular dystrophy dogs

BackgroundCytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins.MethodsGiven the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle.ResultsIn affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs.ConclusionsOur study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines.

Sidestream dark field video microscopy demonstrates shelf-stable blood products preserve the endothelial glycocalyx in a canine hemorrhagic shock model.

To utilize sidestream dark field video microscopic technology to evaluate the endothelium in a canine hemorrhagic shock and resuscitation model. 6 purpose-bred adult dogs were anesthetized, instrumented, and subjected to hemorrhagic shock from September 2021 through June 2022. Each dog was resuscitated with 5 resuscitation strategies in an experimental crossover design study: (1) lactated Ringer's solution (LRS) and hydroxyethyl starch (HES) solution; (2) canine chilled whole blood (CWB); (3) canine fresh frozen plasma (FFP) and packed RBCs (pRBC); (4) canine freeze-dried plasma (FDP) and hemoglobin-based oxygen carrier (HBOC); or (5) HBOC/FDP and canine lyophilized platelets. Sidestream dark field video microscopic evaluation was performed at 5 time points: commencement, after hemorrhage, after shock, after resuscitation (T135), and conclusion (T180). There was a significant difference between the perfused boundary region (PBR) measurements when comparing the LRS/HES resuscitation arm to the CWB and FFP/pRBC resuscitation arms at T180. A significant difference in PBR was appreciated in the LRS/HES arm at T135 and T180 compared to its baseline. No other significant differences in PBR were appreciated when resuscitation arms were compared longitudinally or to each other. Shelf-stable blood products preserved the endothelial glycocalyx similarly to CWB and pRBC/FFP as evaluated by sidestream dark field video microscopy. Lactated Ringer and HES solutions did not adequately preserve the endothelial glycocalyx compared to CWB and pRBC/FFP. Shelf-stable blood products are a viable option to preserve the endothelial glycocalyx when used during hemorrhagic resuscitation in dogs.

Saurian-associated Leishmania tarentolae in dogs: Infectivity and immunogenicity evaluation in the canine model.

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.

Pre-vascularized porous gelatin-coated β-tricalcium phosphate scaffolds for bone regeneration: an in vivo and in vitro investigation.

Vascularization is vital in bone tissue engineering, supporting development, remodeling, and regeneration. Lack of vascularity leads to cell death, necessitating vascularization strategies. Angiogenesis, forming new blood vessels, provides crucial nutrients and oxygen. Pre-vascularized gelatin-coated β-tricalcium phosphate (G/β-TCP) scaffolds show promise in bone regeneration and vascularization. Our study evaluates G/β-TCP scaffolds' osteogenic and angiogenic potential in vitro and a canine model with vascular anastomosis. Channel-shaped G/β-TCP scaffolds were fabricated using foam casting and sintering of a calcium phosphate/silica slurry-coated polyurethane foam, then coated with cross-linked gelatin. Buccal fat pad-derived stem cells (BFPdSCs) were seeded onto scaffolds and assessed over time for adhesion, proliferation, and osteogenic capacity using scanning electron microscopy (SEM), 4,6-diamidino-2-phenylindole (DAPI) staining, Alamar blue, and alkaline phosphatase (ALP) assays. Scaffolds were implanted in a canine model to evaluate osteogenesis and angiogenesis by histology and CT scans at 12 wk. Our studies showed preliminary results for G/β-TCP scaffolds supporting angiogenesis and bone regeneration. In vitro analyses demonstrated excellent proliferation/viability, with BFPdSCs adhering and increasing on the scaffolds. ALP activity and protein levels increased, indicating osteogenic differentiation. Examination of tissue samples revealed granulation tissue with a well-developed vascular network, indicating successful angiogenesis and osteogenesis was further confirmed by a CT scan. In vivo, histology revealed scaffold resorption. However, scaffold placement beneath muscle tissue-restricted bone regeneration. Further optimization is needed for bone regeneration applications.