Immunohistochemical Analysis of Knee Chondral Defect Repair after Autologous Particulated Cartilage and Platelet-Rich Plasma Treatment in Sheep.

This study performs an analysis that will enable the evaluation of the quality, durability, and structure of repaired cartilaginous extracellular matrix tissue using an autologous-based particulated autograft cartilage and platelet-rich plasma treatment (PACI + PRP). A single-blind controlled experiment was conducted on 28 sheep to evaluate the efficacy of the PACI + PRP treatment for cartilage defects. Full-thickness 8 mm diameter defects were created in the weight-bearing area of both knees. The right knees received PACI + PRP. The left knees were treated with Ringer's lactate solution (RLS) or hyaluronic acid (HA) injections. Sheep were euthanized at 9- or 18-months post-surgery. An extensive immunohistochemical analysis was performed to assess collagen types (I, II, III, V, VI, IX, X, XI) and aggrecan positivity. A semiquantitative scoring system provided a detailed evaluation of immunostaining. Collagens and aggrecan scores in the PACI + PRP groups were similar to healthy cartilage. Significant differences were found in collagens associated with matrix maturity (II and V), degradation (IX), structure and mechanics (VI), and hypertrophy (X) between healthy cartilage and RLS- or HA-repaired cartilage. The PACI + PRP treatment advanced the repair cartilage process in chondral defects with mature hyaline cartilage and enhanced the structural and mechanical qualities with better consistent cartilage, less susceptible to degradation and without hypertrophic formation over time.

Particulate cartilage and platelet-rich plasma treatment for knee chondral defects in sheep.

PURPOSE: Articular cartilage is vulnerable to multiple types of damage and it has limited reparative and regenerative capacities due to its absence of vascularity. Although a large number of therapeutic strategies exist to treat chondral defects, they have some limitations, such as fibrocartilage formation. Therefore, the goal of the present study was to evaluate the chondrogenic regenerative properties of an autologous-made matrix of particulated cartilage and platelet-rich plasma (PACI + PRP) implantation for the treatment of full-thickness chondral defects in sheep. METHODS: A full-thickness 8 mm diameter cartilage defect was created in the weight-bearing area of the medial femoral condyle in both knees of 16 sheep. The right knees of all animals were treated with particulated autograft cartilage implantation and platelet-rich plasma, while the left knees were injected with Ringer's lactate solution or hyaluronic acid. The sheep were killed 9 or 18 months after surgery. Macroscopic evaluations were performed using three different scoring systems, and histopathological evaluations were performed using a modified scoring system based on different scoring systems. RESULTS: The PACI + PRP groups showed statistically significant differences in the percentage of defect repair and chondrocytes in the newly formed cartilage tissue at 18 months compared to 9 months. CONCLUSIONS: The results suggest that macroscopic appearance, histological structure and chondrocyte repair were improved when using PACI + PRP treatment for chondral defects, producing an outcome similar to the surrounding healthy cartilage. PACI + PRP is a totally autologous, easy, and unexpensive treatment that can be performed in one-step procedure and is useful as a therapeutic option for knee chondral defects.

Main and Minor Types of Collagens in the Articular Cartilage: The Role of Collagens in Repair Tissue Evaluation in Chondral Defects.

Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.

Preperitoneal ropivacaine infusion versus epidural ropivacaine-morphine for postoperative analgesia in dogs undergoing ovariohysterectomy: a randomized clinical trial.

OBJECTIVE: To assess the effect of continuous wound infusion (CWI) with preperitoneal ropivacaine on postoperative analgesia and compare it with the epidural administration of ropivacaine and morphine in bitches undergoing ovariohysterectomy. STUDY DESIGN: A parallel, randomized, clinical, prospective and nonblinded study. ANIMALS: A group of 38 Greyhound bitches. METHODS: In the catheter group (CathG), CWI with ropivacaine 1% (1 mg kg-1 + 0.8 mg kg-1 hour-1) was applied to the preperitoneal space over the surgical incision. In the epidural group (EpiG), ropivacaine 0.5% (1.3 mg kg-1) and morphine (0.1 mg kg-1) were epidurally administered. Occipital-coccygeal length was used to calculate the volume for the epidural. Pain was scored using a dynamic interactive visual analogue scale (DIVAS) and Glasgow composite measure pain scale-short form (CMPS-SF) before anaesthesia and at 2, 4, 6, 18, 21 and 24 hours after extubation. Incisional sensitivity using a dynamometer (MWTs-incision) was evaluated simultaneously. Plasma ropivacaine and cortisol concentrations, degree of sedation, motor blockade and response to interdigital clamping were measured or assessed. A two-way mixed analysis of variance and a Mann-Whitney U test were used to analyse data; p < 0.05. RESULTS: No differences were detected in the DIVAS (p = 0.301), CMPS-SF (p = 0.600) scores, MWTs-incision measurements (p = 0.257) and cortisol values (p = 0.878) between the groups. Rescue analgesia was required in two dogs, one in each group, at 2 hours. Sedation, motor blockade and negative response to interdigital clamping were detected in EpiG at 2, 4 and 6 hours. Mean plasma ropivacaine values were higher in CathG (0.475 ± 0.164 ng mL-1) than in EpiG (0.184 ± 0.213 ng mL-1; p = 0.001). CONCLUSION AND CLINICAL RELEVANCE: Compared with epidural ropivacaine and morphine, CWI with preperitoneal ropivacaine is an effective analgesic technique for postoperative pain management in bitches undergoing ovariohysterectomy without motor blockade.

Systemic and local effects of lidocaine or mepivacaine when used for intravenous regional anaesthesia of the distal limb in standing sedated horses.

BACKGROUND: Local anaesthetics are being combined clinically with amikacin in intravenous regional limb perfusion (IVRLP), with limited knowledge on the analgesia provided and its onset and duration of action after tourniquet application and release. OBJECTIVE: To evaluate the systemic clinical effect, limb withdrawal to nociceptive stimulation, and plasma and synovial fluid concentrations after IVRLP with lidocaine or mepivacaine in standing sedated horses. STUDY DESIGN: Prospective, controlled, randomised, cross-over study. METHODS: Six healthy adult horses were sedated and received IVRLP with lidocaine, mepivacaine or saline (negative control), or perineural anaesthesia of the medial and lateral palmar and palmar metacarpal nerves (positive control) in one forelimb with a 3-week washout period between trials. Electrical and mechanical stimuli were used to test nociceptive threshold of the limb before and after IVRLP/perineural anaesthesia. For lidocaine and mepivacaine trials, blood was collected from the jugular vein and synovial fluid from the radiocarpal joint before, during and out to 24 hours after IVRLP. Drug concentrations were measured using high-performance liquid chromatography. RESULTS: Nociceptive thresholds for lidocaine, mepivacaine and perineural anaesthesia trials were significantly increased compared with saline and baseline values at 10, 20 and 30 minutes, with no differences between anaesthetic trials. During this time, horses had lower heart rates than IVRLP with saline. After tourniquet release at 30 minutes, nociceptive thresholds for lidocaine and mepivacaine trials gradually returned to baselines, whereas perineural anaesthesia trial remained unchanged out to an hour. Plasma lidocaine and mepivacaine concentrations were ≤50 ng/mL while the tourniquet was in place, significantly increasing 10 minutes after tourniquet release. Maximal lidocaine and mepivacaine concentrations in synovial fluid were reached 25 minutes after IVRLP injection. MAIN LIMITATIONS: Amikacin was not included in the perfusate. CONCLUSION: Similar to perineural anaesthesia, IVRLP with lidocaine or mepivacaine provides anti-nociception to the distal limb in standing sedated horses while a tourniquet is applied with concentrations remaining below toxic levels in plasma and synovial fluid.

Comparison of bispectral index and spectral entropy during isoflurane and medetomidine general anaesthesia in horses.

BACKGROUND: The bispectral index (BIS) has been evaluated as an indicator of central nervous system (CNS) depression in horses during general anaesthesia. The spectral entropy is another electroencephalographic device and it has not been evaluated yet in horses. OBJECTIVES: To determine if spectral entropy can assess anaesthetic depth during the different phases of anaesthesia, define the value of state and response entropy during surgical plane of anaesthesia and compare them with BIS. STUDY DESIGN: Clinical, prospective, non-blinded observational study. METHODS: Thirty-five horses ASA I or II undergoing scheduled surgical procedure were used. BIS and electromyography (EMG) with a BIS monitor and state and response entropy with a spectral entropy monitor were recorded at baseline after receiving 5 µg/kg bwt i.v. of medetomidine (sedation period), during the anaesthetic maintenance with isoflurane and medetomidine (intraoperative period) and once the trachea was extubated (recovery period). A general linear model for repeated measurements was employed. Correlation and agreement between methods were also assessed. Data are presented as mean ± SD. RESULTS: State entropy, response entropy and EMG showed significant differences according to the anaesthetic period (P < .001). There was no significant difference in BIS between baseline and sedation period, but there were differences between the remainder of the periods (P < .001). BIS (53.4 ± 11.2) was significantly higher (P < .001) than response entropy (35.1 ± 7.1) and state entropy (27.4 ± 4.8) during surgical plane of anaesthesia. The ICC between BIS and response entropy was 0.56 and between BIS and state entropy was 0.43, without agreement between them. MAIN LIMITATIONS: The need to shave the skin in contact with the sensors and the difficulty in taking measurements during recovery period. CONCLUSIONS: Spectral entropy can be used to detect the different periods of an anaesthetic protocol, with the lowest values during the intraoperative period. A low correlation and no concordance were observed between both methods.

Anesthetic effects of isoflurane and propofol on cerebrospinal fluid biochemical markers in healthy dogs.

BACKGROUND: The study of brain energy metabolism (BEM) markers in the cerebrospinal fluid (CSF) is a potential diagnostic and prognostic tool for many central nervous system (CNS) diseases. To date, in veterinary medicine, few studies are reporting physiologic ranges for some BEM markers. Recently, the influence of anesthetic drugs on BEM markers has been described in mice; subsequently, the study of CSF-BEM markers has gained increasing attention. OBJECTIVES: The effects of anesthetic agents on BEM are poorly understood in dogs. The aim of this study was to evaluate the influence of propofol, isoflurane, and the duration of anesthesia on CSF-BEM markers in dogs. METHODS: Nine dogs were anesthetized at two different periods, one month apart. In the first period, the dogs were intravenously anesthetized with propofol (PRO-group), and in the second period, the dogs received inhalant anesthesia with isoflurane (ISO-group). In both cases, CSF and blood were collected 15 minutes (T0) and 3 hours after induction (T3) and analyzed for lactate, pyruvate, glucose, creatine kinase, glutamate, and electrolyte concentrations. RESULTS: CSF lactate (CSF-L) showed variation depending on the anesthetic agent and time, being significantly lower after 3 hours of anesthesia in the PRO-group and showing a trend to increase over time in the ISO-group. No changes were detected over time or between groups in CSF glutamate, glucose, or electrolytes. CONCLUSIONS: The results of this study support that the anesthetic drug choice and length of the general anesthesia should be considered when CSF-L analyses are interpreted in dogs.

Comparison of the isoflurane concentration of using dexketoprofen or methadone at premedication during orthopedic surgery in dogs.

Thirty-two dogs were used in this prospective, randomized, clinical and double-blinded study. Dexmedetomidine was administered at 1 µg/kg IV, and randomly each dog received dexketoprofen 1 mg/kg IV (group DK) or methadone 0.2 mg/kg IV (group M). Dogs were induced with propofol and maintained with isoflurane in 100% oxygen. During surgery, the isoflurane concentration was changed depending on clinical signs of depth of anesthesia. Fentanyl and propofol could be used as required. Qualities of sedation and recovery were evaluated. A generalized linear mixed model or Mann-Whiney U test was used, and P<0.05 was considered statistically significant. No significant differences were observed between groups in the qualities of sedation and recovery, isoflurane concentration and in the total amount of fentanyl and propofol used intraoperatively. This study shows that the administration of dexketoprofen at 1 mg/kg IV at premedication required a similar isoflurane concentration to maintain anesthesia as methadone at 0.2 mg/kg IV during orthopedic surgery in dogs. Further analgesia is recommended intraoperatively, because of the need of fentanyl and propofol in same animals in both groups.

Romifidine and low doses of tiletamine-zolazepam in dogs.

OBJECTIVE: To evaluate clinical effects of romifidine and low doses of tiletamine-zolazepam (TZ) in dogs. STUDY DESIGN: Randomized "blinded" cross-over study. ANIMALS: Six healthy beagle dogs (two males, four females). METHODS: In separate preliminary experiments dogs received intravenous (IV) tiletamine-zolazepam (TZ) at 1 and 2 mg kg(-1). For the main trial, dogs received romifidine (R) followed 5 minutes later by IV at six dose regimens: R40TZ1, R60TZ1, R80TZ1 (Romifidine at 40, 60, 80 µg kg(-1) and TZ at 1 mg kg(-1)), R40TZ2, R60TZ2 and R80TZ2 (Romifidine at 40, 60, 80 µg kg(-1) and TZ at 2 mg kg(-1)). Dogs underwent endotracheal intubation, but breathed room air. Cardiorespiratory variables were measured and arterial blood analyzed. Quality of sedation, duration of anaesthesia and time to recovery (TR) were recorded. Data were analysed by anova or Friedman test as relevant. RESULTS: Endotracheal intubation was possible with all romifidine/TZ combinations but not with TZ alone. Mean times (minutes) from TZ injection to return of pedal reflex were 1-3 minutes for TZ alone, and 9-17 minutes for romifidine combinations. In the main trial (romifidine combinations) mean time (minutes) to standing increased with increasing dosage (R40TZ1 13; R80TZ2 32). Five minutes after TZ administration, when compared with baseline arterial blood pressures and arterial carbon dioxide had increased, and respiratory rate, pH and arterial oxygen tensions decreased, these changes becoming statistically significant with the higher dose rates. One dog in R60TZ2 and three dogs in R80TZ2 became hypoxaemic. CONCLUSIONS AND CLINICAL RELEVANCE: Romifidine improves the quality and lengthens the duration of anaesthesia induced by TZ. The combination provides a suitable protocol for induction of or short-term anaesthesia in healthy dogs. However, the higher doses cause cardiovascular stimulation and respiratory depression, and precautions should be taken accordingly.

Histological study of the influence of plasma rich in growth factors (PRGF) on the healing of divided Achilles tendons in sheep.

BACKGROUND: The use of plasma rich in growth factors (PRGF) has been proposed to improve the healing of Achilles tendon injuries, but there is debate about the effectiveness of this therapy. The objective of the present study was to evaluate the histological effects of PRGF, which is a type of leukocyte-poor platelet-rich plasma, on tendon healing. METHODS: The Achilles tendons of twenty-eight sheep were divided surgically. The animals were randomly divided into four groups of seven animals each. The repaired tendons in two groups received an infiltration of PRGF intraoperatively and every week for the following three weeks under ultrasound guidance. The tendons in the other two groups received injections with saline solution. The animals in one PRGF group and one saline solution group were killed at four weeks, and the animals in the remaining two groups were killed at eight weeks. The Achilles tendons were examined histologically, and the morphometry of fibroblast nuclei was calculated. RESULTS: The fibroblast nuclei of the PRGF-treated tendons were more elongated and more parallel to the tendon axis than the fibroblast nuclei of the tendons in the saline solution group at eight weeks. PRGF-treated tendons showed more packed and better oriented collagen bundles at both four and eight weeks. In addition to increased maturation of the collagen structure, fibroblast density was significantly lower in PRGF-infiltrated tendons. PRGF-treated tendons exhibited faster vascular regression than tendons in the control groups, as demonstrated by a lower vascular density at eight weeks. CONCLUSIONS: PRGF was associated with histological changes consistent with an accelerated early healing process in repaired Achilles tendons in sheep after experimental surgical disruption. PRGF-treated tendons showed improvements in the morphometric features of fibroblast nuclei, suggesting a more advanced stage of healing. At eight weeks, histological examination revealed more mature organization of collagen bundles, lower vascular densities, and decreased fibroblast densities in PRGF-treated tendons than in tendons infiltrated with saline solution. These findings were consistent with a more advanced stage of the healing process. CLINICAL IMPLICATIONS: Based on the findings in this animal model, PRGF infiltration may improve the early healing process of surgically repaired Achilles tendons.

A comparison of the effect of convection against diffusion in hemodynamics and cytokines clearance in an experimental model of septic shock.

BACKGROUND: Replacement therapies based on the use of convection have value for the removal of inflammatory mediators. Such therapies have been proposed for the management of septic shock, but diffusion has not proved useful in this scenario, unless high-flow membranes are used. The exact role of diffusion in these cases remains to be clarified because continuous replacement therapies are usually delivered with low-flow membranes and mixed convection-diffusion modalities. However, studies specifically addressing this problem have not been performed. Our aim was to define the efficacy of hemofiltration (convection) and hemodialysis (diffusion) in cytokine clearance and hemodynamic improvement in an experimental model of septic shock. METHODS: Shock was induced in 15 beagle dogs (weight 10-15 kg) by infusion of 1 mg/kg of ultrapure Escherichia coli lipopolysaccharide diluted in 20 mL saline for 10 minutes. Five animals were followed without interventions (controls), five animals were treated with convection (100 mL kg h) for 6 hours, and five animals were treated with diffusion (100 mL kg h) for 6 hours. RESULTS: All subjects in the control group died during the study, whereas all treated subjects survived. Mean arterial pressure, cardiac output, systolic variability volume, systemic vascular resistances, dPMax, and pulmonary compliance improved in treated subjects. However, the differences in mean arterial pressure and cardiac output were significant only in the convection group and not in the diffusion-treated group.Tumor necrosis factor α rose equally in all groups and decreased only in treated subjects. Interleukin 6 rose in the three groups but decreased only in the convection group and remained unchanged in the control and diffusion groups. CONCLUSION: Convection and diffusion improved survival and hemodynamic parameters in a septic shock model. Improvement was more pronounced with convection, a difference that may be explained by convective clearance of cytokines.

Bladder mucosal CO2 compared with gastric mucosal CO2 as a marker for low perfusion states in septic shock.

Recent reports indicate the possible role of bladder CO(2) as a marker of low perfusion states. To test this hypothesis, shock was induced in six beagle dogs with 1 mg/kg of E. coli lipopolysaccharide, gastric CO(2) (CO(2)-G) was measured with a continuous monitor, and a pulmonary catheter was inserted in the bladder to measure CO(2) (CO(2)-B). Levels of CO(2)-B were found to be lower than those of CO(2)-G, with a mean difference of 36.8 mmHg (P < 0.001), and correlation between both measurements was poor (r(2) = 0.16). Even when the correlation between CO(2)-G and ΔCO(2)-G was narrow (r(2) = 0.86), this was not the case for the relationship between CO(2)-B and ΔCO(2)-B (r(2) = 0.29). Finally, the correlation between CO(2)-G and base deficit was good (r(2) = 0.45), which was not the case with the CO(2)-B correlation (r(2) = 0.03). In our experience, bladder CO(2) does not correlate to hemodynamic parameters and does not substitute gastric CO(2) for detection of low perfusion states.

Cardiorespiratory effects of desflurane in dogs given romifidine or medetomidine before induction of anesthesia with propofol.

The objective of this study was to evaluate the use of desflurane after induction of anesthesia with propofol in dogs sedated with romifidine or medetomidine. Each of 8 healthy dogs received intravenously, in random order, 3 preanesthetic protocols: romifidine, 40 microg/kg of body weight (BW) (R40); romifidine, 80 microg/kg BW (R80); and medetomidine, 10 microg/kg BW (MED). Cardiovascular and respiratory variables were recorded during the procedure. Time to extubation, time to sternal recumbency, and time to standing were also recorded. Heart rate and respiratory rate decreased significantly during sedation from baseline values, but there were no differences between the means for the 3 preanesthetic protocols. Mean values for heart rate, mean arterial blood pressure, systolic arterial pressure, diastolic arterial pressure, respiratory rate, tidal volume, arterial oxygen saturation, end-tidal CO2 level, pH, and arterial blood gas values during anesthesia were similar for the 3 protocols. The mean end-tidal desflurane concentration was significantly lower with the R80 protocol than with the R40 protocol. The mean time to extubation was significantly shorter with the R40 protocol than with the R80 and MED protocols.