Transcutaneous treatment with vetdrop(®) sustains the adjacent cartilage in a microfracturing joint defect model in sheep

The significance of the adjacent cartilage in cartilage defect healing is not yet completely understood. Furthermore, it is unknown if the adjacent cartilage can somehow be influenced into responding after cartilage damage. The present study was undertaken to investigate whether the adjacent cartilage can be better sustained after microfracturing in a cartilage defect model in the stifle joint of sheep using a transcutaneous treatment concept (Vetdrop(®)). Carprofen and chito-oligosaccharids were added either as single components or as a mixture to a vehicle suspension consisting of a herbal carrier oil in a water-in-oil phase. This mixture was administered onto the skin with the aid of a specific applicator during 6 weeks in 28 sheep, allocated into 6 different groups, that underwent microfracturing surgery either on the left or the right medial femoral condyle. Two groups served as control and were either treated intravenously or sham treated with oxygen only. Sheep were sacrificed and their medial condyle histologically evaluated qualitatively and semi-quantitatively according to 4 different scoring systems (Mankin, ICRS, Little and O'Driscoll). The adjacent cartilage of animals of group 4 treated transcutaneously with vehicle, chito-oligosaccharids and carprofen had better histological scores compared to all the other groups (Mankin 3.3±0.8, ICRS 15.7±0.7, Little 9.0±1.4). Complete defect filling was absent from the transcutaneous treatment groups. The experiment suggests that the adjacent cartilage is susceptible to treatment and that the combination of vehicle, chitooligosaccharids and carprofen may sustain the adjacent cartilage during the recovery period.

Characterization of the 9L gliosarcoma implanted in the Fischer rat: an orthotopic model for a grade IV brain tumor.

Among rodent models for brain tumors, the 9L gliosarcoma is one of the most widely used. Our 9L-European Synchrotron Radiation Facility (ESRF) model was developed from cells acquired at the Brookhaven National Laboratory (NY, USA) in 1997 and implanted in the right caudate nucleus of syngeneic Fisher rats. It has been largely used by the user community of the ESRF during the last decade, for imaging, radiotherapy, and chemotherapy, including innovative treatments based on particular irradiation techniques and/or use of new drugs. This work presents a detailed study of its characteristics, assessed by magnetic resonance imaging (MRI), histology, immunohistochemistry, and cytogenetic analysis. The data used for this work were from rats sampled in six experiments carried out over a 3-year period in our lab (total number of rats = 142). The 9L-ESRF tumors were induced by a stereotactic inoculation of 10(4) 9L cells in the right caudate nucleus of the brain. The assessment of vascular parameters was performed by MRI (blood volume fraction and vascular size index) and by immunostaining of vessels (rat endothelial cell antigen-1 and type IV collagen). Immunohistochemistry and regular histology were used to describe features such as tumor cell infiltration, necrosis area, nuclear pleomorphism, cellularity, mitotic characteristics, leukocytic infiltration, proliferation, and inflammation. Moreover, for each of the six experiments, the survival of the animals was assessed and related to the tumor growth observed by MRI or histology. Additionally, the cytogenetic status of the 9L cells used at ESRF lab was investigated by comparative genomics hybridization analysis. Finally, the response of the 9L-ESRF tumor to radiotherapy was estimated by plotting the survival curves after irradiation. The median survival time of 9L-ESRF tumor-bearing rats was highly reproducible (19-20 days). The 9L-ESRF tumors presented a quasi-exponential growth, were highly vascularized with a high cellular density and a high proliferative index, accompanied by signs of inflammatory responses. We also report an infiltrative pattern which is poorly observed on conventional 9 L tumor. The 9L-ESRF cells presented some cytogenetic specificities such as altered regions including CDK4, CDKN2A, CDKN2B, and MDM2 genes. Finally, the lifespan of 9L-ESRF tumor-bearing rats was enhanced up to 28, 35, and 45 days for single doses of 10, 20, and 2 × 20 Gy, respectively. First, this report describes an animal model that is used worldwide. Second, we describe few features typical of our model if compared to other 9L models worldwide. Altogether, the 9L-ESRF tumor model presents characteristics close to the human high-grade gliomas such as high proliferative capability, high vascularization and a high infiltrative pattern. Its response to radiotherapy demonstrates its potential as a tool for innovative radiotherapy protocols.

Histomorphometric and Radiographical Changes After Lumbar Implantation of the PEEK Nonfusion Interspinous Device in the BB.4S Rat Model

Study Design. An experimental animal study. Objective. To investigate histomorphometric and radiographical changes in the BB.4S rat model after PEEK (polyetheretherketone) nonfusion interspinous device implantation. Summary of Background Data. Clinical effectiveness of the PEEK nonfusion spine implant Wallis (Abbott, Bordeaux, France; now Zimmer, Warsaw, IN) is well documented. However, there is a lack of evidence on the long-term effects of this implant on bone, in particular its influence on structural changes of bone elements of the lumbar spine. Methods. Twenty-four male BB.4S rats aged 11 weeks underwent surgery for implantation of a PEEK nonfusion interspinous device or for a sham procedure in 3 groups of 8 animals each: 1) implantation at level L4–L5; 2) implantation at level L5–L6; and 3) sham surgery. Eleven weeks postoperatively osteolyses at the implant-bone interface were measured via radiograph, bone mineral density of vertebral bodies was analyzed using osteodensitometry, and bone mineral content as well as resorption of the spinous processes were examined by histomorphometry. Results. Resorption of the spinous processes at the site of the interspinous implant was found in all treated segments. There was no significant difference in either bone density of vertebral bodies or histomorphometric structure of the spinous processes between adjacent vertebral bodies, between treated and untreated segments and between groups. Conclusion. These findings indicate that resorption of spinous processes because of a result of implant loosening, inhibit the targeted load redistribution through the PEEK nonfusion interspinous device in the lumbar spinal segment of the rat. This leads to reduced long-term stability of the implant in the animal model. These results suggest that PEEK nonfusion interspinous devices like the Wallis implants may have time-limited effects and should only be used for specified indications.

Levels of matrix metalloproteinase-9 within cerebrospinal fluid in a rabbit model of coccidioidal meningitis and vasculitis.

Matrix metalloproteinase (MMP)-9 is produced by the central nervous system and inflammatory cells in a variety of inflammatory conditions in both animals and humans. MMP-9 promotes inflammation, breakdown of the blood-brain barrier, and vasculitis. Because vasculitis is seen frequently in patients with coccidioidal meningitis (CM), this study evaluated the presence of MMP-9 within the cerebrospinal fluid (CSF) of rabbits infected intracisternally with Coccidioides immitis arthroconidia. Infected rabbits demonstrated systemic and neurological sequelae to infection, including CSF pleocytosis. Levels of MMP-9 within CSF were assayed by use of zymography and compared with MMP-2 levels, which served as an internal control. Elevated levels of MMP-9 were detectable by day 3, continued to increase through day 10, and declined by day 15 after infection. MMP-9 may contribute to inflammation and vasculitis in this animal model. Future work can focus on evaluation of MMP inhibitors, to gain a better perspective of the role of this MMP in CM.

Reactive oxygen intermediates contribute to necrotic and apoptotic neuronal injury in an infant rat model of bacterial meningitis due to group B streptococci.

Reactive oxygen intermediates (ROI) contribute to neuronal injury in cerebral ischemia and trauma. In this study we explored the role of ROI in bacterial meningitis. Meningitis caused by group B streptococci in infant rats led to two distinct forms of neuronal injury, areas of necrosis in the cortex and neuronal loss in the dentate gyrus of the hippocampus, the latter showing evidence for apoptosis. Staining of brain sections with diaminobenzidine after perfusion with manganese buffer and measurement of lipid peroxidation products in brain homogenates both provided evidence that meningitis led to the generation of ROI. Treatment with the radical scavenger alpha-phenyl-tert-butyl nitrone (PBN) (100 mg/kg q8h i.p.) beginning at the time of infection completely abolished ROI detection and the increase in lipidperoxidation. Cerebral cortical perfusion was reduced in animals with meningitis to 37.5+/-21.0% of uninfected controls (P < 0.05), and PBN restored cortical perfusion to 72.0+/-8.1% of controls (P < 0.05 vs meningitis). PBN also completely prevented neuronal injury in the cortex and hippocampus, when started at the time of infection (P < 0.02), and significantly reduced both forms of injury, when started 18 h after infection together with antibiotics (P < 0.004 for cortex and P < 0.001 for hippocampus). These data indicate that the generation of ROI is a major contributor to cerebral ischemia and necrotic and apoptotic neuronal injury in this model of neonatal meningitis.

Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model

Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial direct current stimulation could reduce the infarct size and the number of depolarizations, focal ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15min on and 15min off) starting 45min after middle cerebral artery occlusion and lasting 4h. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but starting soon after middle cerebral artery occlusion and lasting 6h. In both experiments controls were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p=0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048). Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.

Standard and higher doses of atropine in a canine model of pulseless electrical activity

OBJECTIVE To determine whether standard or increased doses of atropine improve the return of spontaneous circulation (ROSC) rate in a canine model of pulseless electrical activity (PEA). METHODS A prospective, controlled, blinded laboratory investigation was performed using an asphyxial canine cardiac arrest model. After the production of asphyxial PEA, 75 dogs remained in untreated PEA for 10 minutes and then were randomized to receive placebo (group 1) or one of four doses of atropine (group 2, 0.04 mg/kg; group 3, 0.1 mg/kg; group 4, 0.2 mg/kg; group 5, 0.4 mg/kg). All the animals received mechanical external CPR and epinephrine (0.02 mg/kg every 3 minutes) throughout resuscitation. RESULTS The ROSC rates were not significantly different between the groups (group 1, 73%; group 2, 67%; group 3, 40%; group 4, 47%; group 5, 27%; p = 0.06). The heart rates and hemodynamics during resuscitation were not significantly different between the groups. CONCLUSION In this canine model of asphyxial PEA cardiac arrest, standard-dose atropine did not improve ROSC rates, compared with placebo. Increasing doses of atropine tended to decrease ROSC rates, compared with placebo and standard-dose atropine.

The effects of endothelin-1 on coronary perfusion pressure during cardiopulmonary resuscitation in a canine model

OBJECTIVE To study the hemodynamic effects of exogenously administered endothelin-1 (ET-1), a peptide produced by endothelial cells with potent non-adrenergically mediated vasoconstrictor properties. METHODS A prospective drug intervention study was carried out in a resuscitation research laboratory. Fifteen mixed-breed dogs were anesthetized and instrumented for hemodynamic monitoring. Asphyxia arrest was produced by clamping the endotracheal tube. Hemodynamic data were collected continuously. Following loss of aortic fluctuations monitored by thoracic aortic catheter, the animals remained in pulseless electrical activity (PEA) for 10 minutes. After 10 minutes of no-flow PEA, closed-chest CPR was begun and the animals were randomized to one of three treatment groups (EPI, 0.02 mg/kg epinephrine IV every 3 minutes; ENDO, 100 micrograms ET-1 IV at 0 minutes; and EPI/ENDO, a combination of the EPI and ENDO treatments). RESULTS ENDO and EPI alone produced similar coronary perfusion pressures (CPPs). The EPI/ENDO combination produced significantly improved CPP compared with that of either EPI or ENDO alone. In the EPI group, the best mean CPP was 16 +/- 14 mm Hg and occurred 7 minutes after drug administration. In the ENDO group, the best mean CPP was 28 +/- 7 mm Hg and occurred 13 minutes after drug administration. In the EPI/ENDO combination group, the best mean CPP was 61 +/- 37 mm Hg and occurred 7 minutes after drug administration (p < 0.05 compared with the EPI and ENDO groups alone). CONCLUSION ET-1 is a potent vasoconstrictor. The combination of EPI and ENDO significantly improved CPP compared with that for either agent alone. ET-1 should be investigated further as a vasoconstrictor in cardiac arrest.

Preliminary study on carprofen concentration measurements after transcutaneous treatment with Vetdrop® in a microfracture joint defect model in sheep.

BackgroundThe present preliminary study describes concentration time courses of the NSAID carprofen in the plasma and synovial fluid in a microfrature sheep model after transcutaneous treatments with a novel application device (Vetdrop®). To treat circumscribed inflammatory processes a transcutaneous application device could potentially be beneficial. After transcutaneous application normally lower systemic concentrations are measured which may reduce the incidence of side effects, whereas efficacy is still maintained.In this study carprofen was used based on its capacity to provide analgesia after orthopaedic procedures in sheep and it is considered that it may have a positive influence on the healing of cartilage in low concentrations.ResultsIn all transcutaneously treated animals, carprofen plasma concentrations exceeded those of synovial fluid, although plasma levels remained significantly reduced (300-fold) as compared to carprofen administered intravenously. Furthermore, in contrast to the intravenously treated animals, a modest accumulation of carprofen in plasma and synovial fluid was observed in the transcutaneously treated animals over the 6-week treatment period.ConclusionsThe transcutaneously administered carprofen using the Vetdrop® device penetrated the skin and both, plasma- and synovial concentrations could be measured repeatedly over time. This novel device may be considered a valuable transcutaneous drug delivery system.

Evaluation of a new experimental model to study bone healing after ridge expansion with simultaneous implant placement--a pilot study in minipigs.

OBJECTIVE To evaluate the suitability of a minipig model for the study of bone healing and osseointegration of dental implants following bone splitting and expansion of narrow ridges. MATERIAL AND METHODS In four minipigs, the mandibular premolars and first molars were extracted together with removal of the buccal bone plate. Three months later, ridge splitting and expansion was performed with simultaneous placement of three titanium implants per quadrant. On one side of the mandible, the expanded bone gap between the implants was filled with an alloplastic biphasic calcium phosphate (BCP) material, while the gap on the other side was left unfilled. A barrier membrane was placed in half of the quadrants. After a healing period of 6 weeks, the animals were sacrificed for histological evaluation. RESULTS In all groups, no bone fractures occurred, no implants were lost, all 24 implants were osseointegrated, and the gap created by bone splitting was filled with new bone, irrespective of whether BCP or a barrier membrane was used. Slight exposure of five implants was observed, but did not lead to implant loss. The level of the most coronal bone-to-implant contact varied without being dependent on the use of BCP or a barrier membrane. In all groups, the BCP particles were not present deep in the bone-filled gap. However, BCP particles were seen at the crestal bone margin, where they were partly integrated in the new bone. CONCLUSIONS This new minipig model holds great promise for studying experimental ridge splitting/expansion. However, efforts must be undertaken to reduce implant exposure and buccal bone resorption.

A tick-borne encephalitis model in infant rats infected with langat virus

Tick-borne encephalitis virus (TBEV) is the causative agent of human TBE, a severe infection that can cause long-lasting neurologic sequelae. Langat virus (LGTV), which is closely related to TBEV, has a low virulence for human hosts and has been used as a live vaccine against TBEV. Tick-borne encephalitis by natural infection of LGTV in humans has not been described, but one of 18,500 LGTV vaccinees developed encephalitis. The pathogenetic mechanisms of TBEV are poorly understood and, currently, no effective therapy is available. We developed an infant rat model of TBE using LGTV as infective agent. Infant Wistar rats were inoculated intracisternally with 10 focus-forming units of LGTV and assessed for clinical disease and neuropathologic findings at Days 2, 4, 7, and 9 after infection. Infection with LGTV led to gait disturbance, hypokinesia, and reduced weight gain or weight loss. Cerebrospinal fluid concentrations of RANTES, interferon-γ, interferon-β, interleukin-6, and monocyte chemotactic protein-1 were increased in infected animals. The brains of animals with LGTV encephalitis exhibited characteristic perivascular inflammatory cuffs and glial nodules; immunohistochemistry documented the presence of LGTV in the thalamus, hippocampus, midbrain, frontal pole, and cerebellum. Thus, LGTV meningoencephalitis in infant rats mimics important clinical and histopathologic features of human TBE. This new model provides a tool to investigate disease mechanisms and to evaluate new therapeutic strategies against encephalitogenic flaviviruses.

Effect of enamel matrix derivative on periodontal wound healing and regeneration in an osteoporotic model.

BACKGROUND Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis-related periodontal defects. METHODS Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD-treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD-treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro-CT) analysis. The number of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. RESULTS OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by micro-CT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165μm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP-positive osteoclasts did not vary between untreated and EMD-treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMD-treated samples. CONCLUSIONS The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis.

Do different anesthesia regimes affect hippocampal apoptosis and neurologic deficits in a rodent cardiac arrest model?

Background Different anesthesia regimes are commonly used in experimental models of cardiac arrest, but the effects of various anesthetics on clinical outcome parameters are unknown. We conducted a study in which we subjected rats to cardiac arrest under medetomidine/ketamine or sevoflurane/fentanyl anesthesia. Methods Asystolic cardiac arrest for 8 minutes was induced in 73 rats with a mixture of potassium chloride and esmolol. Daily behavioral and neurological examination included the open field test (OFT), the tape removal test (TRT) and a neurodeficit score (NDS). Animals were randomized for sacrifice on day 2 or day 5 and brains were harvested for histology in the hippocampus cornus ammonis segment CA1. The inflammatory markers IL-6, TNF-α, MCP-1 and MIP-1α were assessed in cerebrospinal fluid (CSF). Proportions of survival were tested with the Fisher’s exact test, repeated measurements were assessed with the Friedman’s test; the baseline values were tested using Mann–Whitney U test and the difference of results of repeated measures were compared. Results In 31 animals that survived beyond 24 hours neither OFT, TRT nor NDS differed between the groups; histology was similar on day 2. On day 5, significantly more apoptosis in the CA1 segment of the hippocampus was found in the sevoflurane/fentanyl group. MCP-1 was higher on day 5 in the sevoflurane/fentanyl group (p = 0.04). All other cyto- and chemokines were below detection threshold. Conclusion In our cardiac arrest model neurological function was not influenced by different anesthetic regimes; in contrast, anesthesia with sevoflurane/fentanyl results in increased CSF inflammation and histologic damage at day 5 post cardiac arrest.

Evidence of an association between sleep and levodopa-induced dyskinesia in an animal model of Parkinson's disease.

Levodopa-induced dyskinesia (LID) represents a major challenge for clinicians treating patients affected by Parkinson's disease (PD). Although levodopa is the most effective treatment for PD, the remodeling effects induced by disease progression and the pharmacologic treatment itself cause a narrowing of the therapeutic window because of the development of LID. Although animal models of PD provide strong evidence that striatal plasticity underlies the development of dyskinetic movements, the pathogenesis of LID is not entirely understood. In recent years, slow homeostatic adjustment of intrinsic excitability occurring during sleep has been considered fundamental for network stabilization by gradually modifying plasticity thresholds. So far, how sleep affects on LID has not been investigated. Therefore, we measured synaptic downscaling across sleep episodes in a parkinsonian animal model showing dyskinetic movements similar to LID. Our electrophysiological, molecular, and behavioral results are consistent with an impaired synaptic homeostasis during sleep in animals showing dyskinesia. Accordingly, sleep deprivation causes an anticipation and worsening of LID supporting a link between sleep and the development of LID.

The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.