Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection.

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and assessment using micro-computed tomography. Dunkin-Hartley guinea pigs are robust models for osteoarthritis research as they spontaneously develop osteoarthritis in their knees. Intra-articular drug delivery is a common method to study the effects of an investigational drug in vivo. In humans, therapeutic agents administered via intra-articular injection can offer pain relief and delay further progression of osteoarthritis. As with any species, the introduction of a needle into a joint space has the potential to cause injury, which can result in pain, lameness, or infection. Such adverse events can compromise animal welfare, confound study results, and necessitate additional animals to achieve study objectives. As such, it is imperative to develop proper injection techniques to prevent complications, especially in longitudinal studies that require multiple, repeated intra-articular injections. Using the presented methodology, five guinea pigs received bilateral knee injections under general anesthesia. Seven days after injection, animals were humanely euthanized for analysis of osteoarthritis severity. No adverse events occurred following anesthesia or knee injections, including limping, pain, or infection. X-ray micro-computed tomography analysis of the knee can detect pathologic changes associated with osteoarthritis. Micro-computed tomography data indicates osteoarthritis is more severe in older animals, as indicated by increased bone mineral density and trabecular thickness with age. These results are consistent with histologic changes and Modified Mankin scores, an established and widely used scoring system to assess arthritis severity in these same animals. This protocol can be utilized to refine intra-articular injections in guinea pigs.

Inhibition of complement C3 prevents osteoarthritis progression in guinea pigs by blocking STAT1 activation.

Osteoarthritis (OA) is one of the leading causes of disability, affecting over 500 million adults worldwide. Previous studies have found that various inflammatory factors can contribute to the pathogenesis of OA, including complement factors in the synovial fluid of OA patients. However, the pathogenesis of this disease is still not known, and the only therapy of severe OA is total joint replacements. Total joint replacements are invasive, expensive, and affect quality of life. Here we show that when human articular chondrocytes are stimulated with pro-inflammatory mediator interleukin-1ß (IL-1ß) there is an increase in inflammatory factors including complement component 3 (C3). We also found the transcription factor, signal transducer and activator of transcription 1 (STAT1), is responsible for increased C3 expression after IL-1ß stimulation in human articular chondrocytes. A specific STAT1 inhibitor, fludarabine, attenuates the hyper-expression of C3 and delays/prevents spontaneous OA in Dunkin-Hartley guinea pigs. Since fludarabine is already clinically used for chemotherapy, this study has great translational potential as a unique disease-modifying osteoarthritis drug (DMOAD) in treating primary OA.

Establishing New Isosexual Pairs in Adult Male Guinea Pigs (Cavia porcellus) to Facilitate Social Housing.

Guinea pigs (Cavia porcellus) are a commonly used species in biomedical research. As social creatures, compatible guinea pigs should be housed together unless scientific objectives or veterinary care require otherwise. Extensive literature suggests that adult male guinea pigs are highly aggressive in the presence of females, but data are lacking regarding the compatibility of cohoused adult males in the absence of females. Most studies that use adult males do not report housing densities. We used serial wound scoring and observations of behavior to determine whether unfamiliar adult male guinea pigs will develop stable, prosocial isosexual pairs. Wound scoring was performed before and 24 h after pairing. Serial behavioral observations assessed affiliative and agonistic behaviors at 0.5, 2, 24, and 48 h after pairing. Wound scoring and behavioral observations continued weekly for 1 mo and monthly thereafter. Wound scores were significantly higher at 24 h after pairing as compared with baseline and all other time points. Wounding was rare after week 2, indicating reduced aggression. Furthermore, affiliative behaviors significantly increased over time while agonistic behaviors were rare. Together, these data suggest that unfamiliar adult male guinea pigs establish stable prosocial pairs after an acclimation period. As was done in the present study, providing ample space, separate shelters for each animal, and the absence of female guinea pigs will likely facilitate successful pairing. We recommend consideration of a social housing program for adult male guinea pigs to provide companionship and enrich their housing environment.

Accurate detection of spontaneous seizures using a generalized linear model with external validation.

OBJECTIVE: Seizure detection is a major facet of electroencephalography (EEG) analysis in neurocritical care, epilepsy diagnosis and management, and the instantiation of novel therapies such as closed-loop stimulation or optogenetic control of seizures. It is also of increased importance in high-throughput, robust, and reproducible pre-clinical research. However, seizure detectors are not widely relied upon in either clinical or research settings due to limited validation. In this study, we create a high-performance seizure-detection approach, validated in multiple data sets, with the intention that such a system could be available to users for multiple purposes. METHODS: We introduce a generalized linear model trained on 141 EEG signal features for classification of seizures in continuous EEG for two data sets. In the first (Focal Epilepsy) data set consisting of 16 rats with focal epilepsy, we collected 1012 spontaneous seizures over 3 months of 24/7 recording. We trained a generalized linear model on the 141 features representing 20 feature classes, including univariate and multivariate, linear and nonlinear, time, and frequency domains. We tested performance on multiple hold-out test data sets. We then used the trained model in a second (Multifocal Epilepsy) data set consisting of 96 rats with 2883 spontaneous multifocal seizures. RESULTS: From the Focal Epilepsy data set, we built a pooled classifier with an Area Under the Receiver Operating Characteristic (AUROC) of 0.995 and leave-one-out classifiers with an AUROC of 0.962. We validated our method within the independently constructed Multifocal Epilepsy data set, resulting in a pooled AUROC of 0.963. We separately validated a model trained exclusively on the Focal Epilepsy data set and tested on the held-out Multifocal Epilepsy data set with an AUROC of 0.890. Latency to detection was under 5 seconds for over 80% of seizures and under 12 seconds for over 99% of seizures. SIGNIFICANCE: This method achieves the highest performance published for seizure detection on multiple independent data sets. This method of seizure detection can be applied to automated EEG analysis pipelines as well as closed loop interventional approaches, and can be especially useful in the setting of research using animals in which there is an increased need for standardization and high-throughput analysis of large number of seizures.

Gα12 is required for renal cystogenesis induced by Pkd1 inactivation.

Mutation of PKD1, encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12, hereafter Gα12) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 (Gα12-/-). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 (Mx1Cre+Pkd1f/fGα12+/+) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 (Mx1Cre+Pkd1f/fGα12-/-) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell-matrix and cell-cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.

Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells.

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused ß-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/ß-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.