A Novel Tendon Injury Model, Induced by Collagenase Administration Combined with a Thermo-Responsive Hydrogel in Rats, Reproduces the Pathogenesis of Human Degenerative Tendinopathy.

Patellar tendinopathy is a common clinical problem, but its underlying pathophysiology remains poorly understood, primarily due to the absence of a representative experimental model. The most widely used method to generate such a model is collagenase injection, although this method possesses limitations. We developed an optimized rat model of patellar tendinopathy via the ultrasound-guided injection of collagenase mixed with a thermo-responsive Pluronic hydrogel into the patellar tendon of sixty male Wistar rats. All analyses were carried out at 3, 7, 14, 30, and 60 days post-injury. We confirmed that our rat model reproduced the pathophysiology observed in human patients through analyses of ultrasonography, histology, immunofluorescence, and biomechanical parameters. Tendons that were injured by the injection of the collagenase-Pluronic mixture exhibited a significant increase in the cross-sectional area (p < 0.01), a high degree of tissue disorganization and hypercellularity, significantly strong neovascularization (p < 0.01), important changes in the levels of types I and III collagen expression, and the organization and presence of intra-tendinous calcifications. Decreases in the maximum rupture force and stiffness were also observed. These results demonstrate that our model replicates the key features observed in human patellar tendinopathy. Collagenase is evenly distributed, as the Pluronic hydrogel prevents its leakage and thus, damage to surrounding tissues. Therefore, this model is valuable for testing new treatments for patellar tendinopathy.

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Muscle Precursor Cells Enhance Functional Muscle Recovery and Show Synergistic Effects With Postinjury Treadmill Exercise in a Muscle Injury Model in Rats.

BACKGROUND: Skeletal muscle injuries represent a major concern in sports medicine. Cell therapy has emerged as a promising therapeutic strategy for muscle injuries, although the preclinical data are still inconclusive and the potential clinical use of cell therapy has not yet been established. PURPOSE: To evaluate the effects of muscle precursor cells (MPCs) on muscle healing in a small animal model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 27 rats were used in the study. MPCs were isolated from rat (n = 3) medial gastrocnemius muscles and expanded in primary culture. Skeletal muscle injury was induced in 24 rats, and the animals were assigned to 3 groups. At 36 hours after injury, animals received treatment based on a single ultrasound-guided MPC (105 cells) injection (Cells group) or MPC injection in combination with 2 weeks of daily exercise training (Cells+Exercise group). Animals receiving intramuscular vehicle injection were used as controls (Vehicle group). Muscle force was determined 2 weeks after muscle injury, and muscles were collected for histological and immunofluorescence evaluation. RESULTS: Red fluorescence-labeled MPCs were successfully transplanted in the site of the injury by ultrasound-guided injection and were localized in the injured area after 2 weeks. Transplanted MPCs participated in the formation of regenerating muscle fibers as corroborated by the co-localization of red fluorescence with developmental myosin heavy chain (dMHC)-positive myofibers by immunofluorescence analysis. A strong beneficial effect on muscle force recovery was detected in the Cells and Cells+Exercise groups (102.6% ± 4.0% and 101.5% ± 8.5% of maximum tetanus force of the injured vs healthy contralateral muscle, respectively) compared with the Vehicle group (78.2% ± 5.1%). Both Cells and Cells+Exercise treatments stimulated the growth of newly formed regenerating muscles fibers, as determined by the increase in myofiber cross-sectional area (612.3 ± 21.4 µm2 and 686.0 ± 11.6 µm2, respectively) compared with the Vehicle group (247.5 ± 10.7 µm2), which was accompanied by a significant reduction of intramuscular fibrosis in Cells and Cells+Exercise treated animals (24.2% ± 1.3% and 26.0% ± 1.9% of collagen type I deposition, respectively) with respect to control animals (40.9% ± 4.1% in the Vehicle group). MPC treatment induced a robust acceleration of the muscle healing process as demonstrated by the decreased number of dMHC-positive regenerating myofibers (enhanced replacement of developmental myosin isoform by mature myosin isoforms) (4.3% ± 2.6% and 4.1% ± 1.5% in the Cells and Cells+Exercise groups, respectively) compared with the Vehicle group (14.8% ± 13.9%). CONCLUSION: Single intramuscular administration of MPCs improved histological outcome and force recovery of the injured skeletal muscle in a rat injury model that imitates sports-related muscle injuries. Cell therapy showed a synergistic effect when combined with an early active rehabilitation protocol in rats, which suggests that a combination of treatments can generate novel therapeutic strategies for the treatment of human skeletal muscle injuries. CLINICAL RELEVANCE: Our study demonstrates the strong beneficial effect of MPC transplant and the synergistic effect when the cell therapy is combined with an early active rehabilitation protocol for muscle recovery in rats; this finding opens new avenues for the development of effective therapeutic strategies for muscle healing and clinical trials in athletes undergoing MPC transplant and rehabilitation protocols.

Design and implementation of a microelectrode assembly for use on noncontact in situ electroporation of adherent cells.

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.

Dopaminergic and glutamatergic signaling crosstalk in Huntington's disease neurodegeneration: the role of p25/cyclin-dependent kinase 5.

Altered glutamatergic and dopaminergic signaling has been proposed as contributing to the specific striatal cell death observed in Huntington's disease (HD). However, the precise mechanisms by which mutant huntingtin sensitize striatal cells to dopamine and glutamate inputs remain unclear. Here, we demonstrate in knock-in HD striatal cells that mutant huntingtin enhances dopamine-mediated striatal cell death via dopamine D(1) receptors. Moreover, we show that NMDA receptors specifically potentiate the vulnerability of mutant huntingtin striatal cells to dopamine toxicity as pretreatment with NMDA increased D(1)R-induced cell death in mutant but not wild-type cells. As potential underlying mechanism of increased striatal vulnerability, we identified aberrant cyclin-dependent kinase 5 (Cdk5) activation. We demonstrate that enhanced Cdk5 phosphorylation and increased calpain-mediated conversion of the Cdk5 activator p35 into p25 may account for the deregulation of Cdk5 associated to dopamine and glutamate receptor activation in knock-in HD striatal cells. Moreover, supporting a detrimental role of Cdk5 in striatal cell death, neuronal loss can be widely prevented by roscovitine, a potent Cdk5 inhibitor. Significantly, reduced Cdk5 expression together with enhanced Cdk5 phosphorylation and p25 accumulation also occurs in the striatum of mutant Hdh(Q111) mice and HD human brain suggesting the relevance of deregulated Cdk5 pathway in HD pathology. These findings provide new insights into the molecular mechanisms underlying the selective vulnerability of striatal cells in HD and identify p25/Cdk5 as an important mediator of dopamine and glutamate neurotoxicity associated to HD.