1,25(OH)2D3 ameliorates palmitate-induced lipotoxicity in human primary osteoblasts leading to improved viability and function.

Contributing to bone loss with aging is a progressive reduction in osteoblast number and function leading to decreased bone formation. In aging bone, mesenchymal stem cells decrease in number and their differentiation potential into osteoblasts is reduced. Instead, there is a shift towards adipogenic differentiation and increased lipid accumulation in the marrow of osteoporotic bones. Bone marrow adipocytes produce palmitic acid (PA), a saturated fatty acid, which is toxic to osteoblasts in vitro. Vitamin D (1,25(OH)2D3) stimulates osteoblastogenesis and has known anti-apoptotic effects on osteoblasts, as such it may protect human primary osteoblasts from PA-induced lipotoxicity. Here, the effects of PA (250 µM) or 1,25(OH)2D3 (10-8 M), alone or in combination, on osteoblast differentiation and mineralization, viability and autophagy were investigated. In PA-treated osteoblasts, 1,25(OH)2D3 ameliorated the decrease in the mRNA transcript abundance of representative palmitoylation (ZDHHC1, ZDHHC2 and ZDHHC12) and osteogenic (alkaline phosphatase and osteocalcin) genes. Collectively these gene regulate signaling pathways pertinent to osteoblastogenesis. In osteoblasts treated with PA and 1,25(OH)2D3, the capacity to undergo differentiation and mineralization was recovered and cell viability was increased when compared to osteoblasts treated with PA alone. 1,25(OH)2D3, irrespective of PA treatment, increased the expression of key osteogenic signaling proteins; specifically, SMAD1-3,5, Runx2 and ß-catenin. 1,25(OH)2D3 also attenuated the high level of impaired autophagy induced by PA and potentiated a shift towards activated, functional autophagy and increased flux through autolysosomes. Altogether, these findings provide in vitro evidence regarding the potential of 1,25(OH)2D3 to protect osteoblasts from lipotoxicity by modulating autophagy and facilitating cell differentiation, which may enhance bone formation in an osteoporotic microenvironment with a high level of marrow adipose tissue.

Picolinic Acid, a Catabolite of Tryptophan, Has an Anabolic Effect on Bone In Vivo.

Fractures attributable to osteoporosis have a severe impact on our older population. Reports of side effects with commonly prescribed osteoporosis drugs have led to the investigation of new and safer treatments with novel mechanisms of action. Picolinic acid (PIC), a catabolite of tryptophan, induces in vitro osteogenic differentiation of mesenchymal stem cells. Here we demonstrate that PIC has an anabolic effect on bone in vivo by increasing bone formation, bone mass, and bone strength in normal and ovariectomized C57BL/6 mice. Activation of the osteogenic pathways triggered this osteoanabolic response without any cross-related effects on mineral absorption or calciotropic hormones. Because PIC was also well tolerated and absorbed with no side effects, it is an ideal potential candidate for the treatment of osteoporosis. © 2020 American Society for Bone and Mineral Research.

Mechanisms of palmitate-induced lipotoxicity in osteocytes.

BACKGROUND: Lipotoxicity is defined as cellular toxicity observed in the presence of an abnormal accumulation of fat and adipocyte-derived factors in non-fat tissues. Palmitic acid (PA), an abundant fatty acid in the bone marrow and particularly in osteoporotic bones, affects osteoblastogenesis and osteoblast function, decreasing their survival through induction of apoptosis and dysfunctional autophagy. In this study, we hypothesized that PA also has a lipotoxic effect on osteocytes in vitro. METHODS: Initially, we tested the effect of PA on osteocyte-derived factors DKK1, sclerostin and RANKL. Then, we tested whether PA affects survival and causes apoptosis in osteocytes. Subsequently, we investigated the effect of PA on autophagy by detecting the membrane component LC3-II (Western blot) and staining it and lysosomes with Lysotracker Red dye. RESULTS: PA decreases RANKL, DKK1 and sclerostin expression in osteocytes. In addition, we found that PA induces apoptosis and reduces osteocyte survival. PA also caused autophagy failure identified by a significant increase in LC3-II and a reduced number of autophagosomes/lysosomes in the cytoplasm. CONCLUSION: In addition to the effects of PA on RANKL, DKK1 and sclerostin expression, which could have significant deleterious impact on bone cell coupling and bone turnover, PA also induced apoptosis and reduced autophagy in osteocytes. Considering that apoptosis and cell dysfunction are two common changes occurring in the osteocytes of osteoporotic bone, our findings suggest that PA could play a role in the pathogenesis of the disease. Suppression of these effects could bring new potential targets for therapeutic interventions in the future.

A comparative study of the effect of submicron porous and smooth ultrafine-grained Ti-20Mo surfaces on osteoblast responses.

The surface of an orthopaedic implant plays a crucial role in determining the adsorption of proteins and cell functions. A detailed comparative study has been made of the in vitro osteoblast responses to coarse-grained (grain size: 500 µm), ultrafine-grained (grain size: 100 nm), coarse-porous (pore size: 350 nm), and fine-porous (pore size: 155 nm) surfaces of Ti-20Mo alloy. The purpose was to provide essential experimental data for future design of orthopaedic titanium implants for rapid osseointegration. Systematic original experimental data was produced for each type of surfaces in terms of surface wettability, cell morphology, adhesion, growth, and differentiation. Microscopic evidence was collected to reveal the detailed interplay between each characteristic surface with proteins or cells. Various new observations were discussed and compared with literature data. It was concluded that the coarse-porous surfaces offered the optimum topographical environment for osteoblasts and that the combination of ultrafine grains and considerable grain boundary areas is not an effective way to enhance cell growth and osteogenic capacity. Moreover, pore features (size and depth) have a greater effect than smooth surfaces on cell growth and osteogenic capacity. It proves that cells can discern the difference in pore size in the range of 100-350 nm. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2020-2033, 2018.

Osteochondral repair using an acellular dermal matrix-pilot in vivo study in a rabbit osteochondral defect model.

The aim of this pilot project was to introduce a novel use of acellular dermal matrix (ADM) in combination with infrapatellar fat pad mesenchymal stromal cells (IPFP-MSCs) to effect repair in a rabbit osteochondral defect model. ADM, in a range of surgical procedures, has been shown to promote remodelling of tissue at the site of implantation. Rabbit-derived ADM (rabADM) was prepared from the skin of donor rabbits. Autologous IPFP-MSCs were obtained at the time of knee surgery. Osteochondral defects (4 mm cartilage outer/2 mm central bone defect) were drilled into distal femoral condyles of 12 New Zealand White rabbits. Treatments groups: (i) defect only; (ii) rabADM alone; (iii) IPFP-MSCs alone; and (iv) rabADM with IPFP-MSCs. Condyles were harvested at 12 weeks, and analyzed using histology, immunohistochemistry (types I and II collagen) and histomorphometry to evaluate osteochondral repair. The rabADM only group achieved the highest ratio of type II to non-type II collagen (77.3%) using areal measures (similar to normal cartilage), which indicated a higher quality of cartilage repair. The addition of IPFP-MSCs, with or without rabADM, formed a fibrous collagen cap above the lesion site not seen with rabADM alone. Macroscopically, there was no joint erosion, inflammation, swelling or deformity, and all animals maintained full range of motion. CONCLUSIONS: RabADM alone resulted in neocartilage formation similar to native cartilage. IPFP-MSCs limited osteochondral repair and contributed to fibrosis, even in combination with the rabADM. Further studies using ADM for osteochondral repair are warranted in a more appropriate pre-clinical model of osteochondral repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1919-1928, 2018.

Chondrogenesis of Human Infrapatellar Fat Pad Stem Cells on Acellular Dermal Matrix.

Acellular dermal matrix (ADM) has been in clinical use for decades in numerous surgical applications. The ability for ADM to promote cellular repopulation, revascularisation and tissue regeneration is well documented. Adipose stem cells have the ability to differentiate into mesenchymal tissue types, including bone and cartilage. The aim of this study was to investigate the potential interaction between ADM and adipose stem cells in vitro using TGFß3 and BMP6. Human infrapatellar fat pad-derived adipose stem cells (IPFP-ASC) were cultured with ADM derived from rat dermis in chondrogenic (TGFß3 and BMP6) medium in vitro for 2 and 4 weeks. Histology, qPCR, and immunohistochemistry were performed to assess for markers of chondrogenesis (collagen Type II, SOX9 and proteoglycans). At 4 weeks, cell-scaffold constructs displayed cellular changes consistent with chondrogenesis, with evidence of stratification of cell layers and development of a hyaline-like cartilage layer superficially, which stained positively for collagen Type II and proteoglycans. Significant cell-matrix interaction was seen between the cartilage layer and the ADM itself with seamless integration between each layer. Real time qPCR showed significantly increased COL2A1, SOX9, and ACAN gene expression over 4 weeks when compared to control. COL1A2 gene expression remained unchanged over 4 weeks. We believe that the principles that make ADM versatile and successful for tissue regeneration are applicable to cartilage regeneration. This study demonstrates in vitro the ability for IPFP-ASCs to undergo chondrogenesis, infiltrate, and interact with ADM. These outcomes serve as a platform for in vivo modelling of ADM for cartilage repair.

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro.

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair.

Applying Advanced Imaging Techniques to a Murine Model of Orthotopic Osteosarcoma.

INTRODUCTION: Reliable animal models are required to evaluate novel treatments for osteosarcoma. In this study, the aim was to implement advanced imaging techniques in a murine model of orthotopic osteosarcoma to improve disease modeling and the assessment of primary and metastatic disease. MATERIALS AND METHODS: Intra-tibial injection of luciferase-tagged OPGR80 murine osteosarcoma cells was performed in Balb/c nude mice. Treatment agent [pigment epithelium-derived factor (PEDF)] was delivered to the peritoneal cavity. Primary tumors and metastases were evaluated by in vivo bioluminescent assays, micro-computed tomography, [(18)F]-Fluoride-PET and [(18)F]-FDG-PET. RESULTS: [(18)F]-Fluoride-PET was more sensitive than [(18)F]-FDG-PET for detecting early disease. Both [(18)F]-Fluoride-PET and [(18)F]-FDG-PET showed progressive disease in the model, with fourfold and twofold increases in standardized uptake value (p < 0.05) by the study endpoint, respectively. In vivo bioluminescent assay showed that systemically delivered PEDF inhibited growth of primary osteosarcoma. DISCUSSION: Application of [(18)F]-Fluoride-PET and [(18)F]-FDG-PET to an established murine model of orthotopic osteosarcoma has improved the assessment of disease. The use of targeted imaging should prove beneficial for the evaluation of new approaches to osteosarcoma therapy.

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor.

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL-33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP(+)) cells. Since TGFß boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFß co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports.

Bioactive coatings for orthopaedic implants-recent trends in development of implant coatings.

Joint replacement is a major orthopaedic procedure used to treat joint osteoarthritis. Aseptic loosening and infection are the two most significant causes of prosthetic implant failure. The ideal implant should be able to promote osteointegration, deter bacterial adhesion and minimize prosthetic infection. Recent developments in material science and cell biology have seen the development of new orthopaedic implant coatings to address these issues. Coatings consisting of bioceramics, extracellular matrix proteins, biological peptides or growth factors impart bioactivity and biocompatibility to the metallic surface of conventional orthopaedic prosthesis that promote bone ingrowth and differentiation of stem cells into osteoblasts leading to enhanced osteointegration of the implant. Furthermore, coatings such as silver, nitric oxide, antibiotics, antiseptics and antimicrobial peptides with anti-microbial properties have also been developed, which show promise in reducing bacterial adhesion and prosthetic infections. This review summarizes some of the recent developments in coatings for orthopaedic implants.

Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFß3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFß3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFß3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

Recent advances in nerve tissue engineering.

Nerve injury secondary to trauma, neurological disease or tumor excision presents a challenge for surgical reconstruction. Current practice for nerve repair involves autologous nerve transplantation, which is associated with significant donor-site morbidity and other complications. Previously artificial nerve conduits made from polycaprolactone, polyglycolic acid and collagen were approved by the FDA (USA) for nerve repair. More recently, there have been significant advances in nerve conduit design that better address the requirements of nerve regrowth. Innovations in materials science, nanotechnology, and biology open the way for the synthesis of new generation nerve repair conduits that address issues currently faced in nerve repair and regeneration. This review discusses recent innovations in this area, including the use of nanotechnology to improve the design of nerve conduits and to enhance nerve regeneration.

Differentiation of stem cells from human infrapatellar fat pad: characterization of cells undergoing chondrogenesis.

Hyaline cartilage repair is a significant challenge in orthopedics and current techniques result in formation of fibrocartilage. Human infrapatellar fat pad (hIPFP)-derived mesenchymal stem cells (MSCs) are capable of differentiation into multiple tissue lineages, including cartilage and bone. Chondrogenesis is a crucial part of normal skeletal development but the molecular mechanisms are yet to be completely defined. In this study we sourced hIPFP-derived MSCs utilizing chondrogenic growth factors, transforming growth factor beta-3, and bone morphogenetic protein-6, to form hyaline-like cartilage in micromass cultures and we studied chondrogenic development of 7, 14, and 28 days. The purpose of this study was (1) to characterize chondrogenesis from MSCs derived from hIPFP tissue by conventional techniques and (2) to characterize temporal changes of key molecular components during chondrogenesis using microarray gene expression. Endpoints included histology, immunohistochemistry (IHC), gene expression profiles using a microarray technique, and changes in expression of specific genes using quantitative real-time polymerase chain reaction. Over 14-28 days, clusters of encapsulated chondrocytes formed surrounded by collagen type II and aggrecan in the extracellular matrix (ECM). Collagen type II and aggrecan production was confirmed using IHC and chondrogenic lineage markers were studied; SRY-related transcription factor (SOX9), collagen type II alpha 1 (COL2A1), and aggrecan gene expression increased significantly over the time course. Normalized microarray highlighted 608 differentially expressed genes; 10 chondrogenic genes were upregulated (2- to 87-fold), including COL2A1, COL10A1, COL9A1, COL11A1, COL9A2, COL11A2, COL1A1, COMP, SOX9, and COL3A1. We found that the upregulated genes (twofold or greater) represent significant level of expression (enrichment score) for the ECM structural constituent of the molecular functional at days 7, 14, and 28 during chondrogenesis. Therefore, we have successfully demonstrated in vitro production of hyaline-like cartilage from IPFP-derived MSCs in micromass culture. Microarray has provided information concerning genes involved in chondrogenesis of hIPFP-derived MSCs and our approach offers a viable strategy for generating clinically relevant cartilage for therapeutic use.

In vivo measurement of hippocampal GABAA/cBZR density with [18F]-flumazenil PET for the study of disease progression in an animal model of temporal lobe epilepsy.

PURPOSE: Imbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether [(18)F]-flumazenil ([(18)F]-FMZ) PET could be used to non-invasively characterise GABAA/central benzodiazepine receptor (GABAA/cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE. METHODS: Dynamic [(18)F]-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5-25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an [(18)F]-FMZ PET scan (3.6-4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the [(18)F]-FMZ PET data. KEY FINDINGS: The PSM was found to adequately model [(18)F]-FMZ binding in vivo. There was a significant decrease in hippocampal receptor density in the SE group (p<0.01), accompanied by an increase in apparent affinity (p<0.05) compared to controls. No change in cortical receptor binding was observed. Hippocampal volume reduction and cell loss was only seen in a subset of animals. Histological assessment of hippocampal cell loss was significantly correlated with hippocampal volume measured by MRI (p<0.05), but did not correlate with [(18)F]-FMZ binding. SIGNIFICANCE: Alterations to hippocampal GABAA/cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with [(18)F]-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. [(18)F]-FMZ PET is useful for investigating the role that changes GABAA/cBZR density and binding affinity play in the pathogenesis of TLE.

The osteochondral dilemma: review of current management and future trends.

The management of articular cartilage defects remains challenging and controversial. Hyaline cartilage has limited capacity for self-repair and post-injury cartilage is predominantly replaced by fibrocartilage through healing from the subchondral bone. Fibrocartilage lacks the key properties that characterize hyaline cartilage such as capacity for compression, hydrodynamic permeability and smoothness of the articular surface. Many reports relate compromised function associated with repaired cartilage and loss of function of the articular surface. Novel methods have been proposed with the key aim to regenerate hyaline cartilage for repair of osteochondral defects. Over the past decade, with many exciting developments in tissue engineering and regenerative cell-based technologies, we are now able to consider new combinatorial approaches to overcome the problems associated with osteochondral injuries and damage. In this review, the currently accepted surgical approaches are reviewed and considered; debridement, marrow stimulation, whole tissue transplantation and cellular repair. More recent products, which employ tissue engineering approaches to enhance the traditional methods of repair, are discussed. Future trends must not only focus on recreating the composition of articular cartilage, but more importantly recapitulate the nano-structure of articular cartilage to improve the functional strength and integration of repair tissue.

18F-flumazenil: a γ-aminobutyric acid A-specific PET radiotracer for the localization of drug-resistant temporal lobe epilepsy.

UNLABELLED: Studies report that (11)C-flumazenil (FMZ) PET more specifically localizes the epileptogenic zone in patients with medically refractory focal epilepsy than (18)F-FDG PET. However, practical aspects of (11)C use limit clinical application. We report a phase I/IIa study assessing the clinical use of (18)F-FMZ PET for the localization of the epileptogenic zone in patients with drug-resistant temporal lobe epilepsy (TLE). Receptor binding was quantified using kinetic modeling that did not require arterial sampling. METHODS: Dynamic (18)F-FMZ PET and static interictal (18)F-FDG PET scans were compared in healthy controls (n = 17 for (18)F-FMZ and n = 20 for (18)F-FDG) and TLE patients with mesial temporal sclerosis on MR imaging (MTS, n = 12) and with normal MR imaging (NL TLE, n = 19). Masked visual assessment of images was undertaken. Parametric images of (18)F-FMZ binding potential (BPND) were generated using the simplified reference tissue model. Region-of-interest analysis on coregistered MR images and statistical parametric mapping were used to quantify (18)F-FMZ BPND and (18)F-FDG uptake in the temporal lobe. RESULTS: The visual assessment of static standardized uptake value images showed (18)F-FMZ PET to have high specificity (16/17 [94%]) and moderate sensitivity (21/31 [68%]) for the localization of the epileptogenic zone, with a more restricted abnormality than (18)F-FDG PET. However, the (18)F-FMZ standardized uptake value images were falsely localizing in 3 of 31 patients (10%). Region-of-interest analysis demonstrated reductions in ipsilateral hippocampal (18)F-FMZ BPND in patients with either MTS or NL TLE, compared with controls subjects. Ipsilateral hippocampal (18)F-FMZ BPND was independent of both hippocampal volume and (18)F-FDG uptake, whereas ipsilateral hippocampal volume was correlated with (18)F-FDG uptake (r(2) = 0.69, P < 0.0001). Statistical parametric mapping analysis demonstrated decreased uptake in 14 of 31 (45%) cases with (18)F-FMZ PET and 18 of 29 (62%) with (18)F-FDG PET. Cluster size was significantly smaller on (18)F-FMZ than (18)F-FDG images (37 vs. 160 voxels, P < 0.01). CONCLUSION: (18)F-FMZ PET has potential as a clinical tool for the localization of the epileptogenic zone in the presurgical evaluation of drug-resistant TLE, providing information complementary to (18)F-FDG PET, with a more restricted region of abnormality.

Bioengineering of articular cartilage: past, present and future.

The treatment of cartilage defects poses a clinical challenge owing to the lack of intrinsic regenerative capacity of cartilage. The use of tissue engineering techniques to bioengineer articular cartilage is promising and may hold the key to the successful regeneration of cartilage tissue. Natural and synthetic biomaterials have been used to recreate the microarchitecture of articular cartilage through multilayered biomimetic scaffolds. Acellular scaffolds preserve the microarchitecture of articular cartilage through a process of decellularization of biological tissue. Although promising, this technique often results in poor biomechanical strength of the graft. However, biomechanical strength could be improved if biomaterials could be incorporated back into the decellularized tissue to overcome this limitation.

Can structural or functional changes following traumatic brain injury in the rat predict epileptic outcome?

PURPOSE: Posttraumatic epilepsy (PTE) occurs in a proportion of traumatic brain injury (TBI) cases, significantly compounding the disability, and risk of injury and death for sufferers. To date, predictive biomarkers for PTE have not been identified. This study used the lateral fluid percussion injury (LFPI) rat model of TBI to investigate whether structural, functional, and behavioral changes post-TBI relate to the later development of PTE. METHODS: Adult male Wistar rats underwent LFPI or sham injury. Serial magnetic resonance (MR) and positron emission tomography (PET) imaging, and behavioral analyses were performed over 6 months postinjury. Rats were then implanted with recording electrodes and monitored for two consecutive weeks using video-electroencephalography (EEG) to assess for PTE. Of the LFPI rats, 52% (n = 12) displayed spontaneous recurring seizures and/or epileptic discharges on the video-EEG recordings. KEY FINDINGS: MRI volumetric and signal analysis of changes in cortex, hippocampus, thalamus, and amygdala, (18) F-fluorodeoxyglucose (FDG)-PET analysis of metabolic function, and behavioral analysis of cognitive and emotional changes, at 1 week, and 1, 3, and 6 months post-LFPI, all failed to identify significant differences on univariate analysis between the epileptic and nonepileptic groups. However, hippocampal surface shape analysis using large-deformation high-dimensional mapping identified significant changes in the ipsilateral hippocampus at 1 week postinjury relative to baseline that differed between rats that would go onto become epileptic versus those who did not. Furthermore, a multivariate logistic regression model that incorporated the 1 week, and 1 and 3 month (18) F-FDG PET parameters from the ipsilateral hippocampus was able to correctly predict the epileptic outcome in all of the LFPI cases. As such, these subtle changes in the ipsilateral hippocampus at acute phases after LFPI may be related to PTE and require further examination. SIGNIFICANCE: These findings suggest that PTE may be independent of major structural, functional, and behavioral changes induced by TBI, and suggest that more subtle abnormalities are likely involved. However, there are limitations associated with studying acquired epilepsies in animal models that must be considered when interpreting these results, in particular the failure to detect differences between the groups may be related to the limitations of properly identifying/separating the epileptic and nonepileptic animals into the correct group.

Confounding neurodegenerative effects of manganese for in vivo MR imaging in rat models of brain insults.

PURPOSE: To examine the long-term consequences of manganese exposure due to the use of manganese-enhanced magnetic resonance imaging (MEMRI) in a model of closed head injury, the fluid-percussion injury (FPI) model. MATERIALS AND METHODS: Two groups of adult male Wistar rats (n = 72) were studied with either MEMRI, whereby rats receive MnCl(2) (100 mg/kg intraperitoneally) 24 hours prior to scanning, or standard MRI (sMRI) with no contrast agent. Rats from both groups underwent FPI or sham injury and were longitudinally assessed for 6 months for neurological toxicity using behavioral tests, EEG recording, and MRI scanning. RESULTS: Regardless of whether they received FPI, MEMRI animals showed progressive signs of cerebral toxicity compared with sMRI rats, including significantly reduced weight gain, progressive brain volume decrease, and increased anxiety and depressive-like behaviors. CONCLUSION: Long-term structural and functional consequences of using manganese as a contrast agent for MRI can confound experimental outcomes and must be taken into account when designing longitudinal imaging studies using manganese-enhanced MRI.