Postoperative Administration of Alpha-tocopherol Enhances Osseointegration of Stainless Steel Implants: An In Vivo Rat Model.

BACKGROUND: Alpha-tocopherol, a well-known antioxidative agent, may have a positive effect on bone formation during the remodeling phase of secondary fracture healing. Fracture healing and osseointegration of implants share common biological pathways; hence, alpha-tocopherol may enhance implant osseointegration. QUESTIONS/PURPOSES: This experimental study in rats assessed the ability of alpha-tocopherol to enhance osseointegration of orthopaedic implants as determined by (1) pull-out strength and removal torque and (2) a histomorphological assessment of bone formation. In addition, we asked, (3) is there a correlation between the administration of alpha-tocopherol and a reduction in postoperative oxidative stress (as determined by malondialdehyde, protein carbonyls, reduced and oxidized glutathione and their ratio, catalase activity and total antioxidant capacity) that develops after implantation of an orthopaedic implant? METHODS: This blinded study was performed in study and control groups, each consisting of 15 young adult male Wistar rats. On Day 0, a custom-designed stainless-steel screw was implanted in the proximal metaphysis of both tibias of all rats. On Day 1, animals were randomized to receive either alpha-tocopherol (40 mg/kg once per day intraperitoneally) or saline (controls). Animals were treated according to identical perioperative and postoperative protocols and were euthanized on Day 29. All animals completed the study and all tibias were suitable for evaluation. Implant pullout strength was assessed in the right tibias, and removal torque and histomorphometric evaluations (that is, volume of newly formed bone surrounding the implant in mm, percentage of newly formed bone, percentage of bone marrow surrounding the implant per optical field, thickness of newly formed bone in µm, percentage of mineralized bone in newly formed bone, volume of mature newly formed bone surrounding the implant in mm and percentage of mineralized newly formed bone per tissue area) were performed in the left tibias. The plasma levels of alpha-tocopherol, malondialdehyde, protein carbonyls, glutathione, glutathione disulfide, catalase, and the total antioxidant capacity were evaluated, and the ratio of glutathione to oxidized glutathione was calculated. RESULTS: All parameters were different between the alpha-tocopherol-treated and control rats, favoring those in the alpha-tocopherol group. The pullout strength for the alpha-tocopherol group (mean ± SD) was 124.9 ± 20.7 newtons (N) versus 88.1 ± 12.7 N in the control group (mean difference -36.7 [95% CI -49.6 to -23.9]; p < 0.001). The torque median value was 7 (range 5.4 to 8.3) versus 5.2 (range 3.6 to 6 ) N/cm (p < 0.001). The newly formed bone volume was 29.8 ± 5.7 X 10 versus 25.2 ± 7.8 X 10 mm (mean difference -4.6 [95% CI -8.3 to -0.8]; p = 0.018), the percentage of mineralized bone in newly formed bone was 74.6% ± 8.7% versus 62.1% ± 9.8% (mean difference -12.5 [95% CI -20.2 to -4.8]; p = 0.003), the percentage of mineralized newly formed bone per tissue area was 40.3 ± 8.6% versus 34.8 ± 9% (mean difference -5.5 [95% CI -10.4 to -0.6]; p = 0.028), the glutathione level was 2 ± 0.4 versus 1.3 ± 0.3 µmol/g of hemoglobin (mean difference -0.6 [95% CI -0.9 to -0.4]; p < 0.001), the median glutathione/oxidized glutathione ratio was 438.8 (range 298 to 553) versus 340.1 (range 212 to 454; p = 0.002), the catalase activity was 155.6 ± 44.6 versus 87.3 ± 25.2 U/mg Hb (mean difference -68.3 [95% CI -95.4 to -41.2]; p < 0.001), the malondialdehyde level was 0.07 ± 0.02 versus 0.14 ± 0.03 µmol/g protein (mean difference 0.07 [95% CI 0.05 to 0.09]; p < 0.001), the protein carbonyl level was 0.16 ± 0.04 versus 0.27 ± 0.08 nmol/mg of protein (mean difference -0.1 [95% CI 0.05 to 0.15]; p = 0.002), the alpha-tocopherol level was 3.9 ± 4.1 versus 0.9 ± 0.2 mg/dL (mean difference -3 [95% CI -5.2 to -0.7]; p = 0.011), and the total antioxidant capacity was 15.9 ± 3.2 versus 13.7 ± 1.7 nmol 2,2-diphenyl-1-picrylhydrazyl radical/g of protein (mean difference -2.1 [95% CI -4.1 to -0.18]; p = 0.008). CONCLUSIONS: These results using an in vivo rat model support that postoperatively administered alpha-tocopherol can enhance the osseointegration of an orthopaedic implant, although a cause and effect relationship between the administration of alpha-tocopherol and a reduction in postoperative stress cannot be securely established. CLINICAL RELEVANCE: These findings suggest that postoperative administration of alpha-tocopherol is a promising approach to enhance osseointegration of orthopaedic implants in patients. Further studies with different animal models and/or different implants and those evaluating the alpha-tocopherol dose response are needed before performing clinical trials that will examine whether these promising, preliminary results can be extrapolated to the clinical setting as well.

Paradise lost: Evidence for a devastating metabolic bone disease in an insular Pleistocene deer.

PURPOSE: This communication reports skeletal pathology in a Pleistocene endemic deer from the Mavromouri caves of Crete. MATERIALS: 287 bones and bone fragments from Mavromouri caves are compared to 2986 bones from Liko Cave. METHODS: Bones were evaluated macroscopically, and measurements were made of morphometric characteristics of limb long bones. Representative bone specimens were examined radiographically and histologically. RESULTS: Macroscopic hallmarks were loss of bone mass and increased porosity. The long bones were brittle, some of them having thin cortices, and others reduction of medullary cavities that contain dense Haversian tissue. The flat bones were spongy and fragile. Erosions of the metaphyses and articular surfaces were noted. Histological findings included: sub-periosteal resorption; loss of lamellar bone; enlargement of vascular canals; and remodeling of cortical bone. Two types of fibrous osteodystrophy were recognized in skeletal remains, subostotic and hyperostotic. CONCLUSIONS: The deer of Mavromouri caves were affected by severe metabolic bone disease, likely nutritional secondary hyperparathyroidism. We hypothesize a multifactorial cause, including overgrazing, flora senescence, soil mineral deficiencies, and a prolonged period of climate extremes, degrading the Cretan deer habitat. VALUE: This is the first evidence of a metabolic bone disease causing this level of destructive pathology in an insular fossil deer. LIMITATIONS: The lack of absolute chronometric dates for the site limits potential linking with the prevailing environmental conditions. SUGGESTIONS FOR FURTHER RESEARCH: Investigation of similar skeletal pathologies at other islands or isolated habitats is advised.

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis.

We report the novel synthesis of cyclic PLP139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H147 replaces W144 and F151-K150 replace H147 as TCR contacts, which may explain the difference on each peptide's response.

Neuronal vacuolation and spinocerebellar degeneration associated with altered neurotransmission.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species, such as the domestic dog (Canis lupus familiaris), and many are caused by mutations in the same genes as corresponding human conditions. In the present study, we report an inherited neurodegenerative condition, termed 'neuronal vacuolation and spinocerebellar degeneration' (NVSD) which affects neonatal or young dogs, mainly Rottweilers, which recently has been linked with the homozygosity for the RAB3GAP1:c.743delC allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized predominantly by abnormalities of the nervous system including axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 proteins, respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, autophagy, axonal transport and synaptic transmission. The present study attempts to carry out a detailed histopathological examination of NVSD disease, extending from peripheral nerves to lower brain structures focusing on the neurotransmitter alterations noted in the cerebellum, the major structure affected. NVSD dogs presented with progressive cerebellar ataxia and some clinical manifestations that recapitulate the WARBM phenotype. Neuropathological examination revealed dystrophic axons, neurodegeneration and intracellular vacuolization in specific nuclei. In the cerebellum, severe vacuolation of cerebellar nuclei neurons, atrophy of Purkinje cells, and diminishing of GABAergic and glutamatergic fibres constitute the most striking lesions. The balance of evidence suggests that the neuropathological lesions are a reaction to the altered neurotransmission. The canine phenotype could serve as a model to delineate the disease-causing pathological mechanisms in RAB3GAP1 mutation. .

Cyclic MOG35-55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis.

EAE is induced to susceptible mice using linear peptides of myelin proteins of the central nervous system. Specific peptide motifs within the peptide-binding groove of the MHC peptide-complex determines the affinity of the peptide in each animal and the consequent T-cell receptor recognition and activation of the cell. Altered peptide ligand (APL) vaccination is a novel approach based on an effort to induce T-cell tolerance or alter cytokine profile from pro-inflammatory to anti-inflammatory. In the present study we synthesized the MOG35-55 peptide and altered its 3-dimensional conformation to make it a cyclic one (c-MOG35-55). EAE was induced in C57BL/6 mice and pathology was studied on acute and chronic phase of the disease. Our data indicates that c-MOG35-55 peptide alone induces a mild transient acute phase without chronic axonopathy. Administration of the c-MOG35-55 peptide at a 1:1 ratio during disease induction significantly ameliorates clinical disease and underlying pathology, such as demyelination and axonopathy in the acute and chronic phases. Binding and structural studies revealed milder interactions between the c-MOG35-55 and mouse or human MHC class II alleles (H2-IAb and HLA-DR2). Collectively, we provide data supporting for the first time the concept that the cyclic modification of an established encephalitogenic peptide ameliorates the clinical outcomes and underlying pathological processes of EAE. Such a cyclic modification of linear peptides could provide a novel treatment approach for future, patient-selective, immunomodulative treatments of multiple sclerosis.

Long-term effects of autoimmune CNS inflammation on adult hippocampal neurogenesis.

Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Aging and chronic degenerative disorders have been shown to impair hippocampal neurogenesis, but the consequence of chronic inflammation remains controversial. In this study the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis was used to investigate the long-term effects of T cell-mediated central nervous system inflammation on hippocampal neurogenesis. 5-Bromodeoxyuridine (BrdU)-labeled subpopulations of hippocampal cells in EAE and control mice (coexpressing GFAP, doublecortin, NeuN, calretinin, and S100) were quantified at the recovery phase, 21 days after BrdU administration, to estimate alterations on the rate and differentiation pattern of the neurogenesis process. The core features of EAE mice DG are (i) elevated number of newborn (BrdU+) cells indicating vigorous proliferation, which in the long term subsided; (ii) enhanced migration of newborn cells into the granule cell layer; (iii) increased level of immature neuronal markers (including calretinin and doublecortin); (iv) trending decrease in the percentage of newborn mature neurons; and (v) augmented gliogenesis and differentiation of newborn neural precursor cells (NPCs) to mature astrocytes (BrdU+/S100+). Although the inflammatory environment in the brain of EAE mice enhances the proliferation of hippocampal NPCs, in the long term neurogenesis is progressively depleted, giving prominence to gliogenesis. The discrepancy between the high number of immature cells and the low number of mature newborn cells could be the result of a caused defect in the maturation pathway. © 2016 Wiley Periodicals, Inc.

Seven days post-injury fate and effects of genetically labelled adipose-derived mesenchymal cells on a rat traumatic brain injury experimental model.

Mesenchymal stromal cells (MSC) have been suggested to have beneficial effects on animal models of traumatic brain injury (TBI), owing to their neurotrophic and immunomodulatory properties. Adipose tissue-derived stromal cells (ASCs) are multipotent MSC that can be harvested with minimally invasive methods, show a high proliferative capacity, low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. In the present study ASCs were genetically labelled using the Sleeping Beauty transposon to express the fluorescent protein Venus. Venus+ASCs were transplanted intra-cerebroventricularly (ICV), on a rat TBI model and their survival, fate and effects on host brain responses were examined at seven days post-injury (7dPI). We provide evidence that Venus+ASCs survived, migrated into the periventricular striatum and were negative for neuronal or glial lineage differentiation markers. Venus+ASCs stimulated the proliferation of endogenous neural stem cells (NSCs) in the brain neurogenic niches, the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). It was also evident that Venus+ASCs modify the host brain's cellular microenvironment both at the injury site and at their localization area by promoting a significant reduction of the lesion area, as well as altering the post-injury, pro-inflammatory profile of microglial and astrocytic cell populations. Our data support the view that ICV transplantation of ASCs induces alterations in the host brain's cellular response to injury that may be correlated to a reversal from a detrimental to a beneficial state which is permissive for regeneration and repair.

Bone lesions in a Late Pleistocene assemblage of the insular deer Candiacervus sp.II from Liko cave (Crete, Greece).

Candiacervus sp.II is one of the deer species that inhabited the island of Crete during the Late Pleistocene. The species evolved on the island under a prolonged period of isolation and, as a consequence, developed a high degree of endemism. Fossils of this species have been discovered at many Cretan sites, including Liko cave (an attritional accumulation of several thousand fossils). In this paper, we present the results of a systematic analysis of the prevalence and anatomical distribution of bone lesions of Candiacervus sp.II, from that cave. We identified one metapodial with a healed fracture and nine (various) specimens with moderate to severe degenerative lesions of osteoarthritis. The lesions were evaluated macroscopically and radiographically, and they were classified as traumatic or degenerative. Degenerative lesions that affected adult individuals had prevalence rates below 5% and were attributed to environmental or nutritional causes. Representative bones were sampled for histological evaluation, to provide essential baseline data on possible underlying disorders. The aims of this study are to provide evidence for bone disease contributing to species morbidity, and to shed new light on causes and potential palaeoecological significance.

Evolution of Gyrification in Carnivores.

The order Carnivora is a large and highly diverse mammalian group with a long and well-documented evolutionary history. Nevertheless, our knowledge on the degree of cortical folding (or degree of gyrification) is limited to just a few species. Here we investigate the degree of cortical folding in 64 contemporary and 37 fossil carnivore species. We do so by measuring the length of gyri impressions on endocranial casts. We use this approach because we have found that there is a very good correlation between the degree of cortical folding and the relative length of the gyri that are exposed on the outer surface of the hemispheres. Our results indicate that aquatic and semiaquatic carnivores have higher degrees of cortical folding than terrestrial carnivores. The degree of cortical folding varies among modern families, with viverrids having the lowest values. Furthermore, the scaling of cortical folding with brain size follows different patterns across specific carnivore families. Forty million years ago, the first carnivores had a relatively small cortex and limited cortical folding. Both the size of the cortex and the degree of cortical folding increased independently in each family during evolution.

Neurogenesis in the septal and temporal part of the adult rat dentate gyrus.

Structural and functional dissociation between the septal and the temporal part of the dentate gyrus predispose for possible differentiations in the ongoing neurogenesis process of the adult hippocampus. In this study, BrdU-dated subpopulations of the rat septal and temporal dentate gyrus (coexpressing GFAP, DCX, NeuN, calretinin, calbindin, S100, caspase-3 or fractin) were quantified comparatively at 2, 5, 7, 14, 21, and 30 days after BrdU administration in order to examine the successive time-frames of the neurogenesis process, the glial or neuronal commitment of newborn cells and the occurring apoptotic cell death. Newborn neurons' migration from the neurogenic subgranular zone to the inner granular cell layer and expression of glutamate NMDA and AMPA receptors were also studied. BrdU immunocytochemistry revealed comparatively higher numbers of BrdU(+) cells in the septal part, but stereological analysis of newborn and total granule cells showed an identical ratio in the two parts, indicating an equivalent neurogenic ability, and a common topographical pattern along each part's longitudinal and transverse axis. Similarly, both parts exhibited extremely low levels of newborn glial and apoptotic cells. However, despite the initially equal division rate and pattern of the septal and temporal proliferating cells, their later proliferative profile diverged in the two parts. Dynamic differences in the differentiation, migration and maturation process of the two BrdU-incorporating subpopulations of newborn neurons were also detected, along with differences in their survival pattern. Therefore, we propose that various factors, including developmental date birth, local DG microenvironment and distinct functionality of the two parts may be the critical regulators of the ongoing neurogenesis process, leading the septal part to a continuous, rapid, and less-disciplined genesis rate, whereas the quiescent temporal microenvironment preserves a quite steady, less-demanding neurogenesis process.