Managing Temporomandibular Joint Osteoarthritis by Dental Stem Cell Secretome.

The potential therapeutic role of the Dental Pulp Stem Cells Secretome (SECR) in a rat model of experimentally induced Temporomandibular Joint (TMJ) Osteoarthritis (OA) was evaluated. Proteomic profiling of the human SECR under specific oxygen tension (5% O2) and stimulation with Tumor Necrosis Factor-alpha (TNF-α) was performed. SECR and respective cell lysates (CL) samples were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed with Bioinformatic tools. The anti-inflammatory properties of SECR were assessed via an in vitro murine macrophages model, and were further validated in vivo, in a rat model of chemically-induced TMJ-OA by weekly recording of the head withdrawal threshold, the food intake, and the weight change, and radiographically and histologically at 4- and 8-weeks post-treatment. SECR analysis revealed the presence of 50 proteins that were enriched and/or statistically significantly upregulated compared to CL, while many of those proteins were involved in pathways related to "extracellular matrix organization" and "immune system". SECR application in vitro led to a significant downregulation on the expression of pro-inflammatory genes (MMP-13, MMP-9, MMP-3 and MCP-1), while maintaining an increased expression of IL-10 and IL-6. SECR application in vivo had a significant positive effect on all the clinical parameters, resulting in improved food intake, weight, and pain suppression. Radiographically, SECR application had a significant positive effect on trabecular bone thickness and bone density compared to the saline-treated group. Histological analysis indicated that SECR administration reduced inflammation, enhanced ECM and subchondral bone repair and regeneration, thus alleviating TMJ degeneration.

Birth and death of neurons in the developing and mature mammalian brain.

Although neuron birth and death are two contradictory processes, they serve the same purpose of the formation of the brain. They coexist during brain development, when cytoarchitecture and synaptic contacts are progressively established. It is the highly programmed interplay between these two processes that results in the making of a mature, complex-wired, functional brain. Neurogenesis is the process that begins with the birth of naïve new neurons, which are gradually specified to their prospective cell fate, translocate through migratory streams to the brain area they are destined for, and terminally differentiate into mature neurons that integrate into neuronal networks with sophisticated functions. This is an ongoing process until adulthood, when it mediates brain neuroplasticity. Neuron death is the process through which the fine sculpting and modeling of the brain is achieved. It serves to adjust final neuron numbers, exerting quality control over neurons that birth has generated or overproduced. It additionally corrects early wiring and performs systems matching by negatively selecting neurons that fail to gain neurotransmitter-mediated neuronal activity or receive neurotrophic support for maintenance and function. It is also a means by which organizing centers and transient structures are removed early in morphogenesis. Both processes are evolutionary conserved, genetically programmed and orchestrated by the same signaling factors regulating the cell cycle, neuronal activity/neurotransmitter action and neurotrophic support. This review summarizes and highlights recent knowledge with regard to birth and death of neurons, the two mutually dependent contributors to the formation of the highly evolved mammalian brain.

Hierarchical Porous Carbon-PLLA and PLGA Hybrid Nanoparticles for Intranasal Delivery of Galantamine for Alzheimer's Disease Therapy.

In the present study, poly(l-lactic acid) (PLLA) and poly(lactide-co-glycolide) (PLGA) hybrid nanoparticles were developed for intranasal delivery of galantamine, a drug used in severe to moderate cases of Alzheimer's disease. Galantamine (GAL) was adsorbed first in hierarchical porous carbon (HPC). Formulations were characterized by FT-IR, which showed hydrogen bond formation between GAL and HPC. Furthermore, GAL became amorphous after adsorption, as confirmed by XRD and differential scanning calorimetry (DSC) studies. GAL was quantified to be 21.5% w/w by TGA study. Adsorbed GAL was nanoencapsulated in PLLA and PLGA, and prepared nanoparticles were characterized by several techniques. Their sizes varied between 182 and 394 nm, with an exception that was observed in nanoparticles that were prepared by PLLA and adsorbed GAL that was found to be 1302 nm in size. DSC thermographs showed that GAL was present in its crystalline state in nanoparticles before its adsorption to HPC, while it remained in its amorphous phase after its adsorption in the prepared nanoparticles. It was found that the polymers controlled the release of GAL both when it was encapsulated alone and when it was adsorbed on HPC. Lastly, PLGA hybrid nanoparticles were intranasally-administered in healthy, adult, male Wistar rats. Administration led to successful delivery to the hippocampus, the brain area that is primarily and severely harmed in Alzheimer's disease, just a few hours after a single dose.

Adult neurogenesis and gliogenesis in the dorsal and ventral canine hippocampus.

Dentate gyrus (DG) of the mammalian hippocampus gives rise to new neurons and astrocytes all through adulthood. Canine hippocampus presents many similarities in fetal development, anatomy, and physiology with human hippocampus, establishing canines as excellent animal models for the study of adult neurogenesis. In the present study, BrdU-dated cells of the structurally and functionally dissociated dorsal (dDG) and ventral (vDG) adult canine DG were comparatively examined over a period of 30 days. Each part's neurogenic potential, radial glia-like neural stem cells (NSCs) proliferation and differentiation, migration, and maturation of their progenies were evaluated at 2, 5, 14, and 30 days post BrdU administration, with the use of selected markers (glial fibrillary acidic protein, doublecortin, calretinin and calbindin). Co-staining of BrdU+ cells with NeuN or S100B permitted the parallel study of the ongoing neurogenesis and gliogenesis. Our findings reveal the comparatively higher populations of residing granule cells, proliferating NSCs and BrdU+ neurons in the dDG, whereas newborn neurons of the vDG showed a prolonged differentiation, migration, and maturation. Newborn astrocytes were found all along the dorso-ventral axis, counting however for only 11% of newborn cell population. Comparative evaluation of adult canine and rat neurogenesis revealed significant differences in the distribution of resident and newborn granule cells along the dorso-ventral axis, division pattern of adult NSCs, maturation time plan of newborn neurons, and ongoing gliogenesis. Concluding, spatial and temporal features of adult canine neurogenesis are similar to that of other gyrencephalic species, including humans, and justify the comparative examination of adult neurogenesis across mammalian species.

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.

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.

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.

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.

Time-dependent fate of transplanted neural precursor cells in experimental autoimmune encephalomyelitis mice.

Transplanted Neural Precursor Cells (NPCs) are capable of long-distance migration inside the inflamed CNS, but exhibit limited myelinating capacities in animal models of Multiple Sclerosis (MS). Inflammation seems to be both beneficial for the recruitment and migration of NPCs and restrictive for their terminal differentiation. In the present study, a set of transplantation experiments was applied in order to investigate the migratory potential, the differentiation pattern and long-term survival of NPCs in Experimental Autoimmune Encephalomyelitis (EAE) mice, the animal model of MS. The in vitro differentiation potential of NPCs in the presence of either pro- (TNFa, INFγ) or anti- (TGFb) inflammatory cytokines was also analyzed. According to the in vivo results obtained, at the acute phase of EAE only a small fraction of transplanted NPCs succeed to differentiate, whereas at chronic phase most of them followed a differentiation process to glial cell lineage along white matter tracts. However, this differentiation was not fully completed, since 8 months after their transplantation a number of NPCs remained as pre-oligodendrocytes. Glial differentiation of NPCs was also found to be inhibited or promoted following their treatment with TNFa or TGFb respectively, in vitro. Our findings suggest that inflammation triggers migration whereas the anti-inflammatory component is a prerequisite for NPCs to follow glial differentiation thereby providing myelinating oligodendrocytes. It is speculated that the fine balance between the pro- and anti-inflammatory determinants in the CNS may be a key factor for transplanted NPCs to exhibit a better therapeutic effect in EAE and MS. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Inflammatory changes induced by transplanted neural precursor cells in a multiple sclerosis model.

Recent studies on neural precursor cell (NPC) transplantation in multiple sclerosis animal models reveal that these cells exert their therapeutic effect mainly because of immunomodulation rather than cell replacement. In this study intraventricularly transplanted NPCs in mice, induced experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, improved the clinical symptoms and suppressed inflammation in the brain by enhancing the apoptosis of inflammatory cells. However, the same treatment failed to reduce significantly the inflammatory cells in the spinal cord, the pathology of which predominantly determines the clinical manifestation of experimental autoimmune encephalomyelitis. Our findings suggest that immunosuppression is rather a local phenomenon and thus, bystander neuroprotective mechanisms triggered by NPC intraventricular transplantation should be accountable for their therapeutic effect.

Mast cells populations fluctuate along the spinal dura mater of the developing rat.

The present study reveals developmental changes in the number, the phenotype and the distribution pattern of mast cells (MCs) along the cervical, the thoracic and the lumbar parts of the spinal dura mater. Postnatal infiltration of spinal dura by MCs does not appear to follow a sequential developmental pattern and meningeal MCs are unevenly distributed along the various parts of the examined dura. At each spinal level, areas most densely populated by MCs are the dorsal dura and the dural sleeves of the dorsal (sensory) spinal roots The developmental time course of the total MCs number is characterized by significant fluctuations in all three parts examined, with notable increases at P1, P4, P21 and P60 (peak value) for the cervical part, at P1 (peak value), P7 and P21 for the thoracic part and at P1, P7 (peak value) and P30 for the lumbar part. At P180, MCs number declines to 56%, 33% and 13% of the peak values for the cervical, the thoracic and the lumbar part, respectively. However, a different developmental pattern is followed by each subpopulation of MCs identified on the basis of their staining characteristics, namely connective tissue type mast cells (CTMCs), mucosal type or cells with characteristics of immature mast cells (MTMCs) and mixed type MCs, in each part examined. The findings may be of importance in elucidating physiological and pathological processes in the dura mater and the vertebral column.

Pyramidal neurons in the septal and temporal CA1 field of the human and hedgehog tenrec hippocampus.

The present study examines comparatively the cellular density of disector-counted/Nissl-stained CA1 pyramidal neurons and the morphometric characteristics (dendritic number/length, spine number/density and Sholl-counted dendritic branch points/20 microm) of the basal and apical dendritic systems of Golgi-impregnated CA1 neurons, in the septal and temporal hippocampus of the human and hedgehog tenrec brain. The obtained results indicate that in both hippocampal parts the cellular density of the CA1 pyramidal neurons is lower in human than in tenrec. However, while the human pyramidal cell density is higher in the septal hippocampal part than in the temporal one, in the tenrec the density of these cells is higher in the temporal part. The dendritic tree of the CA1 pyramidal cells, more developed in the septal than in temporal hippocampus in both species studied, is in general more complex in the human hippocampus. The basal and the apical dendritic systems exhibit species related morphometric differences, while dendrites of different orders exhibit differences in their number and length, and in their spine density. Finally, in both species, as well as hippocampal parts and dendritic systems, changes of dendritic morphometric features along ascending dendritic orders fluctuate in a similar way, as do the number of dendritic branch points in relation to the distance from the neuron soma.

Developmental changes of mast cell populations in the cerebral meninges of the rat.

It is known that both the dura and the pia mater attract and support the differentiation of mast cells. The present study shows that unevenly distributed mast cells in the cerebral meninges of the rat can be found in perivascular sites and vessel ramification points, but can also be unrelated to the meningeal vasculature. It also documents changes in the number, localization and staining preferences of the mast cells in the two meninges of the developing and mature rat brain. Quantitative examination of all types of histochemically differentiated meningeal mast cells reveals no major (although some exist) differences between right and left side subpopulations, but strongly suggests a different origin and fate of the dural and the pial mast cells. The number of dural mast cells, already high from postnatal day 0, although declining from postnatal day 21 onwards, remains conspicuous up to postnatal day 180. In contrast, pial mast cells are comparatively very few in the first day of the postnatal life, and despite a transient significant increase in the following two weeks, they reach almost zero levels from postnatal day 21.

Developmental changes in the vascular network of the rat visual areas 17, 18 and 18a.

The present study examines quantitatively the areal and the laminar fluctuations of the vascular network in the visual areas 17, 18 and 18a of the rat cerebral cortex, from postnatal day (P) 1 to P60. For this purpose, the detailed vascular networks of the visual areas, marked after transcardial perfusion of India ink, are analyzed with the use of an image analysis system in order to measure the total vascular density (VD) and the relative density of capillaries (CD), of medium (MD)- and large (LD)-sized vessels in combination with changes in the mean diameter of all three types of vessels. Comparative quantitative microscopy showed that both VD and CD do not exhibit significant interareal differences in the adult rat brain. However, while VD reaches adult values much earlier in area 18a (P21) than in areas 17 and 18 (P60), CD obtains adult values at P31 in areas 17 and 18a, but later (P60) in area 18. Maturation process of laminar VD, CD, MD and LD was not found to follow a simple (i.e. inside-out or mediolateral) sequence, and, in each cortical area, laminar fluctuations of vessels density revealed a complicated developmental pattern, which might be attributed to their changing structural and functional status. Developmental changes in the diameter of capillaries, examined in conjunction with concomitant changes of vascular and capillary density in each area, suggest the existence of angiogenesis in all three visual areas during the third postnatal week of age.

Areal and laminar variations in the vascularity of the visual, auditory, and entorhinal cortices of the developing rat brain.

Understanding of place-specific cortical cerebrovascular changes after insult and injury depends on the detailed knowledge of the areal and laminar variations in cortical vascularity. The present study examines comparatively the developmental changes of the total vascular density and the density of capillaries and medium- and large-sized vessels in the primary visual cortex (Oc1), the primary auditory cortex (Te1), and the lateral entorhinal cortex (EntL) of the developing rat brain. Vascular networks in the three cortical areas were marked after transcardial perfusion of India ink and quantified with an image analysis system. Parameters examined exhibited (i) peculiar developmental time course within individual cortical layers and (ii) area- and age-dependent variations. Angioarchitecture in Te1 layers was stabilized earlier than that in Oc1 layers and the period of postnatal development of the vascularity of neocortical sensory areas Oc1 and Te1 appeared to be more protracted compared to that of the phylogenetically older entorhinal cortex. By the end of the first postnatal month, vascular densities in the three cortical areas established a dorsoventral gradient (Oc1 > Te1 > EntL). Finally, in all areas, layer IV was the first layer to obtain adult values of capillary density.

The upstream regulatory region of the gene for the human homologue of the adhesion molecule TAG-1 contains elements driving neural specific expression in vivo.

Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) exhibit restricted spatial and temporal expression profiles requiring a tight regulatory program during development. The rodent glycoprotein TAG-1 and its orthologs TAX-1 in the human and axonin-1 in chick are cell adhesion molecules belonging to the contactin/F3 subgroup of the IgSF. TAG-1 is expressed in restricted subsets of central and peripheral neurons, not only during development but also in adulthood, and is implicated in neurite outgrowth, axon guidance and fasciculation, as well as neuronal migration. In an attempt to identify the regulatory elements that guide the neuronal expression of TAG-1, we have isolated genomic clones containing 4 kb of the TAX-1 upstream sequence and used them to drive the expression of the LacZ reporter gene in transgenic mice. We demonstrate that this sequence includes elements not only sufficient to restrict expression to the nervous system, but also to recapitulate to a great extent the endogenous pattern of the TAG-1 expression in the developing CNS.

Anatomic organization of the ascending branch of the milk-ejection reflex in sheep: primary afferent neurons.

The present study examines the anatomic characteristics of the primary afferent neurons that innervate the nipples and pseudonipples of ewes and the nipples of lambs. For this purpose, horseradish-peroxidase coupled to wheat germ agglutinin (WGA-HRP) was injected intradermally into the whole extent, the tip, or the base of the nipples and pseudonipples, as well as into a region of the posterior surface of the udder. After survival periods of 72-96 hours, dorsal root ganglia (DRG), segments of the spinal cord and medulla oblongata were sectioned and reacted histochemically with tetramethylbenzidine to reveal the transganglionically transported tracer. Injections of WGA-HRP in the nipples and pseudonipples of the ewe resulted in labeled cells in the second to fifth ipsilateral lumbar spinal ganglia (L(2)-L(5)) and third and fourth (L(3) and L(4)) lumbar spinal ganglia, respectively. Labeled cells after WGA-HRP injections in the nipples of the lamb were found in the ipsilateral L(3)-L(5) spinal ganglia. Central projections of the DRG-labeled cells were found in the medial part of laminae I-III of the ipsilateral L(3) and L(4) spinal segments (ewe and lamb) and in the ipsilateral dorsal column nuclei (ewe). Central projections of the DRG-labeled cells after injections in the pseudonipples of the ewe were located in the medial part of laminae I-III of the ipsilateral L(3) spinal segment. The results of this study demonstrate that, whereas the innervation of the nipples of the ewe originates from four successive lumbar spinal ganglia (L(2)-L(5)), the innervation of the nipples of the lamb and the pseudonipples of the ewe originates from three (L(3)-L(5)) and two (L(3) and L(4)) successive ganglia, respectively.

Parallel development of blood vessels and mast cells in the lateral geniculate nuclei.

The present study examined quantitatively developmental changes of the vasculature in the dorsal (dLGN) and the ventral (vLGN) lateral geniculate nuclei together with concomitant changes in the number of mast cells (MCs), known for their role in angiogenesis. Vascular network, marked after transcardial perfusion of India ink, and MCs detected with conventional histochemical techniques were examined at postnatal days (P) 1, 8, 14, 21, 31, 90 and 300 of Wistar rats. Quantitative analysis by means of an image analysis system showed age-dependent changes in both vascular parameters [vascular area and relative frequency (%) of capillaries and medium- and large-diameter vessels] and mast cells number in the developing dLGN and vLGN. Despite quantitative differences in the vascularization and MC infiltration between the two nuclei at some age points, MC number, vascular area and the percentage frequency of capillaries exhibited similar developmental time courses, especially up to the end of the first postnatal month. Both MC number and the capillary frequency reached maximal levels at P31 and declined thereafter, following a massive or a partial, respectively, decrease up to P300.