Comparisons of the effects of silk elastin and collagen sponges on wound healing in murine models.

Introductions: Silk elastin, a recombinant protein with repeats of elastin and silk fibroin, possesses a self-gelling ability and is a potential wound dressing material. The aim of this study is to elucidate the mechanism of the wound healing-promoting effect of silk elastin by comparing its in vivo behavior in a mouse wound model with that of a collagen sponge. Methods: Skin defects (8 mm in diameter) were created on the backs of C57BL/6J and BKS.Cg- + Lepr/+Lepr db male mice. Silk elastin sponges of 2.5 or 5.0 mm thickness, as well as collagen sponges, were placed on the wounds and secured with a polyurethane film. In the control group, only the polyurethane film was applied. The remaining wound area was grossly evaluated, and tissue samples were collected after 7, 14, and 21 days for histological evaluation, including neoepithelialization, wound contraction, granulation tissue formation, newly formed capillaries, and macrophages. Genetic analysis was conducted using real-time polymerase chain reaction. Results: In the study with C57BL/6J, there were no significant differences between the silk elastin and collagen sponge groups. Similarly, in the study using BKS.Cg- + Lepr/+Lepr db, no significant differences were found in the remaining wound area and granulation tissue formation between the silk elastin and collagen sponge groups. However, on day 14, the 5.0-mm-thick silk elastin sponge group showed increased macrophages, longer neoepithelialization, and more frequent angiogenesis compared to other groups. Gene expression of inducible nitric oxide synthase and arginase-1 was also higher in the 5.0 mm thick silk elastin sponge group. Conclusions: Silk elastin sponges demonstrated superior neoepithelialization and angiogenesis compared to collagen sponges. The results suggest that silk elastin and collagen sponges promote wound healing through different mechanisms, with silk elastin possibly enhancing wound healing by facilitating increased macrophage migration. Further studies are needed, but silk elastin shows great potential as a versatile wound dressing material.

An artificial silk elastin-like protein modifies the polarization of human macrophages line THP-1.

A silk elastin-like protein (SELP) is an artificial compound with silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the influence of SELP on the polarization of human monocytoma cell line (THP-1)-derived macrophages. When the macrophages of inflammation-type (M1) were cultured with different concentrations of SELP solution, the secretion of a pro-inflammatory cytokine, tumor necrotizing factor (TNF) -α was significantly suppressed at the higher concentrations. In addition, the secretion of an anti-inflammation cytokine, interleukin (IL)-10, was significantly enhanced from the macrophage of M0-, M1-, and M2-types. By the incubation with soluble SELP, the morphology of M2-type macrophages changed to be of an extended shape. Following incubation with the sponge of SELP, M0-type macrophages secreted IL-10 with time. It is concluded that the SELP itself in solution has an ability to induce the anti-inflammation of M2-type macrophages.

An Artificial Silk Elastin-Like Protein Modifies the Polarization of Macrophages.

A silk elastin-like protein (SELP) is an artificial compound with silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the influence of SELP on the polarization of mouse bone marrow-derived macrophages. When the macrophages of inflammation-type (M1) were cultured with different concentrations of SELP solution, the secretion of a pro-inflammatory cytokine, tumor necrotizing factor (TNF)-α, was significantly suppressed at the higher concentrations. In addition, the secretion of an anti-inflammation cytokine, interleukin (IL)-10, was significantly enhanced from the macrophage of an original type (M0). By the incubation with soluble SELP, the morphology of M0- and M1-type macrophages changed to be of a round shape with a large size. Following incubation with the sponge of SELP, the M0-type macrophages secreted IL-10 with time. When injected into an air pouch of mice subcutis which had been prepared by the injection of air, the SELP sponge and 5 wt % of SELP solution induced IL-10 secretion to a significantly high extent compared with the saline injection. Cells isolated from the air pouch 24 h after the injection were stained by the CD206 of a M2 marker. It is concluded that the SELP itself in solution has an ability to induce the anti-inflammation M2-type macrophages.

A combination of dietary fat intake and nicotine exposure enhances CB1 endocannabinoid receptor expression in hypothalamic nuclei in male mice.

BACKGROUND: Cannabinoid receptor 1 (CB1R) is a GPCR expressed widely in the brain as well as in peripheral metabolic organs. Although pharmacological blockade of CB1R has been effective for the treatment of obesity and tobacco addiction, precise distribution of CB1R within the brain and potential changes by obesity or nicotine exposure have not been thoroughly addressed. METHODS: To examine CB1R distribution within the central energy center, we performed immunostaining and qPCR analysis of micro-dissected hypothalamic nuclei from male C57BL/6 mice. To address the effect of nicotine on food intake and body weight, and on potential changes of CB1R levels in the hypothalamus, mice kept on a high fat diet (HFD) for four weeks were challenged with nicotine intraperitoneally. RESULTS: Validity of the micro-dissected samples was confirmed by the expression of established nucleus-enriched genes. The expression levels of CB1R in the arcuate and lateral nuclei of the hypothalamus were higher than paraventricular and ventral-dorsal medial nuclei. Nicotine administration led to a significant suppression of food intake and body weight either under standard or high fat diet. Neither HFD nor nicotine alone altered CB1R levels in any nucleus tested. By contrast, treatment of HFD-fed mice with nicotine led to a significant increase in CB1R levels in the arcuate, paraventricular and lateral nuclei. CONCLUSIONS: CB1R was widely distributed in multiple hypothalamic nuclei. The expression of CB1R was augmented only when mice were treated with HFD and nicotine in combination. These data suggest that the exposure to nicotine may provoke an enhanced endocannabinoid response in diet-induced obesity.

Thalamostriatal projections and striosome-matrix compartments.

The neostriatum has a mosaic organization consisting of striosome and matrix compartments. It receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Recent behavioral studies in rats revealed a selectively active medial prefronto-striosomal circuit during cost-benefit decision-making. However, clarifying the input/output organization of striatal compartments has been difficult because of its complex structure. We recently demonstrated that the source of thalamostriatal projections are highly organized in striatal compartments. This finding indicated that the functional properties of striatal compartments are influenced by their cortical and thalamic afferents, presumably with different time latencies. In addition, these afferents likely support the unique dynamics of striosome and matrix compartments. In this manuscript, we review the anatomy of basal ganglia networks with regard to striosome/matrix structure. We place specific focus on thalamostriatal projections at the population and single neuron level.

Quantitative Analyses of the Projection of Individual Neurons from the Midline Thalamic Nuclei to the Striosome and Matrix Compartments of the Rat Striatum.

A fundamental organizing principle of the striatum is the striosome/matrix system that is defined by inputs/outputs and neurochemical markers. The thalamostriatal projection is highly heterogeneous originating in many subnuclei of the thalamus including the midline (ML) and intralaminar (IL) nuclei. We examined the dendritic morphology and axonal trajectory of 15 ML and 11 IL neurons by single-neuron labeling with viral vectors in combination with mu-opioid receptor immunostaining in rat brains. Dendritic and axonal morphology defined ML neurons as type II cells consisting of at least two subclasses according to the presence or absence of striatal axon collaterals. In the striatum, ML neurons preferentially innervated striosomes, whereas parafascicular neurons preferentially innervated the matrix. Almost all single thalamostriatal neurons favoring striosome or matrix compartments also innervated the cerebral cortical areas that supplied cortical input to the same striatal compartment. We thus revealed that thalamostriatal projections are highly organized 1) by the similarity in morphological characteristics and 2) their preference for the striatal compartments and cortical areas. These findings demonstrate that the functional properties of striatal compartments are influenced by both their cortical and thalamic afferents presumably with a different time latency and support selective dynamics for the striosome and matrix compartments.

Ventral medial nucleus neurons send thalamocortical afferents more widely and more preferentially to layer 1 than neurons of the ventral anterior-ventral lateral nuclear complex in the rat.

Not only inhibitory afferent-dominant zone (IZ) of the ventral anterior-ventral lateral thalamic complex (VA-VL) but also the ventral medial nucleus (VM) is known to receive strong inputs from the basal ganglia and send axons to motor areas. We previously reported differences in axonal arborization between IZ neurons and the other VA-VL neurons in rats by single-neuron tracing with viral vectors. In the present study, the axonal arborization of single VM neurons was visualized by the same method, and compared with that of IZ neurons. VM neurons formed fewer axon collaterals in the striatum, but sent axon fibers more widely and more preferentially (79% of fibers) to layer 1 of cortical areas than IZ neurons. Furthermore, the VM seemed to contain at least 2 types of neurons; a major population of VM neurons sent axon fibers principally to motor-associated areas as VA-VL neurons did, and the other population projected mainly to orbital or cingulate areas. Although both VM and IZ neurons receive strong basal ganglia inputs, these results suggest that VM neurons, at a single neuron level, innervate the apical dendrites of cortical pyramidal neurons more intensely and more widely than IZ neurons.

Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

Two types of thalamocortical projections from the motor thalamic nuclei of the rat: a single neuron-tracing study using viral vectors.

The axonal arborization of single motor thalamic neurons was examined in rat brain using a viral vector expressing membrane-targeted palmitoylation site-attached green fluorescent protein (palGFP). We first divided the ventral anterior-ventral lateral motor thalamic nuclei into 1) the rostromedial portion, which was designated inhibitory afferent-dominant zone (IZ) with intense glutamate decarboxylase immunoreactivity and weak vesicular glutamate transporter 2 immunoreactivity, and 2) the caudolateral portion, named excitatory subcortical afferent-dominant zone (EZ) with the reversed immunoreactivity profile. We then labeled 38 motor thalamic neurons in 29 hemispheres by injecting a diluted palGFP-Sindbis virus solution and isolated 10 IZ and EZ neurons for reconstruction. All the reconstructed IZ neurons widely projected not only to the cerebral cortex but also to the neostriatum, whereas the EZ neurons sent axons almost exclusively to the cortex. More interestingly, 47-66% of axon varicosities of IZ neurons were observed in layer I of cortical areas. In contrast, only 2-15% of varicosities of EZ neurons were found in layer I, most varicosities being located in middle layers. These results suggest that 2 forms of information from the basal ganglia and cerebellum are differentially supplied to apical and basal dendrites, respectively, of cortical pyramidal neurons and integrated to produce a motor execution command.

Paucity of enkephalin production in neostriatal striosomal neurons: analysis with preproenkephalin-green fluorescent protein transgenic mice.

Whether or not the striosome compartment of the neostriatum contained preproenkephalin (PPE)-expressing neurons remained unresolved. To address this question by developing a sensitive detection method, we generated transgenic mice expressing enhanced green fluorescent protein (GFP) under the specific transcriptional control of the PPE gene. Eight transgenic lines were established, and three of them showed GFP expression which was distributed in agreement with the reported localization of PPE mRNA in the central nervous system. Furthermore, in the matrix compartment of the neostriatum of the three lines, intense GFP immunoreactivity was densely distributed in the neuronal cell bodies and neuropil, and matrix neurons displayed > 94% co-localization for GFP and PPE immunoreactivities. In sharp contrast, GFP immunoreactivity was very weak in the striosome compartment, which was characterized by intense immunoreactivity for mu-opioid receptors (MOR). Although neostriatal neurons were divided into GFP-immunopositive and -negative groups in both the striosome and matrix compartments, GFP immunoreactivity of cell bodies was much weaker (~1/5) in GFP-positive striosomal neurons than in GFP-positive matrix neurons. A similar reciprocal organization of PPE and MOR expression was also suggested in the ventral striatum, because GFP immunoreactivity was weaker in intensely MOR-immunopositive regions than in the surrounding MOR-negative regions. As PPE-derived peptides are endogenous ligands for MOR in the neostriatum and few axon collaterals of matrix neurons enter the striosome compartment, the present results raised the question of the target of those peptides produced abundantly by matrix neurons.

PRODAN-conjugated DNA: synthesis and photochemical properties.

A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, PDNX (X = U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8. The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational polarizability of the solvent. The PDNX incorporated into DNA also changed the Stokes shift values depending on the DNA structure. In particular, the excitation spectrum of the PDNX-containing duplex shifted to a longer wavelength and gave a smaller Stokes shift value when the base opposite PDNX could form a Watson-Crick base pair with PDNX. A lower energy excitation of PDNX-containing DNA resulted in a strong fluorescence emission selective to the Watson-Crick pairing base. This unique photochemical character was applicable to the efficient typing of single-nucleotide polymorphisms of genes.

Metabotropic glutamate receptor 4-immunopositive terminals of medium-sized spiny neurons selectively form synapses with cholinergic interneurons in the rat neostriatum.

Metabotropic glutamate receptor 4 (mGluR4) is localized mainly to presynaptic membranes in the brain. Rat neostriatum has been reported to contain two types of mGluR4-immunoreactive axon varicosities: small, weakly immunoreactive varicosities that were distributed randomly (type 1) and large, intensely immunoreactive ones that were often aligned linearly (type 2). In the present study, most type 1 terminals formed asymmetric synapses on dendritic spines, whereas type 2 terminals made symmetric synapses on dendritic shafts, showing immunoreactivity for GABAergic markers. After depletion of neostriatal neurons, type 2 but not type 1 varicosities were largely decreased in the damaged region. When medium-sized spiny neurons (MSNs) were labeled with Sindbis virus expressing membrane-targeted green fluorescent protein, mGluR4 immunoreactivity was observed on some varicosities of their axon collaterals in immunofluorescence and immunoelectron microscopies. Furthermore, type 2 varicosities were often positive for substance P but mostly negative for striatal interneuron markers and preproenkephalin. Thus, striatonigral/striato-entopeduncular MSNs are likely to be the largest source of type 2 mGluR4-immunopositive axon terminals in the neostriatum. Next, in the double-immunofluorescence study, almost all choline acetyltransferase (ChAT)-immunopositive and 41% of NK1 receptor-positive dendrites were heavily associated with type 2 mGluR4-immunoreactive varicosities. Neuronal nitric oxide synthase (nNOS)-positive dendrites, in contrast, seemed associated with only a few type 2 varicosities. Conversely, almost all type 2 varicosities were closely apposed to NK1 receptor-positive dendrites that were known to be derived from cholinergic and nNOS-producing interneurons. These findings indicate that the mGluR4-positive terminals of MSN axon collaterals selectively form synapses with neostriatal cholinergic interneurons.

Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum.

The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals immunopositive for vesicular glutamate transporter (VGluT) 2 were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. By contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both compartments. As axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment.

Synthesis and properties of novel PRODAN-labeled uridine.

We have developed a novel base-discriminating fluorescent (BDF) nucleoside, (PDN)U, which contains a PRODAN chromophore connected at the C-5 position of uracil. The Stokes shift (Deltanu) of (PDN)U was highly dependent on the local dielectric property around the fluorophore. On the excitation at 450 nm, the fluorescence spectrum of the duplex containing a (PDN)U/A base pair showed a strong emission at 520 nm. In contrast, the fluorescence intensities of duplexes containing "mismatched" (PDN)U/N base pairs (N = C, G, or T) were considerably lower. Furthermore, the drastic change of fluorescence intensity by the nature of the complementary base is useful for SNP typing.