Yasuo Sugiura

Tissue-engineered bone using mesenchymal stem cells and a biodegradable scaffold.

Bone marrow has been shown to contain a population of rare cells capable of differentiating to the cells that form various tissues. These cells, referred to as mesenchymal stem cells (MSCs), are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to investigate the potential of a new &bgr;-tricalcium phosphate (&bgr;-TCP) as a scaffold and to compare the osteogenic potential between &bgr;-TCP and hydroxyapatite (HA). The &bgr;-TCP and HA loaded with MSCs were implanted in subcutaneous sites and harvested at 1, 2, 4, and 8 weeks after implantation for biochemical and histological analysis. Biochemically, in both &bgr;-TCP and HA composites, the alkaline phosphatase activity in the composites could be detected and was maintained at a high level for 8 weeks. In the histological analysis, active bone formation could be found in both the &bgr;-TCP and HA composites. These findings suggest that &bgr;-TCP could play a role as a scaffold as well as HA. The fabricated synthetic bone using biodegradable &bgr;-TCP as a scaffold in vivo is useful for reconstructing bone, because the scaffold material is absorbed several months after implantation.

US3 protein kinase of herpes simplex virus type 2 plays a role in protecting corneal epithelial cells from apoptosis in infected mice

To clarify the biological role of US3 protein kinase of herpes simplex virus type 2 (HSV-2) in vivo, the expression of the viral antigen, the appearance of apoptotic bodies and DNA fragmentation were examined immunohistologically after corneal infection of mice with three different kinds of HSV-2 strain 186: the wild-type virus, a US3-deficient mutant (L1BR1) and its revertant (L1B-11). In both wild-type 186- and L1B-11-infected mice, viral antigen was diffusely found in the corneal epithelium; no apoptotic changes were detected in the epithelial cells. Whereas, in L1BR1-infected mice, HSV-immunoreactivity was localized around the virus-inoculated sites, and a large number of apoptotic bodies were observed in the corneal epithelium with dual-positive reactions for both HSV-immunostaining and TUNEL staining. These results suggest that the US3 protein kinase plays an important role in protecting HSV-2-infected cells from apoptotic death in vivo.

TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness.

Abstract The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 μl of 4% carrageenan or subjected to an eccentric exercise (ECC) of the gastrocnemius muscle (GM). The Randall–Selitto and von Frey tests were performed on the calves to evaluate mechanical hyperalgesia of the muscle. The changes in expression of four genes and proteins of ion channels in dorsal root ganglia were examined using quantitative PCR and immunohistochemistry, respectively. Effects of antagonists to transient receptor potential (TRP) channels and acid sensing ion channels (ASICs) on the mechanical hyperalgesia induced by carrageenan injection or ECC were evaluated. The mechanical hyperalgesia was observed 6–24 h after carrageenan injection and 1–3 days after ECC in the Randall–Selitto test. Infiltrations of the inflammatory cells in the GM were seen in carrageenan‐injected animals but not in those subjected to ECC. Expressions of genes and proteins in sensory neurons showed no changes. Intramuscular injection of antagonists to TRPV1 showed an almost complete suppressive effect on ECC‐induced muscle hyperalgesia but not a carrageenan‐induced one. Antagonists to TRP channels and ASICs showed suppressive effects for both carrageenan‐ and ECC‐induced muscle hyperalgesia. The carrageenan injection and ECC models are useful models of acute inflammatory pain and delayed onset muscle soreness (DOMS), respectively, and the time course and underlying etiology might be different. TRP channels and ASICs are closely related to the development of muscle mechanical hyperalgesia, and TRPV1 is involved in ECC‐induced DOMS.

Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues

Gangliosides are considered to be essential in the maintenance and repair of nervous tissues; however, the mechanisms for neurodegeneration caused by ganglioside defects are unknown. We examined gene expression profiles in double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes and showed that the majority of complement genes and their receptors were up-regulated in cerebellum in DKO mice. Inflammatory reactions were demonstrated in those tissues by measuring up-regulated inflammatory cytokines, indicating the presence of complement activation and inflammation as reported in Alzheimers disease. Immunoblotting of fractionated membrane extracts by sucrose density gradient revealed that complement-regulatory molecules such as decay-accelerating factor and CD59 were dispersed from glycolipid-enriched microdomain/rafts in DKO cerebellum. Immunohistostaining of these molecules showed disordered membrane localization. These results suggested that dysfunction of complement-regulatory molecules may be due to abnormal glycolipid-enriched microdomain/rafts that triggered complement activation, subsequent inflammation, and neurodegeneration in DKO mice. Generation of the triple KO mice lacking complement activity in addition to the two glycosyltransferases suggested that complement activation is involved in the inflammatory reactions and neurodegeneration caused by the ganglioside deficiency.

Heat and mechanical hyperalgesia in mice model of cancer pain

&NA; We developed a mouse model of cancer pain to investigate its underlying mechanisms. SCC‐7, squamous cell carcinoma (SCC) derived from C3H mice, was inoculated subcutaneously into either the plantar region or thigh in male C3H/Hej mice. Heat and mechanical sensitivity as well as spontaneous behavior were measured at the plantar surface of the ipsilateral hind paw after the inoculation. Inoculated sites were histologically examined, and the expression of capsaicin receptors (TRPV1) was examined in the dorsal root ganglia (DRG) to clarify their potential contribution to pain sensitivity. Inoculation of cancer cells induced marked heat hyperalgesia and mechanical allodynia in the ipsilateral hind paw for two weeks in both plantar‐ and thigh‐inoculation models. Signs of spontaneous pain, such as lifting, licking and flinching of the paw were also observed. However, further growth of the tumor reversed the mechanical allodynia in both plantar‐ and thigh‐inoculation models, and heat hyperalgesia in thigh‐inoculation models. Histologically, no infiltration of the tumor cells into the nerve was observed. TRPV1 immunoreactive cells increased in the L5 DRG on day 7, but returned to the control level on day 15 post‐inoculation. Intraperitoneal administration of the competitive TRPV1 antagonist capsazepine inhibited hyperalgesia induced by tumor cell‐inoculation in either plantar‐ or thigh‐inoculated animals. This study indicated that inoculation of SCC resulted in spontaneous pain, heat hyperalgesia and mechanical allodynia. The altered expression of TRPV1 in the DRG may be involved in behavioral changes in this model.

Changes in P2X3 receptor expression in the trigeminal ganglion following monoarthritis of the temporomandibular joint in rats

&NA; The pathophysiological mechanisms of orofacial deep‐tissue pain is still unclear. Previously, P2X receptors (P2XR) in sensory neurons have been shown to play a role in the signal transduction of cutaneous pain. We investigated the functional significance of P2X3R in relation to orofacial deep‐tissue pain caused by monoarthritis of the temporomandibular joint (TMJ). Monoarthritis was induced by the injection of complete Freunds adjuvant (CFA) into the unilateral TMJ of the rat. The pain associated with monoarthritis was assessed by the pressure pain threshold (PPT), which was defined as the amount of pressure required to induce vocalization. Fifteen days after CFA‐treatment, changes in PPT were examined after injection of P2XR agonists or antagonists into the TMJ. The number of cells expressing P2X3R in trigeminal ganglia (TG) was investigated by immunohistochemistry. Inflamed TMJ showed a continuous decline in PPT during the experimental period (P<0.001). Injection of α,β‐meATP, an agonist of P2X1,3,2/3R, dramatically reduced the bilateral PPTs of both inflamed and non‐inflamed TMJs (P<0.01) although β,γ‐me‐L‐ATP, a selective agonist of P2X1R, did not. The decreased PPTs of inflamed TMJ were reversed either by PPADS, an antagonist of P2X1,2,3,5,1/5,4/5R, or by TNP‐ATP, an antagonist of P2X1,3,2/3,1/5R. Immunohistochemically, the number of P2X3R‐positive cells increased in the small cell group in TG (P<0.01), whereas there was no change in medium or large cell groups after the CFA‐injection. Retrograde tracing confirmed that TMJ neurons in the TG exhibited P2X3R immunoreactivity. Our results suggested that P2X3R plays an important role in orofacial pressure pain caused by monoarthritis of TMJ.

Central projection of unmyelinated (C) primary afferent fibers from gastrocnemius muscle in the guinea pig.

We have demonstrated the central projections of muscle C or group IV afferent fibers in the guinea pig by tracing arborizations in the spinal cord. C afferent fibers from the gastrocnemius muscle (GCM) were electrophysiologically identified by conduction velocity (less than 1 m/second). A single neuron in the lumbar 5 dorsal root ganglion (L5 DRG) was intracellularly labeled with Phaseolus vulgaris leucoagglutinin (PHA‐L). After iontophoretic injection of PHA‐L, we processed the lumbar cord and L5 DRG for PHA‐L immunohistochemistry. Six muscle C afferent fibers from 40 animals were labeled, and whole trajectories were recovered. Labeled fibers were reconstructed by tracing of the arbor in serial parasagittal sections. The GCM C afferents projected rostrocaudally for two or three segments and ran at the surface of the dorsal funiculus, giving off collaterals into laminae I and II and sometimes into parts of lamina III. We determined, based on the branching pattern and form of the terminal plexus, that the branching of muscle C afferent fibers showed an intermediate pattern that fell morphologically between the terminal patterns of somatic and visceral afferents. The numbers and sizes of fiber swellings and terminal swellings were measured on all collateral branches. We found that the area of distribution of the terminal swellings of muscle C afferent fibers is larger than that of somatic terminals but that the density of terminal swellings in the terminal area was lower than that of the somatic terminals. J. Comp. Neurol. 461:140–150, 2003.

Gangliosides are essential in the protection of inflammation and neurodegeneration via maintenance of lipid rafts: elucidation by a series of ganglioside-deficient mutant mice.

J. Neurochem. (2011) 116, 926–935.

Upregulations of P2X3 and ASIC3 involve in hyperalgesia induced by cisplatin administration in rats

&NA; The role of ion channels expressed in sensory neurons on mechanical and thermal hyperalgesia was examined in a rat model of cisplatin‐induced peripheral neuropathy. The rats were injected with 3 mg/kg of cisplatin intraperitoneally once per week for five consecutive weeks. The von Frey test, pin‐prick test and plantar test were performed to examine any noxious sensitivity of the skin. The Randall–Selitto test of the gastrocnemius muscle (GM) and the measurement of grip forces were performed to quantify muscle hyperalgesia. Coordination/motor was assessed by Rota‐rod testing. Expressions of the ion channels TRPV1, TRPV2, P2X3 and ASIC3 were examined in dorsal root ganglion (DRG) neurons and the muscle afferent neurons innervating GM. Effects of antagonists against either P2X3 or ASICs on behavioral responses were evaluated. Mechanical hyperalgesia and allodynia of both skin and muscle were observed in cisplatin‐treated animals. Expressions of TRPV2, P2X3, and ASIC3 increased in all DRG neurons. In addition, expressions of P2X3 and ASIC3 also increased in muscle afferent neurons in DRGs. Antagonists against P2X3,2/3 and ASICs showed a suppressive effect on both skin and muscle hyperalgesia induced by cisplatin administration. Upregulation of TRPV2, P2X3, and ASIC3 may play important roles in the mechanical hyperalgesia induced by cisplatin. Furthermore, cisplatin treatment also induced muscle hyperalgesia in muscle afferent neurons in connection with the upregulation of P2X3 and ASIC3.

Sensory nerve-dominant nerve degeneration and remodeling in the mutant mice lacking complex gangliosides

Gangliosides, sialic acid-containing glycosphingo-lipids, are enriched in the mammalian nervous system. Since mutant mice with disrupted beta1,4-N-acetylgalactosaminyl-transferase (GM2/GD2 synthase) were generated, there have been several studies on the pathology of the mutant mice, i.e. mild functional disorders and Wallerian degeneration in the peripheral nervous system. To further analyze the chronological alteration in the mutant mice, we examined the peripheral and CNS mainly with morphological approaches, such as electron microscopy and immunohistochemistry. Accordingly with the sensory dysfunction, neural degeneration, glial proliferation and synaptic remodeling in the dorsal horn of the spinal cord were found in adult mice. Thick astrocytic processes with densely packed glial filaments were extended among the neuropils and around blood vessels. Morphological changes in the synaptic vesicles and modes of synaptic contacts with central terminals were detected, suggesting synaptic remodeling following the degeneration. These results suggest that complex gangliosides are essential in the maintenance of integrity in architecture and function of the nervous system, lack of which results in neural degeneration in a sensory nerve-dominant manner.