The density and distribution of substance P-like immunoreactive (SP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in rat temporomandibular joint (TMJ) were investigated in whole-mount preparations and frozen sections by immunohistochemistry with the avidin-biotin-peroxidase complex method. Both types of immunoreactive nerves were observed primarily in the joint capsule, the peripheral articular disc, the synovial membrane, and the periosteum. The distribution of CGRP-LI nerves was similar to that of SP-LI nerves. The anterior portion of the joint capsule and disc was most densely innervated, followed by the posterior, lateral, and medial portions. In addition, CGRP-LI nerves were more numerous and more dense in immuno-intensity than SP-LI nerves. In the synovial membrane, many SP- and CGRP-LI nerves terminated in the subsynovial layer, but some branches extended into the superficial synovial lining layer close to the joint cavity. Immunolabeled nerves were prominently located in the disc attachment and peripheral portion of the disc, and occasional nerves were located in the dense collagenous disc band as an actual disc. However, no fibers were detected in the central disc band. Thus, most of the disc was not innervated by any nerves. The present study provides a morphological basis for the possible roles of neuropeptides in endocytosis by synoviocytes, regulation of blood flow in the synovial membrane, nociception mechanisms of the TMJ, and modulation of the inflammatory response in the TMJ.

This study aimed to investigate whether the administration of mononuclear cells derived from human umbilical cord blood cells (UCBCs) could ameliorate hypoxic-ischemic brain injury in a neonatal rat model. The left carotid arteries of 7-day-old rats were ligated, and the rats were then exposed to 8% oxygen for 60 min. Mononuclear cells derived from UCBCs using the Ficoll-Hypaque technique were injected intraperitoneally 6 h after the insult (1.0 × 10⁷ cells). Twenty-four hours after the insult, the number of cells positive for the oxidative stress markers 4-hydroxy-2-nonenal and nitrotyrosine, in the dentate gyrus of the hippocampus in the UCBC-treated group, decreased by 36 and 42%, respectively, compared with those in the control group. In addition, the number of cells positive for the apoptosis markers active caspase-3 and apoptosis-inducing factor decreased by 53 and 58%, respectively. The number of activated microglia (ED1-positive cells) was 51% lower in the UCBC group compared with the control group. In a gait analysis performed 2 weeks after the insult, there were no significant differences among the sham-operated, control and UCBC groups. An active avoidance test using a shuttle box the following week also revealed no significant differences among the groups. Neither the volumes of the hippocampi, corpus callosum and cortices nor the numbers of neurons in the hippocampus were different between the UCBC and control groups. In summary, a single intraperitoneal injection of UCBC-derived mononuclear cells 6 h after an ischemic insult was associated with a transient reduction in numbers of apoptosis and oxidative stress marker-positive cells, but it did not induce long-term morphological or functional protection. Repeated administration or a combination treatment may be required to achieve sustained protection.

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Perinatal hypoxic-ischemic (HI) brain injury occurs in 1 in 1,000 live births and remains the main cause of neurological disability and death in term infants. Cytotherapy has recently emerged as a novel treatment for tissue injury. In particular, mesenchymal stem cells (MSCs) are thought to have therapeutic potential, but little is known about the differences according to their origin. In the current study, we investigated the therapeutic effects and safety of intravenous injection of allogeneic bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs) in a rat model of HI brain injury. HI models were generated by ligating the left carotid artery of postnatal day 7 Wistar/ST rats and exposing them to 8% hypoxia for 60 min. Bone marrow and adipose tissue were harvested from adult green fluorescent protein transgenic Wistar rats, and cells were isolated and cultured to develop BM-MSCs and ADSCs. At passaging stages 2–3, 1 × 105 cells were intravenously injected into the external right jugular vein of the HI rats at 4 or 24 h after hypoxia. Brain damage was evaluated by counting the number of cells positive for active caspase-3 in the entire dentate gyrus. Microglial isotypes and serum cytokines/chemokines were also evaluated. Distribution of each cell type after intravenous injection was investigated pathologically and bio-optically by ex vivo imaging (IVIS®) with a fluorescent lipophilic tracer DiR. The mortality rate was higher in the ADSC group compared to the BM-MSC group, in pups injected with cells 4 h after hypoxia. The number of active caspase-3-positive cells significantly decreased in the BM-MSC group, and the percentage of M1 microglia (a proinflammatory isotype) was also lower in the BM-MSC vs control group in the penumbra of the cortex. Moreover, BM-MSC administration increased anti-inflammatory cytokine and growth factor levels, while ADSCs did not. Each injected cell type was mainly distributed in the lungs and liver, but ADSCs remained in the lungs longer. Pathologically, pulmonary embolisms and diffuse alveolar hemorrhages were seen in the ADSC group. These results indicated that injection of allogeneic BM-MSCs ameliorated neonatal HI brain injury, whereas ADSCs induced severe lung hemorrhage and higher mortality.

To extend our previous light microscopic observations concerning the distribution of trigeminal sensory nerves in the synovium of the rat temporomandibular joint, we investigated the detailed distribution and fine structure of sensory nerve endings at the light and electron microscopic level by the anterograde transport method using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected into the trigeminal ganglion. At the light microscopic level, HRP-labeled nerve fibers were observed in the joint capsule and peripheral portion of the disc. The anterior portion of the disc was more densely innervated than the posterior portion, while no nerves were found in the central portion. At the electron microscopic level, HRP reaction products were observed intra-axonally in the thinly myelinated (A delta) and unmyelinated (C) axons in the anterior portion of the joint capsule, and were also localized in the extracellular space surrounding the unmyelinated fibers and terminals. In the subsynovial layer of the synovial membrane, the majority of labeled axons located near blood vessels or among the collagenous fibrils were covered by Schwann cell sheaths, although some naked axon terminals without sheaths were also found. These unsheathed terminals contained mitochondria, small clear vesicles, and large granular vesicles, and were close to the synovial A and/or B cells near the joint cavity. The minimum distance between the terminals and synovial cells was 75 nm. This is the first demonstration of trigeminal sensory nerve terminals close to synovial lining cells or joint cavity and suggests that neuropeptides such as substance P may be released close to the synovial lining cells or joint cavity.

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