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Parallel plate fixation for distal humeral fractures provides good clinical outcomes. However, few studies have investigated the insertion sequence of the distal screws, although long screw insertion into the distal fragment is technically demanding. The purpose of this study was to investigate a correlation between the insertion sequence of the distal screws and the screw insertion difficulty.Medial and lateral anatomical locking plates were closely fitted to the medial and lateral sides of the 10 intact humerus bone models on the computer. Most distal screws have 2 patterns: the screw was inserted from the lateral side first followed by insertion from the medial side (group 1) or from the medial side first followed by insertion from the lateral side (group 2). We calculated the target area wherein the second screw can be inserted.The length of the first most distal screw in group 2 was significantly longer than that in group 1 (58.4 vs. 49.8 mm, p 0.05). The target areas in both groups were divided into the distal and proximal areas. The distal and proximal areas in group 1 were 91.6 and 61.6 mmIn parallel plate fixation for distal humeral fracture, most distal screws could be more easily inserted from the medial side first followed by insertion from the lateral side than from the lateral side first followed by insertion from the medial side.

Patients suffer from persistent ulnar wrist pain and distal radioulnar joint instability because of chronic triangular fibrocartilage complex (TFCC) foveal tear are treated with TFCC reconstruction. We performed an arthroscopic TFCC reconstruction using a palmaris longus tendon graft that provided a minimally invasive procedure. After confirming the TFCC foveal tear and stability between the TFCC remnant and radius, the bone tunnel was made in the ulna from the ulnar shaft to ulnar fovea. The position of the bone tunnel was checked by fluorography and arthroscopy. Curved bendable 18-gauge needles into which 3-0 nylon sutures were inserted in a loop shape were passed through the tunnel from the ulnar side, and both volar-side and dorsal-side TFCC remnants were penetrated. The nylon suture was extracted from the arthroscopic 4/5 portal, and the palmaris longus tendon graft was introduced into the joint. The graft was passed through the TFCC remnant and ulnar bone tunnel from the arthroscopic portal and fixed to the ulna using an interference screw. This procedure was indicated for TFCC foveal tears with intact radial-side TFCC remnants. If the radial-side tear and instability between the TFCC and radius coexist, this technique is contraindicated, and stabilization of both the radial and ulnar sides of the TFCC should be considered. This arthroscopic technique does not violate the distal radioulnar joint capsule, extensor carpi ulnaris tendon, or tendon sheath. In addition, it helps to stabilize the ulnar carpal complex.

Background: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. Purpose: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. Study Design: Case series; Level of evidence, 4. Methods: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image–based estimation of morphologic and compositional quality of the repair tissue. Results: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. Conclusion: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

Tenascin-X (TNX), an extracellular matrix protein, is abundantly expressed in peripheral nerves. However, the physiological role of TNX in peripheral nerves remains unknown. In this study, we found that actin levels in sciatic nerves of TNX-deficient mice were markedly decreased. Since actin was highly expressed in endothelial cells in wild-type sciatic nerves, we assessed morphological alterations of blood vessels in TNX-null sciatic nerves. The density of blood vessels was significantly decreased and the size of blood vessels was larger than those in wild-type sciatic nerves. Immunofluorescence showed that TNX was expressed by Schwann cells and fibroblasts in sciatic nerves. The results suggest that TNX secreted from Schwann cells and/or fibroblasts is involved in blood vessel formation in peripheral nerves.

The neural pathways underlying the respiratory variation dependent on vigilance states remain unsettled. In the present study, we examined the orexinergic innervation of Kölliker-Fuse nucleus (KFN) neurons sending their axons to the rostral ventral respiratory group (rVRG) and phrenic nucleus (PhN) as well as to the hypoglossal nucleus (HGN) by using a combined retrograde tracing and immunohistochemistry. After injection of cholera toxin B subunit (CTb) into the KFN, CTb-labeled neurons that are also immunoreactive for orexin (ORX) were found prominently in the perifornical and medial regions and additionally in the lateral region of the hypothalamic ORX field. After injection of fluorogold (FG) into the rVRG, PhN or HGN, we found an overlapping distribution of ORX-immunoreactive axon terminals and FG-labeled neurons in the KFN. Within the neuropil of the KFN, asymmetrical synaptic contacts were made between these terminals and neurons. We further demonstrated that many neurons labeled with FG injected into the rVRG, PhN, or HGN are immunoreactive for ORX receptor 2. Present data suggest that rVRG-, PhN- and HGN-projecting KFN neurons may be under the excitatory influence of the ORXergic neurons for the state-dependent regulation of respiration.

The authors report a case of immunoglobulin G4-related disease (IgG4-RD) of the hip. A 60-year-old man diagnosed with osteoarthritis of the right hip was referred to the authors' outpatient clinic for surgical intervention. Laboratory test results revealed elevated C-reactive protein and serum IgG levels. A subsequent laboratory test revealed an IgG4 level of 318 mg/dL. Magnetic resonance imaging revealed an abnormal mass in the right hip joint. The authors suspected IgG4-RD of the hip. The mass was resected during total hip arthroplasty. Immunohistochemical analysis for IgG revealed positive staining of many plasma cells. Most of the IgG-positive plasma cells were positive for IgG4, and the ratio of IgG4/IgG-positive cells was 51%. This case met all criteria for IgG4-RD; thus, the authors made a definitive diagnosis of IgG4-RD of the hip. The C-reactive protein level decreased to a negative value, and the IgG level decreased to a normal range at 3 weeks postoperatively. The IgG4 level gradually decreased to 152 mg/dL at 5 months postoperatively. This is the first reported case of IgG4-RD of the hip joint. [ Orthopedics . 2018; 41(6):e876–e879.]

The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

Few cases in which open reduction and internal fixation was performed for displaced pisiform fractures have been reported. We present a new surgical technique for the treatment of depressed intra-articular pisiform fractures. First, the depressed fragment was reduced by pushing the bone tamp. Then, the fracture void resulting from the reduction of the depressed fragment was filled with a shaped hydroxyapatite block. Finally, the fragments were sutured using braided polyblend polyethylene sutures. The postoperative radiography could achieve a well-reduced articular facet, and this procedure had a good clinical outcome.

The organization of the emotion-related somatic motor behavior, including jaw movements, is governed not only by the cortical limbic system but also by the subcortical limbic system including the amygdala and hypothalamus. GABAergic neurons in the central amygdaloid nucleus (CeA) and glutamatergic neurons in the posterior lateral hypothalamus (PLH) exert inhibitory and excitatory influences, respectively, upon premotor neurons for the motor trigeminal nucleus (Vm) in the parvicellular reticular formation (RFp) of the medulla oblongata. The CeA also has an inhibitory influence on non-dopaminergic (probably GABAergic) neurons in the retrorubral field of the midbrain that send their axons to the RFp. Furthermore, the CeA and lateral hypothalamus including the PLH may also modulate Vm neurons via projections to the mesencephalic trigeminal nucleus which contains the cell bodies of primary afferent neurons conveying inputs from the masticatory muscle spindles or the periodontal ligament receptors to jaw closing motoneurons within the Vm. These pathways from the subcortical limbic structures to the trigeminal motor system in the lower brainstem underlie the regulation of emotional jaw movements.

Background: The use of mesenchymal stem cells from various tissue sources to repair injured tissues has been explored over the past decade in large preclinical models and is now moving into the clinic. Purpose: To report the case of a patient who exhibited compromised mesenchymal stem cell (MSC) function shortly after use of high-dose steroid to treat Bell’s palsy, who recovered 7 weeks after therapy. Study Design: Case report and controlled laboratory study. Methods: A patient enrolled in a first-in-human clinical trial for autologous implantation of a scaffold-free tissue engineered construct (TEC) derived from synovial MSCs for chondral lesion repair had a week of high-dose steroid therapy for Bell’s palsy. Synovial tissue was harvested for MSC preparation after a 3-week recovery period and again at 7 weeks after therapy. Results: The MSC proliferation rates and cell surface marker expression profiles from the 3-week sample met conditions for further processing. However, the cells failed to generate a functional TEC. In contrast, MSCs harvested at 7 weeks after steroid therapy were functional in this regard. Further in vitro studies with MSCs and steroids indicated that the effect of in vivo steroids was likely a direct effect of the drug on the MSCs. Conclusion: This case suggests that MSCs are transiently compromised after high-dose steroid therapy and that careful consideration regarding timing of MSC harvest is critical. Clinical Relevance: The drug profiles of MSC donors and recipients must be carefully monitored to optimize opportunities to successfully repair damaged tissues.

The pre-Bötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during a single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.

In order to overcome potential problems associated with autologous chondrocyte implantation, mesenchymal stem cell-based therapies could be potential alternatives. Conventional stem cell-based therapy accompanies the separation of cells from tissue followed by monolayer culture for the expansion of cell numbers. On the other hand, the cost of cell culture under quality control is high, which could be a potential barrier for industrialization. In order to reduce the cost associated cell culture, culture-free cell-based therapies have been investigated with the use of bone marrow aspirate. In this chapter, we will introduce the three stem cell-based therapies in cartilage repair. The first two procedures are using cell culture methods and the last one with cell-free method. All the three methods have been into the stage of clinical trials and their surgical procedures as well as their preliminary results will be reported.

Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) are a promising cell source for the repair of skeletal disorders. Recently, neural crest cells (NCCs) were reported to be effective for inducing mesenchymal progenitors, which have potential to differentiate into osteochondral lineages. Our aim was to investigate the feasibility of MSC-like cells originated from iPSCs via NCCs for osteochondral repair. Initially, MSC-like cells derived from iPSC-NCCs (iNCCs) were generated and characterized in vitro. These iNCC-derived MSC-like cells (iNCMSCs) exhibited a homogenous population and potential for osteochondral differentiation. No upregulation of pluripotent markers was detected during culture. Second, we implanted iNCMSC-derived tissue-engineered constructs into rat osteochondral defects without any preinduction for specific differentiation lineages. The implanted cells remained alive at the implanted site, whereas they failed to repair the defects, with only scarce development of osteochondral tissue in vivo. With regard to tumorigenesis, the implanted cells gradually disappeared and no malignant cells were detected throughout the 2-month follow-up. While this study did not show that iNCMSCs have efficacy for repair of osteochondral defects when implanted under undifferentiated conditions, iNCMSCs exhibited good chondrogenic potential in vitro under appropriate conditions. With further optimization, iNCMSCs may be a new source for tissue engineering of cartilage.

Various approaches to treat articular cartilage have been widely investigated due to its poor intrinsic healing capacity. Stem cell-based therapy could be a promising approach as an alternative to chondrocyte-based therapy and some of these therapies have been already applied in clinical condition. This review discusses the current development of stem cell-based therapies in cartilage repair, specifically focusing on scaffold-free approaches.

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding behavior as well as in goal oriented behaviors, and MCH-containing neurons are distributed mainly in the lateral hypothalamic area (LHA). The anterior basomedial nucleus (BMA) and anterior cortical nucleus (CoA) of the amygdala form part of a circuit involved in processing olfactory, gustatory and visceral information, and the BMA-LHA and CoA-LHA pathways are suggested to be implicated in the control of feeding behavior. However, it is still unknown whether or not MCH-containing LHA neurons are under the direct influence of the BMA and CoA. Here the organization of projections from the BMA and CoA to MCH-containing LHA neurons was examined. Using a combined anterograde tracing with biotinylated dextranamine and immunohistochemistry for MCH, we first demonstrated that the distribution pattern of BMA fibers was almost similar to that of CoA fibers in the LHA, and a prominent overlapping distribution of these fibers and MCH-immunoreactive neurons existed in the ventral peripeduncular region of the LHA. We further revealed that asymmetrical synapses were made between these fibers and neurons. Using a combination of retrograde tract-tracing with cholera toxin B subunit and in situ hybridization for vesicular glutamate transporter (VGLUT) 2 mRNA, we finally showed that most of the LHA-projecting BMA and CoA neurons expressed VGLUT2 mRNA. These data suggest that the BMA and CoA of the amygdala may exert excitatory influence upon the MCH-containing LHA neurons for the regulation of feeding behavior.

Prosthetic reconstruction in two patients with malignant bone tumors of the distal tibia was conducted. The diagnoses were metastatic bone tumor in one patient and low grade central osteosarcoma in another. The mean duration of follow-up was 5.5 years (3 and 8 years). Reconstruction was achieved using custom-made prosthesis (JMM, Japan Medical Materials), which replaced the distal tibia. In the patient with metastasis, local recurrence occurred 8 months after the primary surgery and the recurrent tumor was resected. Both patients were free from neoplastic disease at the latest follow-up. The average functional scores according to the system of the Musculoskeletal Tumor Society were 25 and 23. Custom-made prostheses allow an early return to functional weight-bearing without major complications. This technique provides a safe and effective method of stabilization for properly selected malignant tumors of the distal tibia.

This study was performed to understand the anatomical substrates for Kolliker–Fuse nucleus (KFN) modulation of respiratory-related tongue movement. After application of cholera toxin B subunit (CTb) to the medial branch of the hypoglossal nerve (HGn) and injection of biotinylated dextran amine (BDA) into the KFN ipsilaterally, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the ventral compartment of the hypoglossal nucleus (HGN) ipsilateral to the application and injection sites. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites of the HGN neurons, some of which were labeled with CTb. Using retrograde tracing combined with in situ hybridization, we demonstrated that almost all the KFN neurons sending their axons to the HGN were positive for vesicular glutamate transporter (VGLUT) 2 mRNA but not glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde and retrograde tracing techniques and immunohistochemistry for VGLUT2, we further demonstrated that the KFN axon terminals with VGLUT2 immunoreactivity established close contact with the HGN motoneurons whose axons constitute the medial branch of the HGn. The present results suggest that glutamatergic KFN fibers may exert excitatory influence upon the HGN motoneurons sending their axons to the medial branch of the HGn for the control of protruder tongue muscles contraction to maintain airway patency during respiration.

We performed this study to understand the anatomical substrates of parabrachial nucleus (PBN) modulation of orexin (ORX)-containing neurons in the hypothalamus. After biotinylated dextranamine (BDA) injection into the lateral PBN and immunostaining of ORX-containing neurons in the rat, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the ORX-immunoreactive (ir) neurons was found in the lateralmost part of the dorsomedial nucleus and adjacent dorsal perifornical area (this overlapping field was referred to as "suprafornical area" in the present study), and the labeled axon terminals made asymmetrical synaptic contacts with somata and dendrites of the ORX-ir neurons. We further revealed that almost all the "suprafornical area"-projecting lateral PBN neurons were positive for vesicular glutamate transporter 2 mRNA and very few of them were positive for glutamic acid decarboxylase 67 mRNA. The present data suggest that ORX-containing neurons in the "suprafornical area" may be under the excitatory influence of the glutamatergic lateral PBN neurons probably for the regulation of arousal and waking.

The retrorubral field (RRF) contains numerous dopaminergic neurons and projects to the parvicellular reticular formation (RFp) of the medullary and pontomedullary brainstem, where many premotor neurons project to the orofacial motor nuclei. To know how the amygdala affects the RRF-RFp pathway in the rat, we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the RFp-projecting RRF neurons by using combined anterograde and retrograde tracing techniques. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and Fluoro-gold (FG) into the RFp, the prominent overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the lateral part of the RRF ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synapses with somata and dendrites of the FG-labeled neurons. Using a combination of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), we secondly demonstrated that the RFp-projecting RRF neurons were immunonegative for TH. Using a combination of anterograde tracing and immunohistochemistry for glutamic acid decarboxylase (GAD), we finally revealed that the CeA axon terminals in the RRF were immunoreactive for GAD. The present results suggest that GABAergic CeA neurons may exert inhibitory influences on non-dopaminergic RRF neurons that project to the RFp in the control of orofacial movements closely related to emotional behavior.

Osteoarthritis (OA) is a common disease defined as degenerative arthritis or joint disease involving degradation of articular cartilage and subchondral bone, and it could potentially affect the quality of life of elderly populations worldwide. The management of OA remains challenging and controversial. Although there are several clinical options for the treatment of OA, regeneration of the damaged articular cartilage has proven difficult due to the limited healing capacity. With the advancements in tissue engineering approaches including cell-based technologies and development of biomaterial scaffolds over the past decade, new therapeutic options for patients with osteochondral lesions potentially exist. This chapter will focus on the feasibility of tissue-engineered biomaterial scaffolds, which can mimic the native osteochondral complex, for osteochondral repair and highlight the recent development of these techniques toward tissue regeneration, which will contribute to osteochondral repair for the patients who are involved with an incurable OA treated by traditional procedures. Moreover, basic anatomy, strategy for osteochondral repair, and the design and fabrication methods of scaffolds as well as the choice of cells, growth factor, and materials will be discussed. Specifically, we focus on the latest preclinical animal studies using large animals and clinical trials with high clinical relevance. Accordingly, this will contribute to an understanding of the latest trends in osteochondral repair and future application of such clinical therapies in patients with OA.

Kölliker-Fuse nucleus (KF) neurons are considered to excite motoneurons in the phrenic nucleus (PhN) during inspiration through its projection to the PhN and/or to the rostral ventral respiratory group (rVRG), which in turn projects to the PhN, probably by releasing glutamate from their axon terminals. Using a combined retrograde tracing and in situ hybridization technique, here we demonstrate that most of the KF neurons projecting to the PhN and rVRG contain vesicular glutamate transporter 2 (VGLUT2) mRNA but not glutamic acid decarboxylase 67 (GAD67) mRNA, providing definitive evidence that these neurons are glutamatergic. Together with previous data by Stornetta et al. [Stornetta, R.L., Sevigny, C.P., Guyenet, P.G., 2003b. Inspiratory argumenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes. J. Comp. Neurol. 455, 113-124], indicating that PhN-projecting rVRG neurons are VGLUT2 mRNA-positive, the present results suggest that the glutamatergic KF-PhN pathway and/or the glutamatergic KF-rVRG-PhN pathway transmit excitatory outputs of KF neurons to the PhN neurons during inspiration.

Summary Objective To assess whether synovial mesenchymal stem cells (SMSCs) from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) can be used as an alternative cell source for cartilage repair using allogenic tissue engineered construct (TEC). Methods Twenty-five patients (17 female, average age 61.8 years) were divided according to their pathology (control trauma group; N = 6, OA group; N = 6) and RA patients were subdivided into two groups to evaluate the impact of biologics in accordance with whether treated with biologics [Bio(+)RA; N = 7] or not [Bio(−)RA; N = 6]. We compared the following characteristics among these groups: (1) The cell proliferation capacity of SMSCs; (2) The influence of passage number on features of SMSCs; (3) The weight and volume of TEC from the same number of SMSCs; (4) Inflammatory cytokine gene expressions levels of TEC; (5) The chondrogenic potential of TEC; and (6) Osteochondral repair using TEC in athymic nude rats. Results SMSCs from the four groups exhibited equivalent features in the above evaluation items. In in vivo studies, the TEC-treated repair tissues for all groups exhibited significantly better outcomes than those for the untreated group and no significant differences among the four TEC groups. Conclusion SMSCs from OA or RA patients are no less appropriate for repairing cartilage than those from trauma patients and thus, may be an effective source for allogenic cell-based cartilage repair.

Because of its limited healing capacity, treatments for articular cartilage injuries are still challenging. Since the first report by Brittberg, autologous chondrocyte implantation has been extensively studied. Recently, as an alternative for chondrocyte-based therapy, mesenchymal stem cell–based therapy has received considerable research attention because of the relative ease in handling for tissue harvest, and subsequent cell expansion and differentiation. This review summarizes latest development of stem cell therapies in cartilage repair with special attention to scaffold-free approaches.

The synaptic organization between and among the insular cortex (IC) axons, central amygdaloid nucleus (ACe) axons and posterolateral hypothalamus (PLH) neurons was investigated in the rat using double anterograde tracing and anterograde tracing combined with postembedding immunogold analysis. After ipsilateral injections of biotinylated dextran amine (BDA) into the IC and Phaseolus vulgaris-leucoagglutinin (PHA-L) into the ACe, the conspicuous overlapping distribution of BDA-labeled axon terminals and PHA-L-labeled axon terminals was found in the PLH region just medial to the subthalamic nucleus ipsilateral to the injection sites. At the electron microscopic level, approximately two-thirds of the IC terminals made synapses with small-sized dendrites and the rest did with dendritic spines of the PLH neurons, whereas about 79%, 16% and 5% of the ACe terminals established synapses with small- to medium-sized dendrites, somata, and dendritic spines, respectively, of the PLH neurons. In addition, the IC axon terminals contained densely packed round clear vesicles and their synapses were of asymmetrical type. On the other hand, most of the ACe terminals contained not only pleomorphic clear vesicles but also dense-cored vesicles and their synapses were of symmetrical type although some ACe terminals contained densely packed round clear vesicles and formed asymmetrical synapses. Most of the postsynaptic elements received synaptic inputs from the IC or ACe terminals, and some of single postsynaptic elements received convergent synaptic inputs from both sets of terminals. Furthermore, almost all the ACe terminals were revealed to be immunoreactive for gamma-aminobutyric acid (GABA), by using the anterograde BDA tracing technique combined with immunohistochemistry for GABA. The present data suggest that single PLH neurons are under the excitatory influence of the IC and/or inhibitory influence of the ACe in the circuitry involved in the regulation of cardiovascular functions.

The authors report a case of cervical myelopathy caused by invagination of the bilaterally separated lamina of the axis. They also present a literature review. The patient was a previously healthy 68-year-old man with a 1-year history of slowly progressive gait disturbance, right-hand clumsiness, and right dominant sensory disturbance in his trunk and extremities. Both MRI and CT showed that the spinal cord was markedly compressed at the C2–3 level, on the right side, by a deeply invaginated anomalous lamina of the axis. A bilaterally separated lamina was also visible. The patient underwent removal of the anomalous invaginated fragment of the separated lamina and the spinous process of the axis. One year after surgery, his myelopathic symptoms had almost completely resolved. Here, the authors present the case of a patient with an extremely rare anomaly of the lamina of the axis. The underlying pathogenesis of this anomaly could be the failure of the 2 chondrification centers on either side to fuse into a single ossification center. Surgical removal of the anomalous invaginated lamina produced a satisfactory outcome.

Migration behaviour and fate of cells originated from the lower rhombic lip (LRL) was examined in the chick embryo hindbrain. LRL-derived cells tangentially migrate along the pial surface of the brainstem and form a transient subpial migratory stream. In the initial stages of migration, LRL-derived cells appose each other or axon-like processes, which is indicative of mode of homophilic chain migration and/or axophilic migration. Some LRL-derived cells relocate rostroventrally towards the pontine region, although the majority of them migrate circumferentially to the ventral medulla oblongata. Depending on the stage of generation, LRL-derived cells undergo transmedian migration; late-generated LRL-derived cells preferentially colonize the contralateral brainstem compared with early generated cells. Thus, latecomer neuron precursors may migrate past their predecessors in the migratory stream. When LRL-derived cells leave the subpial migratory stream, they change their migratory direction to a radial one and relocate inwardly, with a profile that resembles a tangential-to-radial change seen in cerebellar granule cell precursors. After they enter the parenchymal region of the brainstem, they exhibited morphological differentiation, and some differentiate into excitatory neurons. The present results suggest that LRL-derived cells migrate across boundaries such as midline or rhombomere, which may facilitate to build up cellular and functional architectures of the hindbrain.

After ipsilateral injections of biotinylated dextran amine (BDA) into the central nucleus of the amygdala (ACe) and cholera toxin B subunit (CTb) into the motor trigeminal nucleus (Vm) in the rat, numerous BDA-labeled axons with bouton-like varicosities were distributed bilaterally with a clear-cut ipsilateral dominance in the parvicellular reticular formation (RFp), where many CTb-labeled neurons existed bilaterally with slightly ipsilateral dominance. The prominent overlapping distribution of these labeled axons and neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus throughout the caudalmost part of the pons and the rostral half of the medulla oblongata. Within the neuropil of the RFp region in the rostral half of the medulla oblongata, BDA-labeled axons made symmetrical synaptic contacts predominantly with the dendrites and additionally with the somata of RFp neurons, some of which were labeled with CTb. These data suggest that output signals from the ACe may be transmitted disynaptically to the Vm via the RFp neurons in the medulla oblongata for the control of jaw movements.

After ipsilateral injections of biotinylated dextran amine (BDA) into the Kölliker-Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.

Background Although there is a considerable difference in the number of clinical reports of neurologic injury between spinal anesthesia and other regional techniques, there are no animal data concerning a difference in the local anesthetic neurotoxicity between intrathecal and epidural administration. In the current study, the functional and morphologic effects of lidocaine administered intrathecally and epidurally were compared in rats. Methods Male rats were implanted with an intrathecal or epidural catheter through L4-L5 vertebra in the caudal direction. In experiment 1, to determine relative anesthetic potency, 16 rats received repetitive injections of 2.5% lidocaine into intrathecal or epidural space in different volumes and were examined for tail flick test for 90 min. In experiment 2, to ascertain whether the relative potency obtained in experiment 1 would apply to other concentrations of lidocaine, additional rats received saline, 1%, 2.5%, or 5% lidocaine in a volume of 20 or 100 microl through the intrathecal or epidural catheter, respectively. In experiment 3, additional rats that received saline, 2.5% lidocaine, or 10% lidocaine in a volume of 20 or 100 microl through the intrathecal or epidural catheter, respectively, were examined for persistent functional impairment and morphologic damage. Results In experiment 1, the two techniques produced parallel dose-effect curves that significantly differed from each other. The potency ratio calculated was approximately 4.72 (3.65-6.07):1 for intrathecal:epidural lidocaine. In experiment 2, every lidocaine solution produced a similar increase in tail flick latency for the two techniques. In experiment 3, five of eight rats given 10% intrathecal lidocaine incurred functional impairment 4 days after injection, whereas no rats in the other groups did. Significantly more morphologic damage was observed in rats given 10% intrathecal lidocaine than in those given 10% epidural lidocaine. Conclusions Persistent functional impairment occurred only after intrathecal lidocaine. Histologic damage in the nerve roots and the spinal cord was less severe after epidural lidocaine than after intrathecal lidocaine. The current results substantiate the clinical impression that neurologic complications are less frequent after epidural anesthesia than after spinal anesthesia.

The present tract-tracing study in the rat indicated that neurons in the ventrolateral part of the parafascicular thalamic nucleus (PF), where nigral fibers from the dorsolateral part of the substantia nigra pars reticulata (SNr) terminated, sent their axons to the ventrolateral part of the striatum as well as to the rostrolateral part of the lateral agranular cortex (AGl). We further demonstrated that symmetrical synaptic contacts were made between these nigral axons and striatum- or AGl-projecting PF neurons. Since the dorsolateral part of the SNr, ventrolateral part of the striatum and rostrolateral part of the AGl are responsible regions for orofacial behaviors, the nigrothalamostriatal and nigrothalamo-cortical pathways via the ventrolateral part of the PF may play a role in the control of orofacial motor function.

Electrical microstimulation of the jaw region of the rat striatum (SJR) has been reported to provoke clear electromyographic activity in the anterior digastric muscle but not in the masseter muscle (Neurosci. Lett., 252 (1998) 79-82). Thus, in the present study, we examined the sites of origin of cortico-SJR fibers by the retrograde labeling. The SJR, identified by electrical microstimulation, was injected electrophoretically with cholera toxin B subunit. In the cerebral cortex ipsilateral to the injection, there existed two foci of retrograde labeling: One focus was centered on the lateral part of the sensorimotor area, while the other on the insular cortical area around the middle cerebral artery. These foci appeared to correspond to the reported two cortical areas, where two different types of rhythmical jaw movements were induced by repetitive electrical stimulation (Jap. J. Oral Biol., 32 (1990) 57-68).

The organization of corticotectal projections from the medial agranular cortex (AGm), which has been considered to contain rat's frontal eye field, was examined using anterograde and retrograde tracing techniques. When biotinylated dextranamine (BDA) injections were made into the rostral part of the AGm, small numbers of BDA-labeled axons were found in the rostral two-thirds of the superior colliculus (SC) while some labeled axons were seen in the caudal one-third of the SC. These labeled axons were distributed mainly in the lateral part of the stratum griseum intermediale. On the other hand, after BDA injections into the caudal part of the AGm, moderate to dense plexuses of labeled axons were found in the rostral two-thirds of the SC while some labeled axons were seen in the caudal one-third of the SC. These labeled axons were distributed in the ventromedial and dorsolateral marginal zones of the stratum griseum intermediale as well as in the stratum griseum profundum. The corticotectal projections were largely uncrossed. After combined injections of BDA into the caudal part of the AGm on one side and cholera toxin B subunit (CTb) into the paramedian pontine reticular formation on the opposite side or into the interstitial nucleus of Cajal on the same side, the overlapping distributions of BDA-labeled axons and CTb-labeled neurons were found in the ventromedial marginal zone of the stratum griseum intermediale ipsilateral to the site of BDA injection. These results suggest that the caudal part of the AGm plays a more significant role in the oculomotor function than does the rostral part of the AGm.

The parabrachial complex, which plays an important role in respiratory regulation, has been reported to send projection fibers to the phrenic nucleus, but the synaptic organization between the parabrachial fibers and the phrenic motoneurons has not been examined. Using anterograde and retrograde tracing methods, we found in the rat that the parabrachial fibers originating mainly from the Kölliker-Fuse nucleus (KF) terminated not only within the phrenic nucleus but also on the radial dendritic bundles of the phrenic motoneurons. It was further revealed that the KF fibers made asymmetrical synapses predominantly with dendrites and partly with somata of the phrenic motoneurons. These data suggest that output signals from the KF may exert excitatory influence directly upon the phrenic motoneurons.

The projections from the subiculum to the hypothalamus were comprehensively examined in the rat by using the anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde cholera toxin B subunit (CTb) methods. Tracing of efferents with PHA-L indicated that the medial preoptic region received projection fibers from the temporal two-thirds of the subiculum, whereas the anterior, tuberal, and mammillary regions received those from the full longitudinal extent of the subiculum. The subicular projections to the anterior and tuberal hypothalamic regions were also found to be organized in a topographical manner such that the temporal-to-septal axis of origin in the subiculum determined a ventromedial-to-dorsolateral axis of termination in the medial zone of the hypothalamus: Massive labeled fibers from the temporalmost part of the subiculum terminated in the subparaventricular zone and its caudal continuum around the dorsal and medial aspects of the ventromedial nucleus, and those from progressively more septal parts terminated in progressively more dorsolateral parts of the medial zone. In addition, the temporal-to-septal axis of origin in the subiculum tended to determine a medial-to-lateral axis of termination in the preoptic region as well as a ventral-to-dorsal axis of termination in the mammillary region. Furthermore, the temporal-to-septal axis of origin in the septal two-thirds of the subiculum corresponded to a ventrolateral-to-dorsomedial axis of termination in the medial mammillary nucleus. The topographical projections from the subiculum to the medial zone of the hypothalamus were confirmed by CTb experiments, representatively in the subicular projections to the anterior hypothalamic region. These results suggest that different populations of neurons existing along the longitudinal axis of the subiculum may exert their influences on the execution of different hypothalamic functions. J. Comp. Neurol. 419:205–222, 2000. © 2000 Wiley-Liss, Inc.

We examined a direct pathway and an indirect pathway via the reticular thalamic nucleus (RT) from the substantia nigra pars reticulata (SNr) to the parafascicular thalamic nucleus (PF) by using anterograde and retrograde tract tracing methods. After biotinylated dextranamine (BDA) injection into the dorsolateral part of the SNr, many labeled fibers and axon terminals were distributed in the ventral part of the RT, as well as in the ventrolateral part of the PF, bilaterally with an ipsilateral dominance. After BDA injection into the ventral part of the RT, a plexus of labeled axons was found bilaterally with an ipsilateral dominance in the ventrolateral part of the PF. After combined injections of BDA into the dorsolateral part of the SNr and cholera toxin B subunit (CTb) into the ventrolateral part of the PF on the same side, overlapping distribution of BDA-labeled fibers and CTb-labeled neurons was observed in the ventral part of the RT ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synaptic contacts with soma and dendrites of the CTb-labeled neurons.

Injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin were made into individual nuclei of the vestibular nuclear complex of the rat to identify specific projections to the thalamus. The results showed that the superior vestibular nucleus and the medial vestibular nucleus, especially its rostral-to-middle parts, project to the lateral part of the parafascicular thalamic nucleus (corresponding to the centromedian nucleus in primates), the transitional zone between the ventrolateral thalamic nucleus (VL) and the ventral posterolateral thalamic nucleus (VPL) (the region considered to be the nucleus ventralis intermedius of Vogt [Vogt C. 1909. La myeloarchitecture du thalamus du cercopitheque. J Psychol Neurol 12:285-324.]), the lateral part of the centrolateral thalamic nucleus and the dorsal part of the caudal VL; the spinal vestibular nucleus projects to the lateral part of the parafascicular thalamic nucleus, the transitional zone between the VL and the VPL, the caudal part of the ventrobasal complex, and the suprageniculate thalamic nucleus. These results suggest that vestibular information is transmitted not only to the cerebral cortex (mainly area 2V and area 3a) but also to the striatum. They also suggest that vestibular activity may affect gaze control by means of vestibulothalamocortical pathway in addition to vestibulo-ocular and vestibulopremotoneuronal routes.

The axons of the optic nerve layer are known to be myelinated by oligodendrocytes in the chick retina. The development of the retinal oligodendrocytes has been studied immunohistochemically with antibodies against oligodendrocyte lineage: monoclonal antibodies O4 and O1, and an antibody against myelin basic protein. O4 positive (O4+) cells were first detected in the retina on the tenth day of incubation (embryonic day (E)10, stage 36). The labeled cells were located in the optic nerve layer close to the optic fissure. Most were unipolar in shape, extending a leading process with a growth cone toward the periphery of the retina. By E12, unipolar O4+ cells had spread to the middle of the retina. Many O4+ cells close to the optic fissure showed radial arrangement with extension of processes toward the inner limiting membrane. O1+ oligodendrocytes were first observed in the E14 retina positioned just above (interiorly to) retinal ganglion cells. These labeled cells extended fine processes in the optic nerve layer. Limited numbers of myelin basic protein-positive cells were present by E16 and located interiorly to the retinal ganglion cells. In addition to the oligodendrocyte in the optic nerve layer, a limited number of O4+ cells were observed in the inner nuclear layer by E14, and they became O1+ by E18. Furthermore, explant culture experiments showed E10 to be the youngest stage at which the retina contained oligodendrocyte precursors. An intraventricular inj ection of fluorescent dye 1,1',dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (DiI) at E6 yielded O4+/DiI+ cells in the retina at E10, which provided direct evidence to support migration of oligodendrocyte precursor into the retina. The present results demonstrated the sequential appearance of the cells of oligodendrocyte lineage and the detailed morphology of the developing oligodendrocytes in the retina. These morphologic features strongly suggested that retinal oligodendrocytes were derived from the optic nerve and spread by migration through the optic nerve layer.

After Phaseolus vulgaris leucoagglutinin injection into the lateral part of the superior colliculus (SC) in the rat, labeled fibers and axon terminals in the lower brainstem were distributed not only in the medial reticular formation but also in the lateral tegmental field including the parvicellular reticular formation (RFp). More caudally, in the upper cervical spinal cord labeled fibers with bouton-like varicosities were distributed mainly in laminae V, VII and VIII, with relatively sparse distribution in lamina IX. These labeled axons were found bilaterally with a clear-cut contralateral dominance. After combined injections of rhodamine-dextranamine, Fluoro-ruby (FR) into the RFp and Fluoro-gold (FG) into the upper cervical spinal cord on the same side, SC neurons labeled with FR were intermingled with those labeled with FG in the lateral part of the SC contralateral to the injection sites. In the stratum griseum intermediale, some of them were double-labeled with both tracers. Our results suggest that SC neurons innervating both the RFp and the cervical spinal cord may be involved in the coordination of head and mouth movements.

We examined a nigro-tecto-bulbar pathway to the parvicellular reticular formation (RFp), where many premotor neurons for orofacial motor nuclei are known to be distributed, by using a combined anterograde and retrograde tracing method. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the substantia nigra (SNr) and cholera toxin B subunit (CTb) into the RFp, overlapping distributions of BDA-labeled terminals and CTb-labeled neuronal cell bodies were found in the lateralmost part of the superior colliculus (SC) ipsilateral or contralateral to the site of BDA injection or CTb injection, respectively. After contralateral injections of BDA into the SNr and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) injection into the RFp, ipsilateral labeled axon terminals with BDA were found to make symmetrical synaptic contacts with the somata and dendrites of contralateral labeled neurons with WGA-HRP in the lateralmost part of the SC. Furthermore, we demonstrated that BDA-labeled axon terminals were immunoreactive for GABA, by using the anterograde tracing method combined with immunohistochemistry for GABA. Thus, GABA-like immunoreactive fibers originating from the dorsolateral part of the SNr make monosynaptic contacts with the tectal neurons sending their axons to the RFp.

It has been demonstrated that the spinal cord oligodendrocytes in the vertebrates arise in the ventral ventricular zone adjacent to the floor plate in their early development. Because of the similarities of basic structures in the spinal cord and metencephalon, it is probable that the mode of early oligodendrocyte development in the metencephalon is the same as that in the spinal cord. We examined this possibility in chick embryos, using monoclonal antibodies O1 and O4, markers for oligodendrocyte lineage. An O4-positive (O4+) cell focus was observed in the medial ventricular zone of E5 chick ventral metencephalon (the earliest stage examined), adjacent to the floor plate. At E6, O4+ cells were dispersed from the medial to the lateral pons and, at E7, to the cerebellar anlagen. O4+ cells in the E6 brainstem and in the E7 cerebellum were unipolar in shape, whereas one day later, some of the labeled cells were multipolar with a few thin processes. O1+ oligodendrocytes first appeared at E8 in the ventromedial part of the pons and were distributed throughout the pons at E10 and in the cerebellum at E12. Explants from three subdivisions of the metencephalon (medial and lateral pons, and cerebellum) from E5 to E8 chick embryos were separately cultured to confirm the potential for generation of oligodendrocyte lineage. O4+ cells appeared in the culture of the E5 medial pons (the earliest stage examined), in the E6 lateral pons, and in the E7 cerebellum. In addition, E7 was the youngest stage from which cerebellar explants were able to generate O1+ oligodendrocytes. Our results clearly demonstrated the in vivo morphology of oligodendrocyte precursors in the metencephalon and their developmental appearance in a ventral-to-dorsal manner. From the bipolar morphology of O4+ cells and the spacio-temporal continuity of the dispersion, it is inferred that the initial dispersion of O4+ cells may involve oligodendrocyte migration from the focus of the medial pons to the lateral and dorsal parts of the metencephalon.

The authors report a case of cervical myelopathy caused by invagination of the bilaterally separated lamina of the axis. They also present a literature review. The patient was a previously healthy 68-year-old man with a 1-year history of slowly progressive gait disturbance, right-hand clumsiness, and right dominant sensory disturbance in his trunk and extremities. Both MRI and CT showed that the spinal cord was markedly compressed at the C2-3 level, on the right side, by a deeply invaginated anomalous lamina of the axis. A bilaterally separated lamina was also visible. The patient underwent removal of the anomalous invaginated fragment of the separated lamina and the spinous process of the axis. One year after surgery, his myelopathic symptoms had almost completely resolved. Here, the authors present the case of a patient with an extremely rare anomaly of the lamina of the axis. The underlying pathogenesis of this anomaly could be the failure of the 2 chondrification centers on either side to fuse into a single ossification center. Surgical removal of the anomalous invaginated lamina produced a satisfactory outcome.

Melanocortin-4 receptor (MC4R)-expressing neurons are widely distributed in the central nervous system and play a crucial role in a variety of physiological functions including energy and glucose/insulin homeostasis. However, their neural pathways remain to be elucidated. In the present study, we examined a possible pathway from MC4R-expressing neurons in the dorsal motor nucleus of the vagus nerve (DMV) to the intrapancreatic ganglia using transgenic mice that express green fluorescent protein (GFP) under the control of the MC4R-promoter. Using immunofluorescence labeling, we demonstrated that GFP-immunoreactive (ir) nerve fibers were distributed in the intrapancreatic ganglia closely associated with the islets as well as among the acini. These GFP-ir fibers with bouton-like varicosities were frequently observed to surround ganglion cells immunoreactive for vasoactive intestinal polypeptide, a marker for postganglionic parasympathetic neurons. Using the pre-embedding immunoperoxidase method, we clearly showed that GFP-ir terminals formed synapses predominantly with dendrites and additionally with somata of the ganglion cells. Moreover, bilateral subdiaphragmatic vagotomy caused a marked loss of GFP immunoreactivity in the pancreas. Using a combination of retrograde tracing and immunohistochemistry, we finally demonstrated that nearly half of the pancreas-projecting DMV neurons were immunoreactive for GFP. These results suggest that MC4R-expressing DMV neurons may participate in the regulation of glucose/insulin homeostasis through their projections to the intrapancreatic ganglia.

A possible pathway from the substantia nigra pars reticulata (SNr) to the parvicellular reticular formation (RFp) via the red nucleus (RN) was examined light and electron microscopically by combining anterograde and retrograde tracing techniques. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the SNr and cholera toxin B subunit (CTb) into the RFp, many CTb-labelled neurones were distributed contralaterally in the dorsal part of the RN, where numerous BDA-labelled axon terminals originating from the ipsilateral SNr were found. After contralateral injections of BDA into the dorsolateral part of the SNr and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the RFp, ipsilateral axons labelled with BDA were found to make synaptic contacts with the somata and dendrites of contralateral neurones labelled with WGA-HRP in the dorsal part of the RN.

We examined the distribution of nerves containing nitric oxide synthase in the intestine of congenitally aganglionic rats, using a reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemical method for whole-mount and cryostat specimens. A constricted intestinal segment extends from the terminal ileum to the anus in this mutant. No nerve elements with the activity were found in the affected terminal ileum, cecum, and proximal colon. Although intrinsic ganglionic neurons were absent along the constricted intestine, nerve fibers with the activity were found in both the submucous and intermuscular layers distal to the proximal colon. These fibers increased in density towards the rectum, forming hypertrophic nerve bundles and unusual fiber networks. However, positive fibers were never seen within the circular and longitudinal musculature of the constricted lesion. Some of these hypertrophic nerve bundles were continuous with ectopic ganglia that were situated in the adventitial connective tissue around the lower rectum and in the submucosa near the anus. The hypertrophic nerve bundles seemed to have an extrinsic origin; some of them may have originated from ectopic ganglia. These results suggest that the defective distribution of nerves containing nitric oxide synthase may be involved in the pathogenesis of congenital colonic aganglionosis.