Ken Ichi Iyama

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization.

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammons horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.

Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats.

Alveolar re‐epithelialization is necessary in the repair of damaged alveolar epithelium after lung injury. Keratinocyte growth factor (KGF) has been shown to be a potent proliferation and differentiation factor for rat alveolar type II cells. The present study examined whether KGF would prevent bleomycin‐induced lung fibrosis. Adult rats were anaesthetized and recombinant human KGF (rhKGF) (150 μg/kg) or saline was injected intratracheally at 48 h before and 24 h after bleomycin (Bleo, 5 mg/kg) instillation. Seven and 14 days after the last administration, rat lungs were processed for lung physiology, immunohistochemistry, and in situhybridization. Double instillation of KGF prevented the loss of body weight and reduction in total lung capacity (TLC) due to Bleo, and markedly attenuated the protein accumulation and mRNA expression of collagen types I and III and the decreased expression of surfactant protein mRNAs in the fibrotic lesions of Bleo‐treated rats. KGF may play an important role in maintaining alveolar epithelium and repairing the damaged epithelium after lung injury.

The effect of a local application of fibroblast growth factor-2 on tendon-to-bone remodeling in rats with acute injury and repair of the supraspinatus tendon

METHODS We investigated the effect of application of fibroblast growth factor (FGF)-2 on the tendon-to-bone remodeling of repaired supraspinatus tendon in rats subjected to bilateral detachment. FGF-2 (100 mg/kg) in a fibrin sealant or sealant alone was applied on the right and left shoulders, respectively. Twelve animals each at 2, 4, and 6 weeks after surgery were sacrificed for histological analysis (n = 5) and biomechanical Q1 testing (n = 7). RESULTS Histologically, at 2 weeks, FGF-treated specimens had significantly higher tendon-to-bone insertion maturing scores then untreated specimens (P < .002). At 4 and 6 weeks, the scores of FGF-treated and untreated specimens were similar (P > .05). Biomechanically, FGF-treated specimens were stronger at 2 weeks (P = .001); at 4 and 6 weeks, both specimens exhibited similar strength (P > .05). CONCLUSIONS The initial tendon-to-bone remodeling was accelerated by a local application of FGF-2. This may represent a clinically important improvement in rotator cuff repair.

The Effects of Fibroblast Growth Factor-2 on Rotator Cuff Reconstruction With Acellular Dermal Matrix Grafts

PURPOSE Our purpose was to determine whether the local application of fibroblast growth factor (FGF) 2 accelerates regeneration and remodeling of rotator cuff tendon defects reconstructed with acellular dermal matrix (ADM) grafts in rats. METHODS Thirty adult male Sprague-Dawley rats were divided into equal groups undergoing FGF-treated and FGF-untreated repairs. All rats underwent placement of an ADM graft for the supraspinatus defect (3 x 5 mm). FGF-2 (100 microg/kg) in a fibrin sealant was applied to both shoulders in the FGF-treated group, whereas only fibrin sealant was applied in untreated group. At 2, 6, and 12 weeks after surgery, 5 rats (10 shoulders) in each group were sacrificed for histologic analysis (3 shoulders) and biomechanical testing (7 shoulders). The controls were 5 unoperated rats (3 histologic and 7 biomechanical control specimens). RESULTS Unoperated control tendons inserted into the bone by direct insertion; there was a zone of fibrocartilage between the tendon and bone. At 2 weeks, the FGF-treated group had tendon maturing scores similar to those in the untreated group (P > .05). At 6 and 12 weeks, the FGF-treated group had significantly higher scores (P < .05). At 2 weeks, specimens in both the treated and untreated groups exhibited similar strength; the ultimate tensile failure load was 6.0 +/- 4.0 N and 5.8 +/- 2.0 N, respectively (P > .05). At 6 weeks, the FGF-treated specimens were stronger, with an ultimate tensile failure load of 10.2 +/- 3.1 N compared with 7.2 +/- 2.2 N in the untreated group (P = .02). At 12 weeks, the FGF-treated specimens were stronger, with an ultimate tensile failure load of 15.9 +/- 1.6 N compared with 13.2 +/- 2.0 N in the untreated group (P = .0072), and there were no significant differences in strength compared with the controls (17.8 +/- 2.6 N) (P > .05). CONCLUSIONS The remodeling of ADM grafts placed in rat rotator cuff tendon defects was accelerated by the local administration of FGF-2. CLINICAL RELEVANCE The application of FGF-2 may result in improved histologic characteristics and biomechanical strength in ADM graft constructs in humans.

Reconstruction of large rotator-cuff tears with acellular dermal matrix grafts in rats

HYPOTHESIS With the acellular dermal matrix (ADM), it may be possible to bridge large rotator cuff tears and induce tendon regeneration. MATERIALS AND METHODS A 3 x 5 mm defect of the rotator cuff was created on both shoulders of adult male Sprague-Dawley rats. The graft group (n = 15) underwent reconstruction of the rotator cuff defect with an ADM patch graft; in the defect group (n = 15) no repair was performed. We sacrificed 5 rats from each group at 2, 6, and 12 weeks after surgery and harvested both shoulders; 3 specimens were subjected to histological analysis and the other 7 specimens were used for biomechanical testing. The controls were 5 unoperated rats; they were sacrificed to obtain 3 histologic and 7 biomechanical control shoulder specimens. RESULTS At each time points, the graft group had significantly higher modified tendon maturing scores than the defect group (p < 0.002); specimens from the graft group demonstrated a greater mean ultimate force to failure than those from the defect group (p < 0.05). Within 12 weeks, the ADM graft was histologically incorporated into a structure resembling control specimen; the mean ultimate force to failure in control was significantly greater than in specimens from both groups (p < 0.01). DISCUSSION Although the defect was restored in the defect group, histologically and biomechanically specimens from the defect group were inferior to the graft group. CONCLUSION ADM grafts were useful as a scaffold in the reconstruction of large rotator cuff defects in rats. LEVEL OF EVIDENCE Basic science study.

Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver.

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.

Differential distribution and expressions of collagens in the cerebral aneurysmal wall

Abstract To investigate the role of collagens in the formation and rupture of cerebral aneurysms, we examined the distribution and synthesis of vascular collagens in the wall of normal human cerebral main trunks and of cerebral aneurysms using immunohistochemistry and in situ hybridization techniques. Fifteen cerebral aneurysmal walls were resected at operation; control cerebral main trunks were obtained from seven autopsy cases. Semiserial sections from the specimens were subjected to immunofluorescence and immunohistochemical staining with antibodies to collagen types I, III, IV, V, VI, desmin and α-smooth muscle actin. In addition, type III collagen mRNA was examined by in situ hybridization. Immunohistochemical study showed that all collagen types were grossly preserved in the aneurysmal wall, although the distribution patterns were different for each collagen. The distribution of major fibrillar collagen types I and III was more diffuse and homogeneous in the luminal layer of the aneurysmal wall than the media of the control artery, although the intensity of immunohistochemical staining was weaker in the abluminal layer of the aneurysmal wall than the adventitia of the control artery. Collagen types IV and V were distributed more sparsely in the luminal layer of the aneurysmal wall than the media of the control artery. Collagen type VI was noted in the luminal as well as the abluminal layer of the aneurysmal wall, whereas it was located exclusively in the adventitia of the control artery. In situ hybridization showed that the signal for collagen type III mRNA on fibroblastic and smooth muscle cells was higher in the aneurysmal walls than the control arteries, suggesting up-regulation of type III collagen transcription in the cerebral aneurysmal wall. The study of the distribution and synthetic regulation of various types of collagen in the aneurysmal wall may be essential for understanding the formation of the aneurysmal wall and its protection against enlargement or rupture.

Differential expression of CCAAT enhancer binding protein family in rat alveolar epithelial cell proliferation and in acute lung injury.

Abstract Although alveolar reorganization after acute lung injury depends on regeneration of alveolar epithelial cells, there is little knowledge of regulation of pulmonary healing process. Transcription factors may play key roles in this regulation. To investigate whether the CCAAT enhancer binding protein (C/EBP) family, α, β, and δ, were involved in alveolar reorganization after injury, we examined expression of C/EBP proteins and mRNAs in lung injuries induced by lipopolysaccharide (LPS) or bleomycin (Bleo) and in cell proliferation by keratinocyte growth factor (KGF). By immunohistochemistry, we demonstrated that C/EBPα and C/EBPβ were expressed in alveolar type II cells and alveolar macrophages, but C/EBPδ was expressed restrictedly in some of alveolar type II cells in a spatial pattern in the control lungs. Further, these three C/EBP family members were differentially expressed in alveolar cell proliferation and in acute lung injury, in which, interestingly, C/EBPα and C/EBPδ were reciprocally expressed in alveolar type II cell proliferation and in pulmonary fibrosis. However, expressions of their mRNAs by in situ hybridization were dramatically increased in the affected lesions of the lungs by LPS and Bleo, and Northern blot analysis showed an increased abundance of the mRNA for C/EBPβ in LPS-treated lungs and for C/EBPδ in Bleo-treated lungs, compared with those in the control lungs. Thus, differential expression of the C/EBP family may be required to maintain and reorganize the basic integrity of alveolar structure during pathological states, which suggests an important role for the C/EBP family in maintaining normal alveolar architecture and function and in repairing the damaged epithelium after injury.

Loss of alveolar basement membrane type IV collagen α3, α4, and α5 chains in bronchioloalveolar carcinoma of the lung

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct α(IV) chains. This study investigated for the first time the expression of these six α(IV) chains immunohistochemically, using α(IV) chain‐specific monoclonal antibodies, in normal lung and in small (less than 2 cm in diameter) adenocarcinoma of the lung with a bronchioloalveolar growth pattern at the periphery. Small adenocarcinomas were histopathologically classified into three subtypes: bronchioloalveolar carcinoma (BAC) without collapse, BAC with collapse, and adenocarcinoma with bronchioloalveolar features. In normal lung, alveolar BM was composed of α1(IV)/α2(IV) chains and α3(IV)/α4(IV)/α5(IV) chains. In non‐collapsed areas of BAC, alveolar BM was composed of linear α1(IV)/α2(IV) chains and discontinuous α3(IV)/α4(IV)/α5(IV) chains. In collapsed areas of BAC, alveolar BM was composed of linear and thick α1(IV)/α2(IV) chains only, because of the complete loss of α3(IV)/α4(IV)/α5(IV) chains. In invasive areas of adenocarcinoma with bronchioloalveolar features, α1(IV)/α2(IV) chains around the cancer cell nests were disrupted, in addition to the complete loss of α3(IV)/α4(IV)/α5(IV) chains. In conclusion, during the process of stromal invasion of lung adenocarcinoma, type IV collagen of alveolar BM is remodelled from the complete type, composed of α1(IV)/α2(IV)/α3(IV)/α4(IV)/α5(IV) chains, to the incomplete type, composed of only α1(IV)/α2(IV) chains, before the disruption of α1(IV)/α2(IV) chains. These findings may help to clarify the molecular mechanisms of cancer invasion. Copyright

Mice lacking CCAAT/enhancer-binding protein-α show hyperproliferation of alveolar type II cells and increased surfactant protein mRNAs

Abstract. The lung-specific surfactant proteins (SP) are essential for normal respiratory function. Transcription factors may play an important role in the regulation of surfactant proteins. The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors that can stimulate expression of genes in lipid-metabolizing epithelial cells. C/EBPα-deficient mice have been shown to exhibit abnormal pulmonary histopathology. Recently, we demonstrated that C/EBP family members are differentially expressed in alveolar type II cell proliferation and in pulmonary fibrosis. In the present study, to investigate whether the C/EBP family would be involved in the regulation of surfactant proteins, we examined the protein expression of SP-A, and SP-C, and mRNA expression of SP-A, SP-B, and SP-C in the lungs from newborn C/EBPα-deficient mice. Using immunohistochemistry, we demonstrated that positive cells for SP-C, specific to alveolar type II cells, in the lungs were more abundant in the newborn C/EBPα-deficient mice than in control mice, which suggests the hyperproliferation of alveolar type II cells in the lungs of the C/EBPα-deficient mice. In situ hybridization analysis revealed that expression of SP-A, SP-B, and SP-C mRNAs were increased in the lungs of newborn C/EBPα-deficient mice. Northern blot analysis revealed that surfactant protein mRNAs were also increased. Thus, these results suggest that C/EBPα may play a key role in the proliferation of alveolar type II cells and the regulation of genes of surfactant protein.