Analysis of circulating microRNA during early gestation in Japanese black cattle.

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals.

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.

Coupling between the photo-excited cyclic π system and the 4f electronic system in a lanthanide single molecule magnet.

A new type of electronic interaction which couples two angular momenta, i.e. the angular momentum of a localized 4f system (J) and an orbital angular momentum generated in a cyclic π conjugated system by irradiation with a circularly-polarized light, has been identified in a lanthanide single molecule magnet.

Continuous Vocalization during Kendo Exercises Suppresses Expiration of CO2.

One distinctive trait of kendo, the Japanese martial art of fencing, is the execution of sustained, high-effort vocalizations during actions. The purpose of this study was to determine the effect of these vocalizations on respiratory functions. First, the intensity of 3 kendo exercises was quantified by measuring oxygen uptake (V̇O2) and comparing it with V̇O2max measured during treadmill tests of 8 university kendo athletes. Respiratory variables of these 8 athletes were then analyzed using a portable breath gas analyzer during the most intensive kendo exercise, kakari-keiko, with and without vocalization. Breathing frequency (fB) increased regardless of vocalization, but in trials with vocalization, fB and ventilation were significantly lower, and expiration time was significantly longer. Components of expired gases were also affected by vocalization. Although there was no significant difference in oxygen uptake, vocalization yielded a reduction in carbon dioxide output (V̇CO2) and an increase in fraction of end-tidal carbon dioxide (FetCO2). We thus conclude that these vocalizations greatly affect expiration breathing patterns in kendo. Moreover, repetition of kakari-keiko caused a reduction in V̇CO2 and an increase in FetCO2 and CO2 storage. We consider the possibility that the sustained high-effort vocalizations of kendo also increase cerebral blood flow.

Effect of reduced coenzyme Q10 (ubiquinol) supplementation on blood pressure and muscle damage during kendo training camp: a double-blind, randomized controlled study.

AIM: This randomized controlled study was designed to examine the effects of reduced coenzyme Q10 (ubiquinol; CoQ10) supplementation on blood pressure (BP) and exercise-induced muscle damage in kendo athletes during a 4-day kendo training camp. METHODS: In a double-blinded manner, 32 young kendo athletes were randomly assigned to supplement with either placebo or CoQ10 (600 mg) daily for 11 days from 1 week prior to camp to end of camp. BP was measured every morning after waking up during the training camp. Blood samples were taken at 3 time points; 1 week and 1 day prior and upon completion of training camp at 17:30. Statistical analysis was performed by repeated-measures analysis of variance followed by Bonferroni/Dunn post-hoc tests. RESULTS: Before the training camp started, there were no differences in diastolic BP between these groups. However, after kendo training started, diastolic BP in the CoQ10 group was significantly lower than that in the placebo group (P<0.05). Plasma creatine kinase (CK) and myoglobin (Mb) concentrations were significantly increased in both groups during the camp (P<0.05), whereas there were no significant differences in CK and Mb between CoQ10 and placebo groups (CK: P=0.82, Mb: P=0.69). CONCLUSION: Oral supplementation with reduced form of CoQ10 (ubiquinol; Kaneka QHTM) showed a significant hypotensive effect in young male kendo athletes during a 4-day kendo training camp, although it did not significantly ameliorate kendo exercise-induced muscle damage.

Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever.

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor ß (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

Expression of ADAMTS1 mRNA in bovine endometrium and placenta during gestation.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a secreted protease. Through the regulation of extracellular matrix remodeling or developmental processes or both, ADAMTS1 is involved in several biological functions, including ovulation and embryo receptivity. However, the expression and possible role of ADAMTS1 in bovine endometrium is unknown. In this study, we analyzed ADAMTS1 mRNA expression in bovine endometrium during the estrous cycle, peri-implantation period, and at different stages of gestation by using quantitative real-time RT-PCR (qPCR) and in situ hybridization. The qPCR results indicated that the expression of ADAMTS1 mRNA was not affected by the day of the estrous cycle and was similar to cyclic levels on day 35 of gestation; however, the expression was more abundant in cotyledonary tissues of the placenta during late gestation. The in situ hybridization study showed that ADAMTS1 mRNA was detected mainly in uterine luminal epithelia and stromal cells during the estrous cycle and peri-implantation period. A disintegrin and metalloproteinase with thrombospondin motifs 1 mRNA was also expressed in the peri-implantation conceptus as well as in trophoblast cells, which include binucleate cells, and increased during late gestation. Furthermore, treatment of stromal cell with progesterone (300 nM) stimulated the expression of ADAMTS1 mRNA. This study indicates that ADAMTS1 participates in bovine endometrial remodeling, which is required for implantation and placental development in coordination with ovarian steroids.

Dynamic expression of SOLD1 in bovine uteroplacental tissues during gestation.

INTRODUCTION: Secreted protein of Ly-6 domain 1 (SOLD1), a novel member of the Ly-6 superfamily, is present in the extracellular matrix of the mesenchyme in placental cotyledonary villi and is possibly involved in placental construction. OBJECTIVES: We investigated bovine SOLD1 expression in uteroplacental tissues with temporo-spatial patterning throughout gestation. METHODS: Placentomal and endometrial tissues during gestation were analyzed for SOLD1 mRNA levels by using quantitative RT-PCR. Tissue sections of placentomes and intercaruncular endometrium were used for determining SOLD1 mRNA and protein levels respectively with in situ hybridization and immunohistochemistry. RESULTS: SOLD1 mRNA was more strongly expressed in fetal membranes than in endometrial tissues on day 35 of pregnancy, and its expression was maintained throughout pregnancy. SOLD1 mRNA was detected in mononucleate cells at early and mid gestation, and, interestingly, in mononucleate and binucleate trophoblast cells at late gestation. It was also present in endometrial epithelial cells and the stroma surrounding uterine glands. SOLD1 protein was widely distributed in the mesenchyme of the villous tree as the pregnancy progressed. CONCLUSION: Our study shows the temporo-spatial expression patterns of bovine SOLD1 during gestation in uteroplacental tissues. To our knowledge, this is the first report on the involvement of fetal trophoblastic and endometrial cells in the secretion of SOLD1. These results suggest that SOLD1 may play a crucial role not only in the remodeling of the uteroplacental area, but also in that of the endometrium during late gestation, in addition to its role at early gestation in placental construction.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.

Expression of endogenous retrovirus-like transcripts in bovine trophoblastic cells.

UNLABELLED: Endogenous retrovirus envelope elements are considered to participate in trophoblastic cell fusion and multinucleate cell formation in humans, mice, and sheep. However, there is limited information about their roles in the ruminant placenta. OBJECTIVES: We explore and identify the endogenous retrovirus envelope element genes expressed in bovine trophoblasts. METHODS: The NCBI UniGene database (Build #97 Bos taurus) was screened by in silico analysis. After cloning endogenous retrovirus envelope element-like transcript (ERVE), expression profiles were analyzed with quantitative RT-PCR and in situ hybrizaidation. RESULTS: Two UniGene clusters, UniGene ID: Bt.68042 and Bt.85243, were detected, and ERVE-A gene was cloned. Weak expression of this gene was first detected on Day 20 of gestation, and the intensity of its expression increased up to Day 70 of gestation. The intensity of its expression was maintained throughout gestation in the placenta, and its specific expression in trophoblastic binucleate cells was confirmed by in situ hybridization. CONCLUSIONS: bERVE-A has a similar sequence to human syncytin-1, although it lacks an intact envelope sequence, and is specifically expressed in binucleate cells. This is the first evidence that endogenous retrovirus envelope element genes are expressed in bovine binucleate cells.

Temporospatial expression of placental lactogen and prolactin-related protein-1 genes in the bovine placenta and uterus during pregnancy.

The anatomical location of binucleate cells (BNC) influences protein expression but not steroid synthesis in ruminants. In order to determine if BNC in disparate locations differentially express bovine placental lactogen (bPL) and prolactin-related protein-1 (bPRP-1), we quantitated bPL and bPRP-1 transcripts in placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues throughout pregnancy in the bovine using real-time reverse transcription PCR (RT-PCR) and in situ hybridization. Levels of both bPL and bPRP-1 transcripts at peri-implantation were significantly higher (P < 0.01) in the fetal membrane than in caruncular and intercaruncular tissues. Thereafter, mRNA for these related proteins demonstrated different spatial as well as temporal patterns of expression. Levels of bPRP-1 transcripts peaked at day 60 of pregnancy. Between day 60 and 100, bPRP-1 transcripts fell by approximately sevenfold (P < 0.01) in cotyledonary and intercotyledonary tissues, and fourfold in caruncular (P < 0.01) tissue. Levels of bPRP-1 transcripts remained low in the cotyledonary, intercotyledonary, and caruncular tissues until peripartum. In contrast, bPL expression in placentomes increased with progression of gestation (P < 0.01), but decreased in interplacentomal tissue around peripartum. To conclude, disparate patterns of bPRP-1 and bPL genes are transcribed in the placentomal and interplacentomal tissues during gestation in the bovine, suggesting that these prolactin-like hormones serve distinct functions and are regulated differently in the uteroplacental unit in this species.

Cloning and localization of heparanase in bovine placenta.

Heparanase (HPA) degrades heparan sulfate proteoglycan in the extracellular matrix. To understand its role during implantation and placental development in bovine placentae, we cloned and characterized a full-length cDNA encoding bovine HPA and identified HPA localization in placentae. A full-length bovine HPA cDNA was cloned with a 1635 nucleotide open-reading-frame corresponding to a protein of 545 amino acids. The predicted amino acid sequence shares 80.0% and 76.5% identity with human and rat HPA, respectively. In placentomes of 60 and 210 days' gestation, in situ hybridization demonstrated HPA mRNA expression in binucleate cells. Binucleate cells may be a source of HPA throughout gestation in bovine placentae; they may assume specific role(s) in foetal and maternal dialogue. Western blot analysis of bovine placental extracts (day 60) was performed using anti-bovine HPA antibody prepared by immunization of rabbits with synthetic peptide conjugate corresponding to amino acid residues 474-489 of bovine HPA; it showed two immunoreactive proteins with approximate molecular weights of 55kDa and 65kDa. Further, immunofluoresence double staining of HPA and placental lactogen (PL) revealed that binucleate cells expressing HPA had immunoreactivity of PL. These results suggest that HPA is specifically expressed in bovine placental binucleate cells and that it may take migratory roles in placentogenesis for degrading the extracellular matrix.

Expression of prolactin-related protein I at the fetomaternal interface during the implantation period in cows.

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.

Neurohistochemical analysis of regeneration in rat peripheral nerve after end-to-side neurorrhaphy.

We investigated the regenerative capacity of motor nerves repaired by end-to-side or end-to-end neurorrhaphy, using choline-acetyltransferase (ChAT) activity measurement or histological analysis. The right medial gastrocnemius nerves (MGNs) of 62 male Fisher strain rats were transected and divided into three groups. In group 1, the distal ends of the MGN were coapted to the side of the lateral gastrocnemius nerve, using a Y-shaped silicone tube in end-to-side neurorrhaphy. In group 2, the nerve ends were reconnected by the traditional end-to-end technique. In group 3, the nerve ends were separated and remained unrepaired. The MGNs were sampled 1, 2, and 3 months postoperatively for histological examinations and ChAT activity measurement. The medial gastrocnemius muscle (MGM) was also sampled for histological evaluations. Axonal regeneration of MGN and the recovery of MGM to nearly normal histology and weight were observed in groups 1 and 2 3 months postoperatively. Although there were no significant differences in ChAT values between groups 1 and 2, the values were significantly larger than that of group 3 3 months postoperatively. These findings suggested that end-to-side neurorrhaphy would be an alternative treatment for peripheral nerve injury in certain clinical situations.

Expression of heparanase mRNA in bovine placenta during gestation.

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.

Acceleration of thrombomodulin gene transcription by retinoic acid: retinoic acid receptors and Sp1 regulate the promoter activity through interactions with two different sequences in the 5'-flanking region of human gene.

The interactions between retinoic acid- (RA)-dependent transcriptional regulatory sequences of the 5'-untranslated region of the thrombomodulin gene and nuclear RA-responsive proteins were studied using human pancreas BxPC-3 cells. Deletion mutants of pTM-CAT plasmid revealed the presence of distal and proximal RA-responsive regions containing direct repeat with 4 spaces (DR4) and three of four Sp1 sites, respectively. Cotransfection of a pTM-CAT plasmid with expression plasmids of RA receptors (RARalpha, RARbeta, and RARgamma) augmented the promoter activity under the condition of lower retinoid X receptor-alpha (RXRalpha) expression, whereas the activity was greatly diminished when RXRalpha was highly expressed. An electrophoretic mobility shift assay with cDNA containing the DR4 indicated that heterodimers of RAR and RXRalpha interacted with the DR4 site, although the interaction gradually disappeared with the increase in the ratio of RXRalpha/RAR. On the other hand, Sp1 protein interacted especially with the tandem Sp1 site corresponding to the first and second Sp1 sequences of the four Sp1 sites in the proximal RA-responsive region. The binding of Sp1 to Sp1 sites was independent of RAR-RXR heterodimer but increased with the increase in Sp1 concentration in the presence of unknown factor(s) of reticulocyte lysate. Upon treatment of the cells with RA, time-dependent increases in the ratio of RARbeta to RXRalpha and the phosphorylated form of Sp1 were observed. We concluded that two genomic DNA regions, the DR4 site (-1531 to -1516) and the first and second Sp1-binding sites (-145 to -121), were involved in the RA-dependent augmentation of thrombomodulin gene expression through increased interactions of the two regions with heterodimer of RAR-RXRalpha and nuclear Sp1, respectively.

Sensory reconstruction in sensate radial forearm flap transfer.

The authors treated 14 patients (13 men and one woman), using a sensate radial forearm flap. Their ages at operation ranged from 27 to 67 years (mean: 52 years). Preoperative conditions were amputations in 10 cases, degloving injury in three, and crush injury in one. Reconstructive sites involved the thumb in nine cases, the mitten-like hand in two, the index finger in one, the ring and small finger in one, and the palm in one. In all cases, the radial forearm flap, including the lateral antebrachial cutaneous nerve, was harvested. Sensory evaluation was performed using the moving two-point discrimination test (m-2PD). Sensation in the mid-palmar area of 50 forearms was examined in 25 healthy adult volunteers as a control group. Follow-up periods ranged from 12 to 87 months (mean: 39.6 months). The mean m-2PD of the 14 sensory flaps was 13.2 mm, and the mean of 50 forearms in the control group was 18.08 mm. A statistically significant difference was demonstrated between the sensory flaps and the 50 forearms of the control group. The mean m-2PD was much more sensitive in the innervated radial forearm flaps than in the donor forearm. The results suggested that sensory return in the innervated flaps is influenced not by the donor nerve in the flaps, but by the recipient digital nerve.

Flap transfers for the treatment of perichondrial ring injuries with soft tissue defects.

Seven cases (six fresh) of perichondrial ring injury with skin defects were treated using flap transfers. The study included four boys and three girls ranging in age from 2 to 9 years (average 6). They were followed up for an average of 8 years and 10 months. The period from injury to flap coverage was 8-12 days, with an average of 10 days in the fresh cases. Fracture was noted in four cases, with one an epiphyseal fracture. Peroneal flaps were transferred in four cases, latissimus dorsi myocutaneous flaps in two, and gastrocnemius muscle flap in one. Six flaps survived perfectly, and one failed due to venous thrombosis. This latter case was treated with a cross leg flap. Postoperative radiographic assessments confirmed partial growth plate arrest in the chronic case, but all the fresh cases had no postoperative growth disturbance. Flap coverage, for perichondrial ring injuries with wide skin defects, is a useful method not only for skin coverage, but for the prevention of growth disturbances as well.

Free vascularized fibula grafts in surgery of the upper limb.

Twenty patients with intractable diseases in the upper extremity were treated using free vascularized fibula grafts. There were 13 men and seven women. Three patients had traumatic bone defects, five had post-traumatic nonunions, two had congenital pseudoarthroses, seven had defects after tumor resection, and three had other lesions. The reconstructed sites were the humerus in two patients, the radius and/or ulna in 17, and the metacarpal and phalangeal bones in one. The length of the bone defect ranged from 3 to 18 cm (mean: 8.4 cm). Follow-up periods ranged from 6 to 204 months. No patient required additional bone grafts. The mean period required to obtain radiographic bone union was 4.4 months. There were no cases with fractures of the grafted bone, but malunion occurred in four cases. The vascularized fibula graft is indicated in patients with large bone defects or intractable nonunions in the humerus, radius, and/or ulna.