Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.

OBJECTIVE: Abnormal joint instability contributes to cartilage damage and osteophyte formation. We investigated whether controlling joint instability inhibited chronic synovial membrane inflammation and delayed osteophyte formation and examined the role of transforming growth factor-beta (TGF-ß) signaling in the associated mechanism. DESIGN: Rats (n = 94) underwent anterior cruciate ligament (ACL) transection. Anterior tibial instability was either controlled (CAM group) or allowed to continue (SHAM group). At 2, 4, and 8 weeks after surgery, radiologic, histopathologic, immunohistochemical, immunofluorescent, and enzyme-linked immunosorbent assay examinations were performed to evaluate osteophyte formation and TGF-ß signaling. RESULTS: Joint instability increased cartilage degeneration score and osteophyte formation, and cell hyperplasia and proliferation and synovial thickening were observed in the synovial membrane. Major findings were increased TGF-ß expression and Smad2/3 following TGF-ß phosphorylation in synovial membarene, articular cartilage, and the posterior tibial growth plate (TGF-ß expression using ELISA: 4 weeks; P = 0.009, 95% CI [260.1-1340.0]) (p-Smad2/3 expression density: 4 weeks; P = 0.024, 95% CI [1.67-18.27], 8 weeks; P = 0.034, 95% CI [1.25-25.34]). However, bone morphogenetic protein (BMP)-2 and Smad1/5/8 levels were not difference between the SHAM model and the CAM model. CONCLUSIONS: This study showed that the difference between anterior tibial instability caused a change in the expression level of TGF in the posterior tibia and synovial membrane, and the reaction might be consequently involved in osteophyte formation.

Experience of Lymphangiography as a Therapeutic Tool for Lymphatic Leakage After Kidney Transplantation.

INTRODUCTION: Lymphatic leakage after kidney transplantation is a relatively frequent complication but sometimes resistant to treatment, and there is no fixed treatment algorithm. The effectiveness of therapeutic lymphangiography for postoperative lymphatic or chyle leakage has been reported, but few reports are available regarding patients who have undergone kidney transplantation. In this study, we report our experience with lymphangiography as a therapeutic tool for lymphatic leakage after kidney transplantation. PATIENTS AND METHODS: Intranodal lymphangiography for lymphatic leakage was performed in 4 patients (3 male, 1 female; age range, 38 to 70 years old) after living kidney transplantation at the Osaka City University Hospital in Japan. The amount of drainage before lymphangiography was 169 to 361 mL/day. The procedure for intranodal lymphangiography was as follows: the inguinal lymph node was punctured under ultrasound guidance, and the tip of the needle was instilled at the junction between the cortex and the hilum, after which Lipiodol was slowly and manually injected. RESULTS: Lymphangiography was technically successful in 3 out of the 4 patients. In all successful cases, the amount of drainage decreased and leakage finally stopped without additional therapy such as sclerotherapy or fenestration. In 2 cases, we were able to directly detect the leakage site using lymphangiography. The time between lymphangiography and leakage resolution ranged from 8 to 13 days. There were neither complications of lymphangiography nor recurrence of lymphatic leakage in the successful cases. CONCLUSIONS: Intranodal lymphangiography may be not only a diagnostic tool but also an effective, minimally-invasive, and safe method for treatment of lymphatic leakage resistant to drainage after kidney transplantation.

Clinical Experience of Late Conversion From Antimetabolites With Standard Exposure Calcineurin Inhibitors to Everolimus With Calcineurin Inhibitor Minimization in Stable Kidney Transplant Recipients With Good Renal Function.

INTRODUCTION: This study describes our clinical experience of late conversion from antimetabolites with standard exposure calcineurin inhibitors (CNIs) to everolimus with CNI minimization in stable kidney transplant recipients with good graft function. PATIENTS AND METHODS: A 1-year retrospective pilot study of 26 kidney recipients converted from antimetabolites with standard exposure CNIs to everolimus with CNI minimization was performed. The recipients enrolled in this study had normal or slightly impaired renal function defined as a serum creatinine value <2.0 mg/dL, and normal or slightly increased albuminuria defined as a urinary albumin excretion rate <100 mg/g creatinine. RESULTS: The median time from transplant to conversion was 39.5 months posttransplant (range, 3-275). Treatment with everolimus was stopped owing to adverse events in 11 patients (42.3%). In the analysis of the patients in whom everolimus was maintained, the mean estimated glomerular filtration rate (eGFR) significantly increased from 50.7 ± 11.9 mL/min/1.73 m(2) at baseline to 53.6 ± 13.9 mL/min/1.73 m(2) at 1 year after conversion. In the patients in whom everolimus was stopped during the observation period, there was no difference in eGFR between baseline and 1 year after conversion. CONCLUSIONS: This study demonstrated that, among the patients converted to everolimus at a late stage, there was no deterioration in renal function whether everolimus was maintained or stopped within 1 year after conversion.

Effects of Granulocyte and Monocyte Adsorptive Apheresis in Renal Transplantation Recipients With Concomitant Cytomegalovirus Infection.

BACKGROUND: Granulocyte and monocyte adsorptive apheresis (GMAA) is widely used as a treatment for active ulcerative colitis (UC) in Japan. Much attention has been paid to the possibility of GMAA for the treatment and control of cytomegalovirus (CMV) reactivation in patients with refractory UC and concomitant CMV infection. In this study, the effects of the combination of GMAA and antiviral therapy were examined in renal transplant recipients with concomitant CMV infection. METHODS: Combination therapy of GMAA and antiviral drugs was performed 9 times in 7 renal transplant recipients with concomitant CMV infection. Four of the cases were positive for CMV-IgG, and 3 were negative. The clinical presentation of CMV infection was viremia in 6 cases and disease (CMV retinitis) in 1 case. CMV infection was diagnosed by using an antigenemia assay (C7-HRP). GMAA session was performed once, and the duration of the session was 120 min. Immediately after the GMAA session, ganciclovir was administered at 5 mg/kg/body weight. CMV infection was monitored based on C7-HRP and CMV-DNA in the peripheral blood samples. RESULTS: All cases became negative for C7-HRP and CMV-DNA within 21 days (median, 14 days; range, 3-21 days) and 17 days (median, 6 days; range, 3-17 days), respectively, after starting the combination therapy. No side effects of GMAA were observed. CONCLUSIONS: This case series found that GMAA in combination with antiviral drugs may shorten the duration of treatment against CMV infection in renal transplant recipients. Further studies in a larger number of patients are required to confirm these results.

Investigation of urinary angiotensinogen in renal transplant recipients.

BACKGROUND: Recent studies have indicated that angiotensinogen (AGT) is also locally produced in the kidney and that urinary AGT is a marker of local renal renin-angiotensin system activation. Because urinary AGT levels are significantly higher in patients with chronic kidney disease (CKD) than in patients without CKD and correlate with urinary albumin and other levels, urinary AGT is increasingly recognized as a marker for CKD monitoring, prognosis, and treatment. In this study, we investigated urinary AGT levels in renal transplant recipients. METHODS: Among the patients who were treated as outpatients at the Department of Urology of Osaka City University Hospital from March 2012 to April 2013, 146 stable renal transplant recipients and 50 donors who gave informed consent were studied. Urinary AGT and creatinine (Cr) levels were measured. The urinary AGT-to-Cr ratio was calculated, and its correlation with clinical parameters was examined. RESULTS: The urinary AGT-to-Cr ratio of the renal transplant recipients was significantly higher than that of the renal transplant donors (P = .0143). Furthermore, the urinary AGT-to-Cr ratio had a significantly positive correlation with the urinary albumin-to-Cr ratio (ACR; r = 0.39, P < .0001), while on the other hand, it had a significantly negative correlation with estimated glomerular filtration rate (eGFR; r = -0.31, P = .0002). Multiple linear regression analysis of factors associated with eGFR showed that urinary AGT was a significant and independent factor after adjusting for age, sex, and ACR. CONCLUSIONS: Our results indicated that urinary AGT levels were elevated in renal transplant recipients. In addition, urinary AGT significantly correlated with renal function and degree of albuminuria.

Effects of conversion from a twice-daily tacrolimus to a once-daily tacrolimus on glucose metabolism in stable kidney transplant recipients.

INTRODUCTION: The adverse effects of tacrolimus are known to play major roles in new-onset diabetes after transplantation. The purpose of this study was to investigate the effects of conversion from a twice-daily tacrolimus (Tac-BID) to a once-daily tacrolimus (Tac-OD) on glucose metabolism in stable kidney transplant recipients. PATIENTS AND METHODS: Twenty-six patients were converted from Tac-BID to Tac-OD on a 1:1 mg basis and examined for its effects on glucose metabolism. Unless rejection or tacrolimus toxicity was suspected, we did not perform dose adjustments of Tac-OD or reconversion to Tac-BID until 4 weeks after conversion. Subsequent dose adjustments were allowed to maintain tacrolimus target trough concentration within the. Changes in clinical parameters were compared between baseline and 24 weeks after conversion. RESULTS: Conversion from Tac-BID to Tac-OD on a 1:1 mg basis resulted in a significant decrease in tacrolimus trough level at 4 weeks after conversion. Because dose adjustments were performed, the trough level did not differ significantly between baseline and 24 weeks after conversion. At 4 and 24 weeks after conversion, the homeostasis model assessment of pancreas ß-cell function (HOMA-ß) increased significantly. CONCLUSIONS: Although there was no change in tacrolimus trough level between baseline and 24 weeks after transplantation, HOMA-ß at 24 weeks after conversion was significantly higher than that at baseline. These results indicated that conversion from Tac-BID to Tac-OD may improve pancreas ß-cell function in kidney transplant recipients.

Glucose intolerance is associated with increased intimal-medial thickness of the carotid artery and increased pulse-wave velocity in renal transplant recipients.

BACKGROUND: Although new-onset diabetes after transplantation has been demonstrated to have a significant negative impact on allograft and patient survival, the role of glucose intolerance (impaired fasting glucose [IFG] and/or impaired glucose tolerance [IGT], as asymptomatic hyperglycemia and borderline diabetes, has not been identified in renal transplant recipients. METHODS: We enrolled 32 renal transplant recipients (at least 1 year after transplantation) without prior evidence of diabetes at our institution in this study. Transplant recipients were divided into 2 groups (normal glucose tolerance group and glucose intolerance group) according to the results of their oral glucose tolerance test with 75 g of glucose. Glucose intolerance included IFG, IGT, and IFG/IGT. Normal glucose tolerance was detected in 19 patients, and glucose intolerance in 13: had 6 IGT, 2 IFG, and 5 IGT/IFG. Bilateral brachial-ankle pulse-wave velocity (baPWV) and intimal-media thickness (IMT) measured as markers of atherosclerosis were compared between the 2 groups. Insulin resistance was estimated with the homeostasis model assessment of insulin resistance (HOMA-R), and pancreatic ß-cell function evaluated by the homeostasis model assessment of ß-cell function and insulinogenic index. RESULTS: The patients in the glucose intolerance group showed significantly greater baPWV and IMT than those in the normal glucose tolerance group. HOMA-R in the glucose intolerance patients was significantly higher than in the normal glucose tolerance patients. Linear regression analysis showed the increased IMT in the renal transplant recipients to be significantly correlated with HOMA-R. CONCLUSIONS: Renal transplant recipients with glucose intolerance had increased IMT and baPWV, suggesting that glucose intolerance in renal transplant recipients may induce atherosclerosis and that the rise in insulin resistance may contribute to the increased IMT in renal transplant recipients.

Insulin resistance and insulin secretion in renal transplant recipients with hepatitis C.

BACKGROUND: Several reports have suggested an association between hepatitis C virus (HCV) infection and new-onset diabetes after transplantation (NODAT). NODAT is a common complication after renal transplantation, and it has been associated with increased long-term morbidity and mortality. HCV-positive recipients may have abnormal glucose metabolism, even though NODAT has never been previously diagnosed. The aim of this study was to analyze the pathogenic factors responsible for glucose metabolism in a series of HCV-positive renal transplant recipients. METHODS: The study population comprised 16 renal transplant patients who received their grafts from deceased or living donors with anti-HCV antibodies. HCV-negative transplant recipients were individually matched with these HCV-positive recipients by year of transplantation, sex, age, serum creatinine levels, and type of calcineurin inhibitors. None of the patients had been diagnosed with diabetes. Insulin secretion and insulin resistance were determined by a 75-g oral glucose tolerance test (OGTT) and compared between the 2 groups. Categories of glucose tolerance were defined according to World Health Organization criteria. RESULTS: Glucose intolerance (impaired fasting glucose, impaired glucose tolerance, diabetes mellitus) as assessed by OGTT was detected in 7 of the HCV-positive recipients (43.8%) and 3 of the HCV-negative recipients. The homeostasis model assessment of insulin resistance was greater in the HCV-positive recipients than in the HCV-negative recipients. The homeostasis model assessment of ß-cell function was higher in the HCV-positive recipients than in the HCV-negative recipients. CONCLUSIONS: The frequency of glucose intolerance tended to be higher in HCV-positive recipients. Furthermore, insulin resistance was greater and insulin secretion higher in HCV-positive recipients, which indicated that the increase in insulin secretion compensated for insulin resistance observed in these patients. However, HCV-positive renal transplant recipients may ultimately develop NODAT as this compensation diminishes with time.

Neuroanatomical technique for studying long axonal projections in the central nervous system: combined axonal staining and pre-labeling in parasagittal gerbil brain slices.

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system.

Conversion of stable kidney transplant recipients from a twice-daily prograf to a once-daily tacrolimus formulation: a short-term study on its effects on glucose metabolism.

BACKGROUND: The adverse effects of tacrolimus are known to play major roles in posttransplantation diabetes mellitus (PTDM). In the present study, we investigated the effects of conversion from a twice-daily (Tac BID) to a once-daily prolonged release of tacrolimus formulation (Tac OD) on glucose metabolism in stable kidney transplant recipients. PATIENTS AND METHODS: In this prospective study, 26 patients converted from Tac BID to the same milligram-milligram daily dose of Tac OD were examined for the effects on renal function, drug trough levels, and glucose metabolism over a 4-week period. RESULTS: Conversion from Tac BID to Tac OD on a 1:1 mg basis resulted in a significant decrease in tacrolimus trough levels, but no significant changes in renal function. At 4 weeks after conversion, a homeostasis model assessment of ß-cell function, and hemoglobin A1c (HbA1c) decreased significantly. CONCLUSIONS: This study demonstrated a significant reduction in tacrolimus trough levels after switching from Tac BID to Tac OD, which increased insulin secretion and decreased HbA1c, suggesting that it may decrease the frequency of PTDM among stable renal transplant recipients.

Excellent outcomes of ABO-incompatible kidney transplantation: a single-center experience.

INTRODUCTION: Due to the severe shortage of deceased donors in Japan, ABO-incompatible living donor kidney transplantation has been performed since the late 1980s. Excellent long-term outcomes have been achieved; the rates of graft survival among these patients are currently similar to those of recipients of ABO-compatible grafts. Our single-center experience describing the immunosuppressive protocols, complications, and grafts survivals is documented in this study. PATIENTS AND METHODS: Among 123 patients with end-stage renal disease who underwent living donor kidney transplantation between January 1999 and December 2010, 25 cases were ABO-incompatible grafts. All of these patients were followed until August 2011. Analyzing these patients, we focused on their immunosuppressive protocols, complications, and graft survivals. RESULTS: Patient and graft survival rates were 100%. One patient experienced antibody-mediated rejection and an intractable acute cellular rejection episode, 1 patient an antibody-mediated rejection, and 6 patients had acute cellular rejection episodes. However, there were no severe complications. CONCLUSION: Although ABO-incompatible kidney transplantation is a high-risk procedure, a short-term graft survival rate of 100% may be expected due to recent significant improvements in desensitization and recipient management.

Desensitization protocol in highly HLA-sensitized and ABO-incompatible high titer kidney transplantation.

BACKGROUND: A positive crossmatch indicates the presence of donor-specific alloantibodies and is associated with a graft loss rate of >80%; anti-ABO blood group antibodies develop in response to exposure to foreign blood groups, resulting in immediate graft loss. However, a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation has not yet been established. METHODS: We treated 6 patients with high (≥1:512) anti-A/B antibody titers and 2 highly HLA-sensitized patients. Our immunosuppression protocol was initiated 1 month before surgery and included mycophenolate mofetil (1 g/d) and/or low-dose steroid (methylprednisolone 8 mg/d). Two doses of the anti-CD20 antibody rituximab (150 mg/m(2)) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6-12 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation, combined with 2 doses of basiliximab. RESULTS: Of the 8 patients, 7 subsequently underwent successful living-donor kidney transplantation. Follow-up of our recipients showed that the patient and graft survival rates were 100%. Acute cellular rejection and antibody-mediated rejection episodes occurred in 1 of the 7 recipients. CONCLUSIONS: These findings suggest that our immunosuppression regimen consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression may prove to be effective as a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation.

[Myers technique and patch enlargement of ascending aorta for diffuse supravalvular aortic stenosis].

A 6-year-old boy was diagnosed with aggravation of diffuse supravalvular aortic stenosis (SVAS). The pressure gradient between the sinus of Valsalva and ascending aorta was 48 mmHg. The diameter of the sino-tubular junction was 7 mm and the ascending aorta was hypoplastic. We performed Myers operation because 3 sinus reconstructions had resulted in superior hemodynamics and reductions in both mortality rate and need for reoperation. We avoided using autologous pericardium because of the possibility of shrinkage and aneurysm. We could easily perform patch enlargement of the ascending aorta by selective cerebral perfusion. The postoperative course was excellent and there was no SVAS or aortic regurgitation (AR). A catheterization showed the pressure gradient was 5 mmHg with trivial AR upon follow-up at 1 year.

[Stenosis of left coronary artery ostium by a hypoplastic cusp].

Occlusion of a coronary ostium due to fusion of the aortic cusp to the aortic wall is a rare but noteworthy anomaly, because it may cause a sudden death. We report a 9-year-old girl with severe stenosis of the left coronary ostium by the aortic cusp that fused to the aortic wall. The left coronary flow was restored by excision of the adherent left aortic cusp, and aortic valve replacement was performed with the technique of bilateral enlargement of the aortic valve ring (Yamaguchi's method) to prevent prosthesis-patient mismatch in the future. The patient had an uneventful recovery and was discharged 14th postoperative day (POD). Postoperative angiography showed no coronary ostial stenosis.

[Primary biventricular repair for interrupted aortic arch with left ventricular outflow tract obstruction and tricuspid valve regurgitation; report of a case].

An interrupted aortic arch was diagnosed in a 10-day-old girl weighing 3.3 kg, as was perimembranous ventricular septal defect (VSD) and severe tricuspid valve regurgitation (TR). The subaortic diameter was 3.6 mm and the aortic valve (3.7 mm in diameter) was bicuspid. We chose definitive repair, modified Yasui procedure, because of severe TR and no straddling of mitral valve. In primary biventricular repair, we undertook extended aortic arch anastomosis. Left ventricular outflow tract reconstruction consisted of intracardiac rerouting from the VSD to the pulmonary artery by using expanded-polytetrafluoroethylene (ePTFE) and Damus-Kaye-Stansel (DKS) anastomosis. Right ventricular outflow tract reconstruction was performed by the Rastelli procedure with an ePTFE valved conduit. Moreover, we carried out semicircular annuloplasty for severe TR.

[Sandwich technique for closing of the large muscular Swiss-cheese type ventricular septal defects].

A 9-year-old girl, who has had a diagnosis as a Taussig-Bing anomary, underwent an original Jatene procedure (o-J) 2 months after birth. This time, she had a diagnosis of the muscular multiple ventricular septal defects (mVSD) and pulmonary stenosis after o-J. The mVSD was Swiss-cheese type and was large from the proximal of the infundibular septum to the apex and posterior of the septum. It was closed by the sandwich technique using a pair of felt patches, which of one was placed at right ventricular side and the other was at left ventricular side, slightly larger than the whole area of the mVSD. The patch fixation was placed with 1 stitch at the center between the patches and a few stitches around the right ventricular side patch to the ventricular septum. Postoperative cardiac function was uneventful regardless of the very large patches placed at the ventricular septum and the cardiac catheterization after 1 year postoperatively showed the pulmonary/systemic blood flow ratio was 1.0. This technique for the closure of the large Swiss-cheese type mVSD can be considered to be very effective.

[Successful staged repair of a symptomatic neonate of tetralogy of Fallot with severe absent pulmonary valve syndrome].

Tetralogy of Fallot with absent pulmonary valve has been classified to a few groups. The most severe one is characterized by symptomatic onset immediately after birth. The others are no or slightly symptomatic at least during their neonatal period. In a severely symptomatic 12-day-old neonate of tetralogy of Fallot with absent pulmonary valve who had required intubation immediately after birth, tight pulmonary banding and left side modified Blalock-Taussig shunt were performed on emergency basis. Consequently, prior massive pulmonary regurgitation was decreased significantly. Forty-five days after this first stage operation, he weaned from respiratory management. At 1-year-old, radical repair based on conotruncal repair, which consisted of patch closure of ventricular septal defect preserving the tricuspid septal leaflet function, resection of anterior wall of enlarged left pulmonary artery, and right ventricular outflow tract reconstruction using autologous tissue and a pericardial patch was performed. Bicuspid pulmonary valve, posterior one of procured autologous pulmonary wall and anterior one of polytetrafluoroethylene (PTFE) respectively, was created to minimize deterioration of the pulmonary insufficiency. Although postoperative cardiac function was kept feasible showing his central venous pressure of 7 mmHg in the main, postoperative general course was eventful especially regarding the respiratory function. The patient was weaned from the prolonged ventilator management 5 months after this radical repair eventually. Generally, to diminish the massive pulmonary regurgitation in early lifetime period could reduce a progressive airway obstruction and minimize pulmonary tissue damage. However, even after the total correction in this case, considerable peripheral segmental pulmonary obstructive lesions were persistent according to the perfusion lung scanning with 99mTc macroaggregated albumin and 99mTechnegas ventilation lung scanning studies. This persistent, supposed to be innate, pulmonary obstructive lesions might prevent ordinal recovery after cardiac radical repair for this most severe subtype of absent pulmonary valve syndrome.

[Valvuloplasty for mitral regurgitation immediately following Kawasaki disease without abnormal coronary arteries lesion; report of a case].

It is not uncommon that valve disease is complicated with Kawasaki disease (KD). However, it is rare to show normal coronary arteries simultaneously. We experienced a case of valvuloplasty towards the mitral regurgitation (MR) followed immediately after KD showing normal coronary arteries. A 3 year-old-female, with a diagnosis of KD at 4 months after birth, was referred to our hospital 5 months after birth. The echocardiography detected a moderate MR. The preoperative catheterization at 2.5 years old showed grade III MR, enlargement of left atrium and left ventricle, pulmonary capillary wedge pressure (PCWP) = 12 mmHg, left ventricular ejection fraction (LVEF) = 675, and normal coronary arteries. Pulmonary hypertention was not revealed. The operative findings showed mitral valve prolapse due to the elongation of the chordae of the anterior leaflet. She underwent artificial chordal reconstruction using expanded polytetrafluoroethylene sutures and mitral annuloplasty by Kay-Reed method. The postoperative course was uneventful, and she was discharged on postoperative day 19.

Intracellular and extracellular labeling of neurons in fresh slice preparations combined with multiple tract tracing.

We describe methods for studying axo-dendritic projections, one of the forms of neural connection involved in the complex circuits of the central nervous system, including brainstem auditory pathways. This form of neural connection is often difficult to visualize by conventional tract tracing techniques. Retrogradely identified cells were filled intracellularly with a mixture of fluorescent Lucifer yellow and nonfluorescent HRP in live slice preparations to reveal the detailed morphological features of these cells with special attention to the distal dendrite that may receive projections from suspected or known input axons. Extracellular or intracellular labeling of cells with axons that project to the distal dendrite of the identified cells was accomplished in the same live slice preparation. Using a live slice rather than a fixed slice allows accurate, visually controlled placement of anterograde tracer, which requires living axons for transport, into the source of input to the identified cells within the slice. Live slices also permit one to characterize the identified cells electrophysiologically. Intracellular labeling of cells in a potential source of local input to the identified cells also provides conclusive information concerning with connections of the cells involved.