Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5.

OBJECTIVE: In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). DESIGN: In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. RESULTS: In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: -51.7, -37.1], 51.5% [-80.5, -22.5], 62.3% [-96.6, -28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8, -8.6]) further with IL-1ß (44.5% [-36.9, -23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8, -5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9, -0.8]), histological damage ([-5.5, -0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d. CONCLUSIONS: This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Expressions of conventional vitellogenin and vitellogenin-like A in worker brains are associated with a nursing task in a ponerine ant.

In eusocial insect colonies, non-reproductive workers often perform different tasks. Tasks of an individual worker are shifted depending on various factors, e.g., age and colony demography. Although a vitellogenin (Vg) gene play regulatory roles in both reproductive and non-reproductive division of labours in a honeybee, it has been shown that the insect Vg underwent multiple gene duplications and sub-functionalisation, especially in apical ant lineages. The regulatory roles of duplicated Vgs were suggested to change evolutionarily among ants, whereas such roles in phylogenetically basal ants remain unclear. Here, we examined the expression patterns of conventional Vg (CVg), Vg-like A, Vg-like B and Vg-like C, as well as Vg receptor, during the task shift in an age-dependent manner and under experimental manipulation of colony demography in a primitive ant Diacamma sp. Expressions of CVg and Vg-like A in a brain were associated with a nursing task. It is suggested that associations of brain expressions of these Vgs with worker tasks were acquired in the basal ant lineage, and that such Vg functions could have sub-functionalised in the derived ant lineage.

NF-kB decoy oligodeoxynucleotide preserves disc height in a rabbit anular-puncture model and reduces pain induction in a rat xenograft-radiculopathy model.

While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes' expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the "pain sensor" nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

A vasodilating ß1 blocker celiprolol inhibits muscular release of uric acid precursor in patients with essential hypertension.

Although nonvasodilating ß1 blockers increase the levels of uric acid in serum, it is not known whether vasodilating ß1 blockers have a similar effect. In the present study, we evaluated the effect of celiprolol on the release of hypoxanthine, a uric acid precursor, from muscles after an exercise. We used the semi-ischemic forearm test to examine the release of lactate (ΔLAC), ammonia (ΔAmm), and hypoxanthine (ΔHX) before and 4, 10, and 60 min after an exercise in 18 hypertensive patients as well as 4 normotensive subjects. Before celiprolol treatment, all the levels of ΔHX and ΔAmm, and ΔLAC were increased by semi-ischemic exercise in hypertensive patients, and the increases were remarkably larger than those in normotensive subjects. Celiprolol decreased both systolic and diastolic pressure. It also decreased the levels of ΔHX and ΔAmm without changes in ΔLAC after an exercise. These findings also were confirmed by summation of each metabolite (ΣΔMetabolites). Celiprolol caused a marginal decrease of serum uric acid, but the difference was not statistically significant. On the other hand, nonvasodilating ß1 blockers did not suppress the levels of ΔHX and ΔAmm, whereas they significantly increased ΔLAC after an exercise. Celiprolol improved energy metabolism in skeletal muscles. It suppressed HX production and consequently did not adversely affect serum uric acid levels.

The gamete fusion process is defective in eggs of Cd9-deficient mice.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans.

AIMS: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. METHODS AND RESULTS: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD(50) value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. CONCLUSIONS: The silkworm-C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.

Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

Spatiotemporal Ca2+ signalling in the cytoplasm is currently understood as an excitation phenomenon by analogy with electrical excitation in the plasma membrane. In many cell types, Ca2+ waves and Ca2+ oscillations are mediated by inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channels in the endoplasmic reticulum membrane, with positive feedback between cytosolic Ca2+ and IP3-induced Ca2+ release creating a regenerative process. Remarkable advances have been made in the past year in the analysis of subcellular Ca2+ microdomains using confocal microscopy and of Ca2+ influx pathways that are functionally coupled to IP3-induced Ca2+ release. Ca2+ signals can be conveyed into the nucleus and mitochondria. Ca2+ entry from outside the cell allows repetitive Ca2+ release by providing Ca2+ to refill the endoplasmic reticulum stores, thus giving rise to frequency-encoded Ca2+ signals.

Respiratory Sarcopenia and Sarcopenic Respiratory Disability: Concepts, Diagnosis, and Treatment.

The condition of muscle fiber atrophy and weakness that occurs in respiratory muscles along with systemic skeletal muscle with age is known as respiratory sarcopenia. The Japanese Working Group of Respiratory Sarcopenia of the Japanese Association of Rehabilitation Nutrition narratively reviews these areas, and proposes the concept and diagnostic criteria. We have defined respiratory sarcopenia as "whole-body sarcopenia and low respiratory muscle mass followed by low respiratory muscle strength and/or low respiratory function." Respiratory sarcopenia can be caused by various factors such as aging, decreased activity, undernutrition, disease, cachexia, and iatrogenic causes. We have also created an algorithm for diagnosing respiratory sarcopenia. Respiratory function decreases with age in healthy older people, along with low respiratory muscle mass and strength. We have created a new term, "Presbypnea," meaning a decline in respiratory function with aging. Minor functional respiratory disability due to aging, such as that indicated by a modified Medical Research Council level 1 (troubled by shortness of breath when hurrying or walking straight up hill), is an indicator of presbypnea. We also define sarcopenic respiratory disability as "a disability with deteriorated respiratory function that results from respiratory sarcopenia." Sarcopenic respiratory disability is diagnosed if respiratory sarcopenia is present with functional disability. Cases of respiratory sarcopenia without functional disability are diagnosed as "at risk of sarcopenic respiratory disability." Functional disability is defined as a modified Medical Research Council grade of 2 or more. Rehabilitation nutrition, treatment that combines rehabilitation and nutritional management, may be adequate to prevent and treat respiratory sarcopenia and sarcopenic respiratory disability.

Amyloid Signature Proteins in Feline Amyloidosis.

In human amyloidoses, amyloid signature proteins (ASPs), such as serum amyloid P component (SAP) and apolipoprotein E (ApoE), are deposited in tissues together with amyloid fibrils and are implicated in the pathogenesis of amyloidosis. Few reports describe ASPs in animals. In this study, we examined feline amyloidosis and performed immunohistochemical and proteomic analyses of SAP, ApoE, apolipoprotein A-I (ApoAI) and apolipoprotein A-IV (ApoAIV). Ten cases of systemic amyloidosis, three cases of amyloid-producing odontogenic tumour and three cases of islet amyloidosis were used for immunohistochemistry (IHC) and/or proteomic analyses. IHC showed that ApoE was present in amyloid deposits in all samples. ApoAI and ApoAIV differed in the degree of co-deposition with amyloid depending on the type of amyloid and the affected organ. SAP was negative in all amyloid deposits. Proteomic analysis showed that ApoE was present in all samples, but ApoAI and ApoAIV were detected only in some samples and SAP was not detected in any samples. The observation that ApoE was detected in all types of amyloid suggests the involvement of ApoE in the development of feline amyloidosis. ASPs in feline amyloidosis are significantly different from those in human amyloidosis, suggesting that the involvement of ASPs in the pathological condition differs between animal species.

Inositol 1,4,5-trisphosphate-induced calcium release and guanine nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs.

Periodic increases in intracellular free calcium occur upon fertilization of golden hamster eggs (Miyazaki et al. 1986. Dev. Biol. 118:259-267). To investigate the underlying mechanism, inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides were microinjected into the egg while Ca2+ transients were monitored by aequorin luminescence and/or hyperpolarization in the membrane potential, which indicates the exact timing and spatial distribution of the Ca2+ rise. Injection of IP3 induced an immediate Ca2+ transient of 13-18 s in the entire egg. The critical concentration of IP3 was 80 nM in the injection pipette (2 nM in the egg, assuming uniform distribution); the effect was all-or-none. The Ca2+ rise occurred even in Ca-free external medium. Injection of 5 mM GTP or 0.33 mM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) (calculated intracellular concentration, 200 or 12 microM, respectively) caused a similar Ca2+ transient with a delay of 160-200 s. More than 50 microM GTP gamma S produced recurring and attenuating Ca2+ transients in a local area of the cytoplasm, with an initial delay of 25-40 s and intervals of 45-60 s. In Ca-free medium the first one to two Ca2+ transients occurred but succeeding ones were absent. Preinjection of guanosine-5'-O-(2-thiodiphosphate) inhibited the occurrence of both GTP gamma S-induced and sperm-induced Ca2+ transients in a dose-dependent manner. Neither pertussis nor cholera toxins had effect. It was proposed that sperm-egg interaction activates a GTP-binding protein that stimulates production of IP3, causing the first one to two Ca releases from internal stores, and also stimulates a pathway for elevation of Ca2+ permeability in the plasma membrane, thereby sustaining the repeated Ca2+ releases.

Development of excitability in embryonic muscle cell membranes in certain tunicates.

During the course of development of muscle cells in certain tunicates, a sign of regenerative membrane response appears in the gastrula stage. In the early tadpole larva, the action potential consists of a spike followed by a plateau. The latter-disappears in fully differentiated cells, conceivably in association with the establishment of delayed rectification.

Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane.

Ionic dependence of regenerative responses of the embryonic cell mnembrane has been studied successively at each stage of development from the unfertilized egg to the differentiated striated muscle in the tadpole larva of the tunicate. The unfertilized egg cell itself showed a type of regenerative response dependent on both sodium and calcium ions, while the spike potentials exclusively dependent on calcium ions were elicited in the differentiated muscle cell.

Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs.

The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2+ release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca(2+)-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.

The effects of seasonal and interannual variability of oceanic structure in the western Pacific North Equatorial Current on larval transport of the Japanese eel Anguilla japonica.

As the North Equatorial Current (NEC)-bifurcation is known to be related to El Niño-Southern Oscillation (ENSO) events, the influence of the position of the NEC bifurcation on transport success of the larval Japanese eel Anguilla japonica was investigated. Using a Lagrangian modelling approach, larval transport was simulated and the relative influence of El Niño and La Niña events and the NEC-bifurcation position on the success of particle transport analysed. The number of particles transported from the NEC to the Kuroshio tended to be lowest during El Niño years, and differences between La Niña and regular years were small. The transport success observed in simulations showed some relationships to annual A. japonica glass eel recruitment to Tanegashima Island over 1993 to 2001, but not in 2002. The study shows that particle tracking simulations can be used to improve knowledge of the oceanic migration of A. japonica but further studies are required, including comparisons with the effects on larval survival of fluctuations in temperature and food availability.

[Simulation of thoracoscopic esophagectomy for esophageal cancer by three-dimensional computed tomography and usefulness for preservation of bronchial arteries].

Thoracoscopic esophagectomy for esophageal cancer was simulated preoperatively by 3-dimensional computed tomography (3D-CT). The anatomical structures such as major vessels, bone, trachea and bronchi, esophagus, lymph nodes, and broncial arteries were extracted from multi-detector row CT scanning and integrated to build the virtual operative field by multi-volume fusion. The virtual thoracoscopy was helpful to understand the location of lymph nodes and bronchial arteries in relation to the adjacent anatomical structures. Preservation of bronchial arteries is important to avoid tracheobronchial ischemia which is a fatal complication in salvage esophagectomy after definitive chemoradiotherapy. 3D-CT revealed anatomical variations of bronchial arteries and was useful for identification and preservation of bronchial arteries in thoracoscopic esophagectomy.

[Perioperative management and prevention of complication for salvage esophagectomy after definitive chemoradiotherapy].

Salvage esophagectomy has been increasing as a second-line treatment after failure of definitive chemoradiotherapy (CRT) for esophageal cancer. A number of patients who have received CRT, especially those who have residual tumors and shorter waiting times until operation, have developed malnutrition and problems in their immunologic condition because of decreasing oral intake and bone marrow suppression. Because high-dose radiation causes inflammation, fibrosis and peripheral circulatory disturbance of various tissues in the treatment fields, salvage surgery can be a technically difficult operation. In our previous experience, postoperative complications of salvage esophagectomy are more frequent and more serious than that of planned esophagectomy. For example, we have experienced necrosis of the reconstructed gastric tube, esophago-tracheal fistula, mediastinal abscess, hard-to-treat acute respiratory distress syndrome (ARDS) and so on. In particular, respiratory tract necrosis or perforation is the most critical complication and frequently becomes lethal. Patients who undergo a salvage esophagectomy have a significantly higher risk of pulmonary and cardiac complication, and have high rates of repeated surgery. Rapid diagnosis and appropriate treatment of complications are necessary to reduce postoperative mortality. To improve the overall outcome, it is very important to better understand the condition of patients after CRT, so appropriate surgery can be carefully planned. Furthermore, it is absolutely essential to perform the operation with great care and to meticulously manage the perioperative care for salvage esophagectomy.