Post-surgical chronic pain and quality of life in children operated for congenital heart disease.

BACKGROUND: Advances in medical technology have resulted in an increased life expectancy in pediatric patients with congenital heart diseases. Assessment of health-related quality of life is crucial to improving their healthcare status. We aimed to assess post-surgical pain prevalence and its impact on health-related quality of life in pediatric patients who underwent cardiac surgery during childhood. METHODS: This cross-sectional study recruited patients aged 4 years or older who underwent cardiac surgery for congenital heart disease at least 1 year prior, during the age of 0-10 years, and were admitted for post-surgical follow-up at our institute. The prevalence, intensity, and location of pain and health-related quality of life were assessed in an interview. Perioperative information was collected from the patients' medical records. Health-related quality of life was assessed using the Pediatric quality of life inventory 4.0 (PedsQL). RESULTS: Pain was reported by 24 (17%) of the 141 participants. One-third of them reported moderate to severe pain that required medical intervention. After adjustment for several confounding factors, multivariable linear regression analysis demonstrated that the presence of pain and the number of surgeries were associated with lower total PedsQL scores. CONCLUSIONS: Pain was present in 17% of the patients who underwent cardiac surgery during childhood. Presence of pain had a negative impact on long-term health-related quality of life after pediatric cardiac surgery.

Synergistic activation of ERK1/2 between A-fiber neurons and glial cells in the DRG contributes to pain hypersensitivity after tissue injury.

Background Intense nociceptive signaling arising from ongoing injury activates primary afferent nociceptive systems to generate peripheral sensitization. ERK1/2 phosphorylation in dorsal root ganglion can be used to visualize intracellular signal activity immediately after noxious stimulation. The aim of this study was to investigate spatiotemporal characteristics of ERK1/2 phosphorylation against tissue injury in the primary afferent neurons. Methods Plantar incisions were made in the hind paws of Sprague-Dawley rats (n =150). Levobupivacaine was injected into the plantar aspect of the paws and ankles, Mitogen-activated protein kinase kinase (MEK) inhibitor was injected into the paw, and carbenoxolone, dual inhibitor of the gap junction and pannexin channel, was intraperitoneally injected. Pain hypersensitivity was investigated by a behavioral study, while phosphorylated ERK1/2 was detected in dorsal root ganglion and hind paw using immunohistochemistry and Western blot. Results Phosphorylated ERK1/2 was induced in dorsal root ganglion (26.8 ± 2.9% at baseline, 65.6 ± 3.6% at 2 min, and 26.3 ± 3.4% at 2 h) after the incision. NF-200 positive A-fiber neurons and satellite glial cells were positive for phosphorylated ERK1/2. Injury-induced pain hypersensitivity was abolished by MEK inhibitor. Levobupivacaine treatment inhibited phosphorylated ERK1/2 induction, carbenoxolone treatment inhibited glial phosphorylated ERK1/2 at 2 min after the injury, and carbenoxolone inhibited pain hypersensitivity and neuronal phosphorylated ERK1/2 at 1 h after the injury. Conclusion ERK1/2 phosphorylation in A-fiber neurons and satellite glial cells immediately after injury contributes to the generation of pain hypersensitivity. Signal communication between neurons and satellite glial cells expands the duration of neuronal ERK1/2 phosphorylation and pain hypersensitivity at 1 h after tissue injury.

IGF1-driven induction of GPCR kinase 2 in the primary afferent neuron promotes resolution of acute hyperalgesia.

Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.

A 3D model of CYP1B1 explains the dominant 4-hydroxylation of estradiol.

CYP1A1 and CYP1A2 exhibit catalytic activity predominantly for the 2-hydroxylation of estradiol, whereas CYP1B1 exhibits catalytic activity predominantly for 4-hydroxylation of estradiol. To understand why CYP1B1 predominantly hydroxylates the 4-position of estradiol, we constructed three-dimensional structures of CYP1A1 and CYP1B1 by homology modeling, using the crystal structure of CYP1A2, and studied the docking mode of estradiol with CYP1A1, CYP1A2, and CYP1B1. The results demonstrated that two particular amino acid residues for each CYP, namely Thr124 and Phe260 of CYP1A2, Ser122 and Phe258 of CYP1A1, and Ala133 and Asn265 of CYP1B1, play an important role in estradiol recognition.

Selective inhibition of methoxyflavonoids on human CYP1B1 activity.

Cytochrome P450 (CYP) 1B1 catalyzes 17beta-estradiol (E(2)) to predominantly carcinogenic 4-hydroxy-E(2), whereas CYP1A1 and 1A2 convert E(2) to non-carcinogenic 2-hydroxy-E(2). Hence, selective inhibition of CYP1B1 is recognized to be beneficial for the prevention of E(2) related breast cancer. In this study, we first evaluated the structure-property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin O-deethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between C-positions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited CYP1B1 activity. In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1. To understand why selective inhibition was observed, we carried out a molecular docking analysis of these methoxyflavonoids with the 2-3 double bond and CYP1s. The results suggested that chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser-122 in CYP1A1 and Thr-124 in CYP1A2. In conclusion, our results demonstrate: (1) strong inhibitory effects of flavonoids on CYP1 activities require the 2-3 double bond on the C-ring; (2) methoxyflavonoids with the 2-3 double bond had strong and selective inhibition against CYP1B1, suggesting chemopreventive flavonoids for E(2) related breast cancer; and (3) binding specificity of these methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues.

Spinal and Peripheral Mechanisms Individually Lead to the Development of Remifentanil-induced Hyperalgesia.

The present study was performed to determine neuronal loci and individual molecular mechanisms responsible for remifentanil-induced hyperalgesia. The effect of methylnaltrexone (MNX) on remifentanil-induced behavioral hyperalgesia was assessed to distinguish contributions of the peripheral and/or central nervous system to remifentanil-induced hyperalgesia. Phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) in the dorsal root ganglion (DRG) neurons after remifentanil infusion, and the effect of a p38MAPK inhibitor on remifentanil-induced hyperalgesia were analyzed to investigate involvement of p38MAPK in the peripheral mechanisms of remifentanil-induced hyperalgesia. Spinal levels of prodynorphin mRNA after remifentanil infusion, and the effect of the BK2 bradykinin receptor antagonist on remifentanil-induced hyperalgesia were investigated to assess potential spinal mechanisms. The effects of MNX and BK2 antagonists on remifentanil-induced exacerbation of post-incisional hyperalgesia were also investigated using behavioral analysis. Remifentanil infusion induced hyperalgesia in the early (4 h to 2 days) and late (8-14 days) post-infusion periods. MNX inhibited hyperalgesia only during the early post-infusion period. p38MAPK phosphorylation was observed in the DRG neuron, and the p38MAPK inhibitor inhibited hyperalgesia during the early post-infusion period. Prodynorphin expression increased in the spinal cord, and a BK2 antagonist inhibited hyperalgesia during the late post-infusion period. Remifentanil-induced exacerbation of incisional hyperalgesia was inhibited by MNX and the BK2 antagonist. The present study demonstrated that remifentanil activates peripheral and spinal neurons to promote chronologically distinctive hyperalgesia. p38MAPK phosphorylation in the DRG neuron leads to peripherally-driven hyperalgesia during the early post-infusion period, while spinal dynorphin-bradykinin signaling promotes hyperalgesia during the late post-infusion period.

Peripheral nerve block combined with general anesthesia for lower extremity amputation in hemodialysis patients: case series.

BACKGROUND: Anesthetic management of lower extremity amputation in chronic hemodialysis (HD) patients can be challenging because of their poor cardiovascular status. As previously reported, peripheral nerve block (PNB) may be beneficial in these complicated cases. We report the effects of PNB combined with general anesthesia on hemodynamic stability in HD patients undergoing elective lower extremity amputation. METHODS: We retrospectively analyzed 13 HD patients who underwent lower extremity amputation. Patients received general anesthesia (GA group, n = 7) or general anesthesia combined with PNB (GA with PNB group, n = 6), as decided by the anesthesiologists. Mean blood pressure (MBP), systolic blood pressure (SBP), lowest BP, heart rate (HR), blood loss, fluid and blood infusion volumes, and doses of vasopressors required were compared for hemodynamic assessment. The coefficient of variation ([Formula: see text]) of MBP (CVMBP) and SBP (CVSBP) was calculated to compare hemodynamic stability. Intraoperative opioid use and postoperative pain scores at rest using a numerical rating scale (NRS) on postoperative days 0 and 1 were compared for pain assessment. We also assessed 30-day mortality. RESULTS: CVMBP in the GA group was significantly higher than that in the GA with PNB group (0.15 ± 0.05 and 0.08 ± 0.04, respectively, p = 0.03). The CVSBP in the GA group was also significantly higher than that in the GA with PNB group (0.16 ± 0.02 and 0.09 ± 0.01, respectively, p = 0.03). No significant differences in other hemodynamic parameters were observed. Intraoperative fentanyl doses were significantly lower in the GA with PNB group (GA 210.7 ± 99.9 µg vs. GA with PNB 113.0 ± 75.6 µg, p = 0.04). There were no significant differences in other pain parameters and 30-day mortality between the groups. CONCLUSION: Our results suggest that PNB combined with general anesthesia contributes to intraoperative hemodynamic stability through better pain control in HD patients undergoing lower extremity amputation.

Endoplasmic Reticulum Stress in the Dorsal Root Ganglion Contributes to the Development of Pain Hypersensitivity after Nerve Injury.

Little is known about the functional relationship between endoplasmic reticulum (ER) stress and development of pain hypersensitivity after nerve injury. The aim of this study was to investigate the role of ER stress in the development of pain hypersensitivity in the dorsal root ganglion (DRG) after spinal nerve ligation (SNL). SNL was performed in male Sprague-Dawley rats. Real-time PCR and immunohistochemistry were performed to investigate ER stress markers including glucose-regulated protein (GRP) 78, C/EBP homologous protein (CHOP), and spliced form of the X-box binding protein 1 (sXBP1) in L4 and L5 DRG. Behavioral assessment with von Frey filaments, radiant heat, and acetone stimulation was performed to investigate pain hypersensitivity. The ER stress inhibitor salubrinal was administered prior to and 1, 3, and 5 days after SNL treatment. Separately, the ER stress inducer tunicamycin was applied to L5 DRG. GRP78, CHOP, and sXBP1 mRNA and protein expression in L5 DRG was increased 1 and 3 days after SNL but returned to baseline 7 days after SNL. In L4 DRG, ER stress markers showed no remarkable change. Immunohistochemistry demonstrated that GRP78 expression was detected in the majority of DRG neurons and in satellite glial cells. Treatment with salubrinal inhibited CHOP expression in L5 DRG and alleviated pain hypersensitivity for 5 days after SNL. Tunicamycin induced ER stress in the DRG and pain hypersensitivity 2 h after treatment. These results demonstrated that ER stress is induced in the injured DRG and contributes to the development of pain hypersensitivity after nerve injury.

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro.

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).

In vitro and in vivo estrogenic activity of chlorinated derivatives of bisphenol A.

The estrogenic activity of bisphenol A (BPA) and its chlorinated derivatives, 2-(3-chloro-4-hydroxyphenyl)-2-(4-hydroxyphenyl)propane (3-ClBPA) and 2,2-bis(3-chloro-4-hydroxyphenyl)propane (3,3'-diClBPA) was assessed by determining their relative binding affinity for the human estrogen receptor-alpha and -beta (ERalpha and ERbeta) and also their uterotrophic activity in ovariectomized female rats. BPA and its chlorinated derivatives were active in competing with [3H]17beta-estradiol for their binding to the human ERalpha and ERbeta proteins. While 3-ClBPA and 3,3'-diClBPA competed more effectively for ERalpha binding than BPA (IC50 values of 2.48x10(-5), 1.28x10(-5), and 1.08x10(-4)M, respectively), they had similar activity as BPA for competing the binding to ERbeta (IC50 values of 1.43x10(-5), 1.87x10(-5), and 2.59x10(-5)M, respectively). To determine the uterotropic activity, three doses (10, 50 and 100 mg/kg/day) of BPA and its derivatives were given to mature ovariectomized Sprague-Dawley rats for 3 consecutive days. Treatment of animals with 50 and 100 mg/kg/day of BPA or its chlorinated derivatives caused a significant increase in the uterine wet weight and the endometrial area. The results of our present study demonstrated that the affinities of 3-ClBPA and 3,3'-diClBPA for ERalpha were higher than the affinity of BPA, although the in vivo estrogenic activity of the two chlorinated BPAs in ovariectomized female Sprague-Dawley rats appeared to be comparable to that of BPA.

Effects of dietary lipids on daunomycin-induced nephropathy in mice: comparison between cod liver oil and soybean oil.

Although it is well known that dietary lipids affect the course of glomerulonephritis in rats and humans, the precise mechanisms involved have not been fully elucidated. The aim of this study was to investigate the effects of different types of dietary lipids (fish oil and vegetable oil) on daunomycin (DM)-induced nephropathy in mice fed on soybean oil (SO) or cod liver oil (CLO). Urinary protein excretion, serum albumin, creatinine, total cholesterol, and TG were measured, and glomerular histological changes were evaluated. Antioxidant enzymes were also measured, along with the levels of lipid peroxide, GSH, thromboxane (Tx) B2, and 6-keto prostaglandin F1alpha in renal cortical tissue. Dietary CLO significantly reduced urinary albumin excretion and ameliorated the histological changes induced by DM. The increase of tissue lipid peroxide levels seen in SO-fed mice was suppressed in CLO-fed mice, whereas CLO-fed mice showed higher GSH levels than SO-fed mice throughout the experiment. In addition, renal tissue GSH peroxidase activity was significantly higher at 72 h after DM injection in CLO-DM mice than in SO-DM mice. Both renal cortical TxB2 and 6-keto PGF1alpha levels were significantly lower in CLO-DM mice than in SO-DM mice. These results suggest that inhibition of oxidative damage by dietary CLO played an important role in the prevention of DM nephropathy in this mouse model. The effect of CLO was closely associated with the inhibition of Tx synthesis.

Quercetin-3-O-glucronide inhibits noradrenaline binding to α2-adrenergic receptor, thus suppressing DNA damage induced by treatment with 4-hydroxyestradiol and noradrenaline in MCF-10A cells.

Risk factors for breast cancer include estrogens such as 17ß-estradiol (E2) and high stress levels. 4-Hydroxyestradiol (4-OHE2), a metabolite of E2 formed preferentially by cytochrome P450 1B1, is oxidized to E2-3,4-quinone, which reacts with DNA to form depurinating adducts that exert genotoxicity and carcinogenicity. Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. Here, we found that treatment with 4-OHE2 (3 µM) and NA (3 nM) significantly induced the phosphorylation of histone H2AX (γ-H2AX), one of the earliest indicators of DNA damage, and apurinic (AP) sites via the α2-adrenergic receptor (α2-AR) in human mammary epithelial MCF-10A cells. As an inverse association between a higher intake of flavonoids and breast cancer risk has previously been suggested from epidemiological studies, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin in the blood, on 4-OHE2- and NA-induced γ-H2AX and AP sites. Q3G (0.1 µM) suppressed their induction and inhibited the binding of [(3)H]-NA to α2-AR. These results suggest that Q3G acts as an α2-AR antagonist and that it could be used as a chemopreventive agent for stress-promoted breast cancer.

Breast cancer and flavonoids - a role in prevention.

Endogenous estrogens, such as 17ß-estradiol (E2), are implicated in the development of breast cancer. The putative mechanisms by which estrogens exert the carcinogenic effects have been recognized to involve the redox cycling of estrogen metabolites and subsequent estrogen-DNA adduct formation as well as the estrogen receptor-dependent pathway of estrogen-induced cell growth. The former pathway is regulated by phase I enzymes, mainly cytochrome P450 (CYP) 1A1, 1A2, and 1B1. Among them, CYP1B1 predominantly catalyzes the C4-position of E2 and forms carcinogenic 4-hydroxy-E2 (4-OHE2), whereas CYP1A1 and CYP1A2 convert E2 to noncarcinogenic 2-hydroxy-E2. Formed 4-OHE2 is further oxidized to semiquinones and quinones, which form DNA adducts, leading to mutagenic lesions. Consequently, CYP1B1 is highly expressed, and 4-OHE2 is predominantly detected in estrogen target neoplastic tissues. Moreover, invasion and metastasis are also involved in the development of breast cancer. Epidemiological studies suggest an inverse association between a higher intake of flavonoids and breast cancer risk. Flavonoids, which are widely distributed in the plant kingdom, have been recently reported as candidate compounds that can exert chemopreventive effects in estrogen-dependent or independent breast cancer. In this review, we provide a comprehensive overview of breast cancer and chemoprevention by flavonoids, mainly focusing on ER-mediated hormonal regulation, redox cycling of estrogen metabolites, and selective inhibition of CYP1B1.

A methoxyflavonoid, chrysoeriol, selectively inhibits the formation of a carcinogenic estrogen metabolite in MCF-7 breast cancer cells.

A 17beta-estradiol (E(2)) is hydrolyzed to 2-hydroxy-E(2) (2-OHE(2)) and 4-hydroxy-E(2) (4-OHE(2)) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE(2) is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3'-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E(2) hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE(2), which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E(2) without any effects on the formation of 2-methoxy E(2). In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE(2) from E(2.).

Inhibitory effects of chrysoeriol on DNA adduct formation with benzo[a]pyrene in MCF-7 breast cancer cells.

Cytochrome P450 (CYP) 1 families including CYP1A1, 1A2 and 1B1 are well known to be deeply involved in the initiation of several cancers, due to the fact that they activate environmental pro-carcinogens to form ultimate carcinogens. Benzo[a]pyrene (BaP) is one of the major classes of prototypical pro-carcinogen. It is activated by the CYP1 family to its ultimate carcinogenic forms, mainly BaP-7,8-diol-9,10-epoxide (BPDE), and it forms adducts with DNA. This has been recognized to be a major initiation pathway for cancer. Our previous study demonstrated that chrysoeriol, which is a dietary methoxyflavonoid, selectively inhibited CYP1B1 enzymatic activity and might protect the CYP1B1 related-diseases such as breast cancer. In the present study, we further examined the effects of chrysoeriol on the other initiation pathway of cancer relating to the CYP1 family with BaP in human breast cancer MCF-7 cells. The effects of chrysoeriol on the formation of BPDE-DNA adducts were analyzed specifically using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. When MCF-7 cells were incubated with 2 microM BaP for 24h, three types of BPDE-dG adducts, especially (+)-trans-BPDE-dG as the dominant adduct, were detected. Co-treatment of MCF-7 cells with 10 microM chrysoeriol and BaP remarkably reduced (+)-trans-BPDE-dG formation. Chrysoeriol (1-10 microM) dose-dependently inhibited both EROD activity and the gene expressions of CYP1A1, 1B1 and 1A2 stimulated by treatment with BaP. In addition, the same amounts of chrysoeriol significantly inhibited the binding of BaP to the aryl hydrocarbon receptor (AhR), which is the key factor concerning the induction of the CYP1 families. In conclusion, our results clearly indicate that chrysoeriol inhibited the formation of BPDE-DNA adducts via regulation of the AhR pathway stimulated by BaP. As a consequence chrysoeriol may be involved in the chemoprevention of environmental pro-carcinogens such as BaP.

A novel convenient method for high bacteriophage titer assay.

The recent various applications of phages (bacteriophages) including phage therapy have brought about a revival of phage investigation. The phage titer assay is indispensable for phage experiments. However, the conventional standard method is a plaque counting method which requires a little skill with tedious repeating operation. Furthermore, it is not directly applicable to high phage titers. In this paper, we describe a novel convenient "cross streak and paper disk assay method" for high titer concentration without plaque counting.