Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BACKGROUND: Retinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats. METHODS: High intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 µL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised. RESULTS: The ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 µm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001). CONCLUSION: Treatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.

Value of postoperative radiation therapy for regional control after dissection in head and neck squamous cell carcinoma cases.

OBJECTIVE: We aimed to define clinicopathologic risk factors associated with regional recurrence (RR) and thus the effectiveness of postoperative radiotherapy (PORT) for neck control for head and neck squamous cell carcinomas (HNSCCs) with differing cervical lymph node status. METHODS: A retrospective study was performed in 196 HNSCC patients with pathologically positive neck node (N+) to evaluate the high-risk factors for RR and to define the role of PORT in control after neck dissection and postoperative radiotherapy (PORT). RESULTS: Overall, the RR rate after neck dissection and PORT was 29%. Extracapsular spread (ECS) was confirmed to be the only independent risk factor for RR. There were no significant risk factors associated with RR in the ECS- group. The 5-year disease-specific survival rate was 45%, which descended to 10% with the emergence of RR. CONCLUSIONS: ECS remains a determined risk factor for RR after neck dissection and PORT in patients with N+. PORT alone is not adequate for preventing RR in the neck with ECS after neck dissection. More intensive postoperative adjuvant therapies, especially combined chemotherapy and radiotherapy, are needed to prevent regional failure in HNSCC patients with ECS.

[Outcomes of salvage laryngectomy after initial radiation failure in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate survival outcomes of salvage surgery preformed for laryngeal squamous cell carcinoma that recurred or progressed after radiotherapy alone. METHODS: A review of 72 patients who underwent salvage laryngectomy for laryngeal cancer failed in initial radiation therapy between 1996 and 2005 was performed. The tumor persistence occurred in 50 cases and recurrence in 22 cases. All patients received salvage total laryngectomy and radical neck dissection. Survival analysis was performed by using Kaplan-Meier method, Log-rank test and Cox proportional hazard model. RESULTS: Thirty patients developed a postoperative complication after salvage surgery. Pharyngocutaneous fistula occurred in 15(20.8%) patients. During 5 years after salvage surgery, the rates of tumor recurrence, distant metastasis and second malignancy were 34.7%, 22.2% and 6.9%, respectively. Kaplan-Meier analysis showed that overall 3 year and 5 year survival rates of those patients after operation were 45.8% and 36.1%, respectively. Univariate analysis indicated that N restage, tumor persistence/recurrences after radiotherapy, surgical margin status, level of tumor invasion, pathologic N stage, extracapsular nodal spread and invasion of nonlymphotic structures were significantly associated with overall survival. Multivariate analysis showed the most significant prognostic factors were tumor persistence or recurrence after radiotherapy, surgical margin status and level of tumor invasion. CONCLUSIONS: Surgical salvage remains the gold standard for management after failure of initial radiation therapy. The extent of tumor invasion must be assessed sufficiently before operation, and the surgical margin status must be identified in operation by using a frozen sectioning approach, especially in patients with tumor invasion to muscle/cartilage and tumor persistence after radiotherapy.

[Clinicopathologic aspects of locoregional recurrence of hypopharyngeal cancer and their implication on the survival of patients].

OBJECTIVE: To investigate the clinicopathologic aspects of locoregional recurrence and their implication on the survival in patients with hypopharyngeal cancer. METHODS: A retrospective review of 101 patients with hypopharyngeal cancer that were treated with surgical management and postoperative radiotherapy from 1998 to 2004 was performed. The clinicopathologic risk factors for locoregional recurrence were evaluated by using univariate chi(2) tests and multiple stepwise Logistic regression models. Survival analysis was performed by using Kaplan-Meier method, Log-Rank test and Cox proportional hazard model. RESULTS: The overall incidence of locoregional recurrence in this series of hypopharyngeal cancer was 43.6% (44/101). The rates of recurrence were local 13.9% (14/101), regional 31.7% (32/101). In a multivariate Logistic regression analysis, it was confirmed that number of levels with pathologic lymph node (OR = 0.334, P = 0.0242) was the most significant risk factors for locoregional recurrence. The same for regional recurrence was capsule rupture of pathologic lymph node (OR = 0.353, P = 0.0389) and multilevel positive nodal involvement in neck (OR = 0.259, P = 0.0069). The overall survival of hypopharyngeal cancer was 23.2%, and reduced to 11.4% for locoregional recurrence. The recurrences were managed by salvage surgery in 12 cases, which included local recurrences after partial pharyngolaryngectomy, ipsilateral regional recurrences after selective neck dissection, and contralateral regional recurrences at initially uninvolved neck. The overall survival of those patients sustained 33.3%. A multivariate Cox regression analysis revealed that the factors impacting the prognosis were recurrent time (RR = 0.317, P < 0.0001) and treatment modality after locoregional recurrence (RR = 6.119, P = 0.0001). CONCLUSIONS: Locoregional recurrences are frequent and have a very negative impact on patient survival in hypopharyngeal cancer, which most commonly have a regional recurrence. Salvage surgery in suitable cases seems to improve survival rates. Much attention should be paid to cervical pathological factors for locoregional recurrences in hypopharyngeal cancer, especially multilevel and capsule rupture of pathologic lymph node.

[Clinicopathologic features and prognostic factors of cervical lymph node metastasis in oral squamous cell carcinoma].

BACKGROUND & OBJECTIVE: The cervical lymphatic meta-static rate of oral squamous carcinoma is 50%-59%. The management of cervical lymphatic metastasis relates with prognosis of patients with oral squamous carcinoma. This study was to investigate the clinicopathologic features and prognosis factors of cervical lymph node metastasis in oral squamous cell carcinoma. METHODS: Clinicopathologic data of 191 patients with oral squamous cell carcinoma were retrospectively reviewed. The relationship between histopathologic factors and cervical lymph node metastasis was evaluated by univariate Chi(2) test. Cox regression model was used to analyze impacts of pathologic factors of cervical lymph nodes, such as metastatic status, size and number of positive neck nodes,levels of positive nodes, the lowest involved nodal level, on prognosis. RESULTS: The overall 5-year survival rate of the 191 patients was 48.7%. Univariate Chi(2) test confirmed that only depth of primary tumor infiltration correlated to cervical lymph node metastasis. Cox regression analysis revealed that clinical N stage, cervical lymphatic metastatic status, number of involved levels, and the lowest involved nodal level affected prognosis of patients, the most significant prognostic factors of cervical lymph node were clinical N stage, and the lowest involved nodal level. CONCLUSIONS: Cervical lymph node metastasis is a significant prognostic factor of oral squamous cell carcinoma. The identification of metastatic patterns of cervical lymph nodes, and the management targeting pathologic factors of cervical lymph node affecting survival are critical for improving the treatment effect on oral squamous cell carcinoma.

[Clinical analysis of 71 cases of multiple primary cancers in head and neck squamous carcinomas].

OBJECTIVE: To study the location, treatment, life status of multiple primary cancers (MPCs) in head and neck squamous cell carcinomas. METHODS: The clinical data of 71 head and neck squamous carcinoma patients with MPCs were retrospectively analyzed. RESULTS: MPCs were seen in head and neck regions in 27 cases and in remote organs in 42 cases, two of which were triplicate primary cancers. Four cases were synchronous MPCs, including one patient with synchronous triplicate primary cancer. Other 67 cases were heterochronous MPCs, including one patient with heterochronous triplicate primary cancer. Of 67 heterochronous MPCs, the time interval between index tumor presentation and diagnosis of MPCs was eight months to twelve years. MPCs occurred in seventy percent index oral cavity squamous cancers, which were located in head and neck regions, and in sixty-two percent index hypopharynx cancers and seventy-nine percent index laryngeal cancers, which were located in remote organs. The incidence of MPCs in esophagus and lung was higher than that in other remote organs. Among the various MPCs in this serials, the incidence of the disease appeared to be the highest in esophagus, accounting for twenty-four percent of all cases. The total three- and five-year survival rates were 32.4% and 22.5%, respectively. Of all MPCs patients, the three-year survival rate for patients who received different therapies for their MPCs was obviously higher than that of untreated patients (P < 0.01, Chi-square test). CONCLUSIONS: Esophageal carcinoma is the most common second primary cancer among the various MPCs of the head and neck squamous carcinomas. Oral cavity cancers tend to have more MPCs in the head and neck regions, and laryngeal and hypopharyngeal cancers are easily to be associated with MPCs in the remote organs. Regular follow-up and early diagnosis with effective treatment can help to improve the survival of MPC patients in head and neck squamous cell carcinomas.

[Analysis of risk factors in the prediction of distant metastases of head and neck squamous cell carcinomas].

OBJECTIVE: To investigate the risk factors related with distant metastases (DM) from head and neck squamous cell carcinomas (HNSCC). METHODS: A retrospective study was carried out to review the histopathological data from 532 HNSCC patients treated in Bethune International Peace Hospital from February 1978 to February 1998. The incidence and the risk factor for DM were evaluated in a model that included the following factors: sex, age, clinical staging, T and N staging, site of primary tumor, depth of primary tumor infiltration, histological grade of primary tumor, presence of cervical lymph node metastasis, number of positive neck nodes and levels involved, and presence of extracapsular nodal spread. Univariate chi2 test and multivariate stepwise logistic regression model were used for the analysis. Statistical analysis of overall survival was performed using Kaplan-Meier method. RESULTS: Sixty cases (11.3%) presented distant metastases in 532 patients of head and neck squamous cell carcinomas. In a univariate analysis, it was confirmed that the following variables correlated to DM, i.e., clinical staging (P = 0.0126), T classification (P = 0.0082), site of primary tumor (P = 0.0011), depth of primary tumor infiltration (P = 0.0005) , presence of cervical metastasis (P = 0.0057), number of positive neck nodes (P = 0.0149) and levels involved (P = 0.0034), presence of extracapsular nodal spread (P = 0.0118). In a multivariate analysis, the most significant risk factors for DM were the site of primary tumor and the depth of primary tumor infiltration. Kaplan-Meier analysis showed that overall survival rates of 60 HNSCC patients who presented distant metastases were 51.7% at 1 year, 13.3% at 3 years, 6.5% at 5 years, respectively. CONCLUSION: The site of primary tumor and the depth of primary tumor infiltration are the key risk factors in determining the development of DM in HNSCC patients. Patients with laryngeal and hypopharyngeal carcinomas and patients with primary tumor infiltrating muscular, bone or cartilage level have the highest risk of developing DM.

[Clinical pathology feature and prognostic factors of cervical lymph node metastases in hypopharyngeal carcinoma].

OBJECTIVE: To investigate the risk clinicopathological factors of primary tumor in the prediction of cervical lymph node metastases and the cervical lymph node prognostic factors in hypopharyngeal squamous cell carcinoma. METHODS: A retrospective study was carried out to review the histopathological data from 98 hypopharyngeal squamous cell carcinoma patients. The relationship between histopathological parameters and cervical lymph node metastases were evaluated by means of a univariate chi2 test and multivariate stepwise logistic regression model. And the Cox regression model was used to define possible pathological parameters of neck node affecting survival including N staging, presence of cervical lymph node metastases and extracapsular nodal spread, size and number of positive neck nodes, and levels of positive neck nodes. RESULTS: The overall 5-year survival rate of patients with hypopharyngeal carcinoma was 28.6%. In a univariate and multivariate analysis, it was confirmed that size and growth pattern of primary tumor correlated to cervical lymph node metastases. In a multivariate Cox regression analysis, the most significant prognostic factors of cervical lymph node were the size of positive neck nodes and level involved. CONCLUSIONS: Cervical lymph node metastases were one of the most significant prognostic factors of hypopharyngeal carcinoma. The identification of patients at risk for cervical lymph node metastases and the management of the neck by coping with pathological factors of cervical lymph node affecting survival are very important to improve the treatment and prognosis of hypopharyngeal carcinoma.