Article type: Original article
Title:Nomogram for Predicting the Overall Survival of Nasopharyngeal Carcinoma treated with Intensity Modulated Radiation Therapy
Authors: S. X. Wu1,2,3, B. Xia3, F. Han4,5,6, R. F. Xie3, T. Song7, L. X. Lu4,5,6, W. Yu7, X. W. Deng4,5,6, Q. C. He7, C. Zhao4,5,6, C. H. Xie1,2,*
Author’s affiliation:
1.Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan
University, Wuhan, Hubei, P. R. China
2.Hubei Cancer Clinical Study Center, Wuhan University, Wuhan, Hubei, P. R.
China
3.Department of Radiation Oncology, Hangzhou Cancer Hospital, Hangzhou,
Zhejiang, P. R. China
4.State Key Laboratory of Oncology in Southern China, Guangzhou, P. R. China
5.Department of Radiation Oncology, Sun Yat-Sen University Cancer Center,
Guangzhou, P. R. China
6.Collaborative Innovation Center of Cancer Medicine, Guangzhou, P. R. China
7.Department of Radiation Oncology, Wenzhou Medical College Cancer Center,
Wenzhou, Zhejiang, P. R. China
*Full address for correspondence:
Prof. Cong-Hua Xie, Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan University, 169 Donghu Road, Wuhan, 430071, P. R. China; Tel: +86-027-********; E-mail: chxie_65@https://www.doczj.com/doc/484325575.html,.
Abstract
Purpose
Distant metastases has become the predominant mode of treatment failure in patients with non-metastatic nasopharyngeal carcinoma (NPC) after intensity-modulated radiation therapy (IMRT), underlining the need of optimizing systemic chemotherapy. The aim of this study was to define possible predictors of poor prognosis and develop a predictable model for survival probability in NPC patients treated with IMRT. Patients and Methods
We reviewed the records of 533 patients with non-metastatic NPC who underwent IMRT with/without concurrent chemotherapy at the department of radiation oncology of Sun Yat-sen University from 2002 to 2009, which was used to construct nomogrambased on Cox regression. None of these patients received induction or adjuvant chemotherapy. The performance of monogram was determined by concordance index(C-index) and calibration curve, and compared with currently used staging systems onNPC. The results were also validated in an external cohort of 442 patients from the department of radiation oncology of Wenzhou Medical College at the same period.
Results
Factors with the greatest influence on survival in the model included primary gross tumor volume, age, tumor stage and nodal stage (2002 Union for International Cancer Control [UICC] staging system). The C-index of the nomogram for predictingsurvival
was 0.748 (95% CI 0.704-0.785), which was statistically higher than that of 2002 UICC staging system (0.684,P< 0.001). The calibration curve showed good model calibration with high correlation of nomogram-predicted and observed probability of overall survival. In the validation cohort, the nomogramalso showed good discrimination and calibration.
Conclusion
The nomogram as proposed in this study accurately predicted the prognosis for NPC patients after IMRT with/without concurrent chemotherapy, which will aid in patient counseling and systemic therapy advice for patients with a predicted poor prognosis in endemic regions.
Key words:nasopharyngeal carcinoma, intensity-modulated radiation therapy, nomogram, survival probability
Background
Nasopharyngeal carcinoma (NPC) is a leading cancer in Southeast Asia, the Arctic, and the Middle East/North Africa, especially in southern China although it is uncommon in the world. Globally NPC resulted in 65,000 deaths in 2010 up from 45,000 in 1990[1].Radiation therapy (RT) is the mainstay of curative treatment for NPC for decades.Currently, the Intensity-modulated radiotherapy (IMRT) is the preferred standard of care in non-metastatic NPC, which significantly improves coverage of tumor target and spare normal structures, leading to higher local-regional
control and less RT-related sequel [2-6].With IMRT, 3-year local-regional control rates of exceeding 90% has been documented,distant metastases (DM)has become the main treatment failure with a 3-year DM rates of 20%-34% [7-13].Although the addition of induction or adjuvant chemotherapyin NPC is a reasonable option for patients at high risk of developing DM, the survival benefits of which are not clear; considerable toxicities related to concurrent chemoradiotherapy and poorly selected patients administrated systemic therapy were confusing factors possibly [14-17]. Accurate prognostic model of generating individualized predictions will be propitious to identify and stratify patients with regard to receive systemic chemotherapy on or off a clinical trial.Although Union for International Cancer Control (UICC) TMN staging provides useful estimates for recurrence risk and survival outcomes, significant variation within each prognostic group has been observed because of the heterogeneityof tumor biology andpatient characteristics, indicating that more relevant variables should be integrated to improve theestimates of patientoutcome.Furthermore, IMRT has been used extensively in the last decades, the suitability of the currently staging system based on data from two-dimensional radiation techniques remains challenge.
Nomogramis a graphical depiction of prediction modelcapable ofmultiple variablesthat provide the probability of a specific outcome for an individual patient [18, 19].The purpose of this study was to develop a nomogramto predictOSin patients with NPC treated with IMRT.
METHODS
Study Population
The retrospective study was conducted in patients treated with IMRT between September 2002 and December 2009at two NPC endemic areas in China (department of radiation oncology, Sun Yat-sen University, training cohort;department of radiation oncology, Wenzhou Medical College, validation cohort). To collect data for nomogramdevelopment, an electronic survey form was designed to select patients and gather relational information before initiation of the study.
Inclusion criteria included the following: histopathologically proven NPC, received IMRT with curative intention, no history of previous anticancer therapy, and no history of other malignancies. Exclusion criteria were as follows: evidence of DM at diagnosis, received induction/adjuvant chemotherapy, received non-cisplatin alone concurrent chemotherapy, and incomplete information of follow-up.
The techniques of planning and delivery of IMRT used in the two cohorts were described previously [11, 13].The prescribed dose was 68-70 Gy/28-30 fractions to the gross target volume, 56-60 Gy/28-30 fractions to the high-risk clinical target volume, and 45-54 Gy/23-30 fractions to the low-risk clinical target volume. Generally, concurrent chemotherapy was delivered withcisplatin alone (80 mg/m2/d on Days 1 and 22 or 25mg/m2/d weekly).
Statistical Analysis
OSwas the predicted outcome thatall causes of death were included. OS was estimated using the Kaplan-Meier method, and comparison was assessed using the
log-rank test. Multivariate Cox proportional hazards model was used to analysis the risk factors associated with OS. The variables used to construct the nomogram were selected a priori, including sex, age, clinical tumor (T) stage, clinical nodal (N) stage, primary gross tumor volume (GTV) and administration of concurrent chemotherapy (yes/no), based on prior research and data availability[20].Stage data was retrieved from the original chart reports and was unified to 2002 UICC stage system.The final model selection was performed by a backward step down selection process with the Akaike information criterion [21] using the package of rms in R version 3.0.1.
The performance of the nomogram was measured by concordance index (C-index), which can be applied to continuous outcome and censored data. Calibration of the nomogram was assessed by plotting the observed rates against the nomogram predicted probabilities. Bootstraps with 1000 re-sample were used for these activities. Comparison between the nomogram andUICC staging system was evaluated by the C-index.During the external validation of the nomogram, the survival probability of each patient in the validation cohort was calculated according to the established nomogram, which were used to plot receiver operating characteristic (ROC) curves by SPSS 19.0 for windows. Generally, areas under the curve (AUC) of 0.7 to 0.8 represent reasonable discrimination.
RESULTS
Nomogram development
The clinical and treatment characteristics of patients in the training cohort (N=533)
are listed in supplementaryTable S1, available at Annals of Oncology online. Three hundred and seventy-one patients were stage III/IV, of which 280 (76%) patients were administrated with concurrent chemotherapy. The median follow-up time was 84.2 months (range 4.2-141.8 months) for all patients. There were 382 (72%) patients with at least 5 years of follow-up time. Of the 533 patients, 35 (7%) and 84 (16%) patients experienced local-regional recurrence and DM, 118 (22%) patients died at last follow-up; the 3- and 5-year OS rates were89.1%(95%CI86.6%-91.6%)and 81.7% (95%CI78.4%-85.0%), respectively.
SupplementaryTable S2, available at Annals of Oncology online,presents univariate and multivariate analyses of OS for the 533 patients in the training cohort. Age, T stage, N stage and GTV were significant associated with OS in Kaplan-Meier analyses (Figure 1).Using concurrent chemotherapy showed a marginally significant correlated with improved OS in univariate analysis but disappeared in multivariate analysis. Considered that patients with high T stage may have a large GTV, the interaction of T stage and GTV was analyzed in Cox proportional hazards modeland the results showed a positive effect (P =0.009). Therefore, we separated the T stage by 4 levels, and the interaction of T stage and GTV were presented as four scale bars in the development of nomogram for OS (Figure 2).Internally validation using bootstrapping showed a c-index of 0.748 (95%CI0.704-0.785), which was significantly higher than that of 2002 UICC staging system (0.684 95%CI0.640-0.728,P< 0.001). Calibration for 5-year probability of OS for the nomogram showed good model calibration with high correlation of
nomogram-predicted and observed probability of OS (Figure 3a).
External V alidation
There were 442 patients in the external validation cohort (Table S1). Overall, patients with stage III/IV were less likely to be treated with concurrent chemotherapyin the validation cohort (13%).The median follow-up time was 30.5 months (range3.1-119.6 months)for all patients. There were 77(17%) patients with at least 5 years of follow-up time. Of the 442 patients with NPC, 24 (5%) and 54(12%) patients experienced local-regional recurrence and DM, 64 (15%) patients died at last follow-up; the 3- and 5-year OS rates were 85.3% (95%CI 81.4%-89.2%) and77.9%(95%CI 72.4%-83.4%), respectively.
ROC curves were drawn to assess the discrimination of the nomogram in the external population. The overall predictive accuracy of the nomograms for 5-year OS, as measured by the AUC was 0.784(95%CI 0.704-0.865)(supplementaryFigureS4, available at Annals of Oncology online). Actual 5-year OS was also plotted against the calculated predicted 5-year probabilities of OS for each patient, which showed good calibration for the events(Figure3b).
Discussion
With the extensive application of IMRT in clinic, DM has become the predominant failure mode in NPC. Incorporation of systemic chemotherapy is often routinely used for patients with poor prognosis and high-risk recurrence. In this study, we have developed a prediction model of OS based on clinical parameters that outperformed
conventional risk model (TNM system). External cohort validated the developed nomogram with good ability of discrimination, allowing the models to aid clinicians when counselingpatients, and to recommendwhetherto receive systemic chemotherapy with its potential adverse effects or not inpatients treated with IMRT at endemic regions.
The current staging system used for NPC is the seventh edition of UICC/AJCC TNM system [22],the major limitations are obvious that exclude some of theimportantprognostic factors including tumor burden.The primary tumor volume represented a significant independent prognostic factor in most of malignant tumors. Recently published data demonstrated that primary tumor volume of NPC was an independent prognostic indicator for treatment outcome in patients treated with IMRT, and all of those studies suggested that the incorporation of GTVinto NPC clinical stage system could provide more information to adjust treatment strategy [23-26].In this study, we also found that large tumors were associated with poor prognosis, and incorporatingtumor volume into TMN stage system significantly improved the ability of identifying patients prone to distant metastasis. Larger tumor often signifies increased number of clonogenic tumor cells as well as radiotherapy resistance associated with tumor hypoxia; whereas clinical T stage seems more likely to signify the tumor capacity of infiltration to surrounding tissue [27].Both of the two factors represent different biological characteristics of the tumor. Therefore, T stage in combination with different level of tumor volume can better prediction of OS, and both of which were incorporated in the final nomogram. As showed in the Figure 2,
GTV has more weight in T1 stage than that in T4 stage, indicating that the prognosis of patients with low T stage was mainly depended on the level of tumor volume.
The Meta-analysis of chemotherapy in NPC Study including eight randomized clinical trials showed that the addition of chemotherapy improved the absolute OS benefit of 6% in 5 years [14]. Neither inductionnor adjuvant chemotherapy improved OS compared with RT alone; the reduced risk of death was mainly attributed to the administration of concurrent chemotherapy by the mechanism of radiosensitization. However, with the emergence of new RT techniques (IMRT) and more advanced imaging equipment, high local-regional control was obtained; the value of concurrent chemotherapy encounters substantial uncertainty and challenges. In the Hongkong study NPC 9901 [28],the control arm used RT alone (half of patients administrated with 3-DCRT) for patients with regional advanced NPC achieved a 3-year OSrate of 78%, which was comparable to the concurrent chemotherapy arm, indicating that the survival benefit from chemotherapy confirmed in two dimensional era could be considered as compensation for poor RT delivery.In this study, no positive correlation of concurrent chemotherapy and OS was observed, indicating that the role of concurrent CT with these new techniques needs to be tested. In the final nomogram of OS, the factor of concurrent chemotherapy was not included.
This study was performed using retrospective data, and treatment was not assigned in a randomized fashion. In order to reduce the impact on prediction of OS came from divergent regimens and cycles of introduction or adjuvant chemotherapy, only those with concurrent chemotherapy of cisplatin alone were included in this study. The
3-year OS rates were 89.1% and 85.3% for training cohort and validation cohort, respectively; which is comparable to the results of 83%-94% reported in recent series using IMRT with/without induction or adjuvant chemotherapy [3, 4, 8, 10].Moreover, only 13% of the patients with stage III/IV were administrated with concurrent chemotherapy in the validation cohort; however, it didn’t lead to inferior local control and poor prognosis. The finding proposed that the good outcomes of patients treated with IMRT-only could be attributed to more modern and aggressive radiation techniques, which may have negated the benefit from concurrent chemotherapy. NPC population in endemic areas has different racial composition, histological subtype and other potential factors in etiology when compared to those in the west. Analysis of the SEER data showed that racial differences exist among NPC patients in the U.S, and Asians had the best 5-year survival rates after stratified by stage and histologic type [29].Non-keratinizing type tumors are predominant of the NPC population in southern China, which are generally associated with Epstein–Barr virus positivity and a favorable prognosis was reported [30].Therefore, the generalizability of nomogram developed in this study is limited. It can be used for patient-clinician communicationand therapy advicein Asian endemic areas. Whether it can be applied to the west population and those with keratinizing type tumor is still to be determined. The nomogram developed here does not specifically provide treatment decision; it simply provides a means to evaluate individual patient risk for disease recurrence after IMRT. However, the strength of the nomogram included the number of patients involved, the length of follow-up and the 5-year results comparable to contemporary
series. In addition, introducing molecular markers of tumor biology in NPC may further enhance the performance of the nomogram in future.
In conclusion, the nomogram as proposed in this study accurately predicted the prognosis for NPC patients treat with IMRT with or without concurrent chemotherapy, which will aid in patient counseling and systemic therapy advice for patients with a predicted poor prognosis in endemic regions. Additional studies are required to determine whether it can be applied to other patient groups.
Disclosure
The authors have declared no conflicts of interest.
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Table Legends
Table S1 clinical and treatment characteristics of patients with NPC
Table S2Univariate and multivariate analyses of overall survival for patients with nasopharyngeal carcinoma in the training cohort (N = 533)
Figure legend
Figure 1 Kaplan-Meier survival plots of overall survivalstratified by age (≤43 or > 43 years), sex (male or female), T stage (1, 2, 3 or 4), N stage (0, 1, 2 or 3), gross tumor volume (GTV, ≤22.4 or > 22.4 mm3) and concurrent chemotherapy (yes or no) for nasopharyngeal carcinoma treated with intensity modulated radiation therapy in the training cohort (N=533). The log-rank test is provided for each comparison. Figure 2 Nomogram developed for 5-year prediction of overall survival. Point score for gross tumor volume (GTV) was identified based on the level of T stage. To estimate risk, calculate points for each variable by drawing a straight line from patient’s variable value to the axis labeled “Points.” The sum of scores is converted to a probability in the lowest axis.
Figure S3 Calibration curves for 5-year probabilities of overall survival in the training cohort of 533 patients(A)and the validating cohort of 442 patients (B).
Patients were grouped byquartilesof predicted risk. Nomogram-predicted probability is plotted on the x-axis; observed probability is plotted on the y-axis (Kaplan-Meier estimates).
Figure 4 Receiver operating characteristic curves applied to the validation cohort of 442 patients using the developed nomogram predicting5-year overall survival.
PII S0360-3016(99)00253-9 CLINICAL INVESTIGATION Esophagus MULTI-INSTITUTIONAL RANDOMIZED TRIAL OF EXTERNAL RADIOTHERAPY WITH AND WITHOUT INTRALUMINAL BRACHYTHERAPY FOR ESOPHAGEAL CANCER IN JAPAN T OMOHIKO O KAWA ,M.D.,*T AKUSHI D OKIYA ,M.D.,?M ASAMICHI N ISHIO ,M.D.,? Y OSHIO H ISHIKAWA ,M.D.,§K OZO M ORITA ,M.D.,?AND J APANESE S OCIETY OF T HERAPEUTIC R ADIOLOGY AND O NCOLOGY (JASTRO)S TUDY G ROUP *Department of Radiology and Oncology,Tokyo Women’s Medical University,Tokyo,Japan;?Department of Radiology,Tokyo Medical Center,Tokyo,Japan;?Department of Radiology,Sapporo National Hospital,Hokkaido,Japan;§Health and Welfare Department,Hyogo Prefectural Government,Hyogo,Japan;and ?Department of Radiation Oncology,Aichi Cancer Center,Aichi,Japan Purpose:With the aim of improving the results of treatment of esophageal cancer,we designed this multi-institutional,randomized trial to establish the optimal irradiation method in radical radiation therapy for esophageal cancer by clinically evaluating external irradiation alone and in combination with intraluminal brachytherapy. Methods and Materials:The study population consisted of patients with squamous cell carcinoma who were expected to be successfully treated with radical radiation therapy.The patients who could be given intraluminal brachytherapy at the end of external irradiation of 60Gy were strati?ed into 2groups.Patients assigned to receive external irradiation alone received boost irradiation of 10Gy/week on a schedule similar to the previous one,and with the same or smaller irradiation ?eld.Intraluminal brachytherapy was performed,as a rule,with the reference dose point set at a depth of 5mm of the esophageal submucosa,and a total of 10Gy was irradiated at a daily dose of 5Gy,on a once-weekly schedule with low-dose-rate or high-dose-rate brachytherapy equipment. Results:A total of 103patients were registered,94of whom were analyzable,with 8ineligible,and 1for whom complete information was unavailable.The overall cumulative survival rate was 20.3%at 5years.The cause-speci?c survival rate was 31.8%at 5years.The cause-speci?c survival rate at 5years was 27%in the external irradiation alone group and 38%in intraluminal brachytherapy combined group.There was no signi?cant difference between the 2groups (p ?0.385).However,in the patients with 5cm or less tumor length,the cause-speci?c survival rate was 64%at 5years in the intraluminal brachytherapy combined group,which showed a signi?cant improvement over 31.5%in the external irradiation alone group (p ?0.025).In the patients with Stage T1and T2disease,cause-speci?c survival rates tended to be better in the intraluminal brachytherapy combined group than in the external irradiation alone group (p ?0.088).In the patients with more than 5cm tumor length or Stage T3–4disease,there were no signi?cant differences between the two groups by treatment methods (p ?0.290).The incidence of early and late complications did not differ according to whether intraluminal brachytherapy was used. Conclusion:For the purpose of establishing the usefulness of intraluminal brachytherapy,further prospective randomized studies are necessary to evaluate the ef?cacy in tumors with short length and those with shallow invasion,or to assess the usefulness of intraluminal brachytherapy,as additional irradiation in large advanced tumors have been shown to have disappeared by diagnostic imaging after chemoradiotherapy with 60Gy/6w external irradiation.?1999Elsevier Science Inc. Esophageal cancer,Radiotherapy,Intraluminal Brachytherapy. INTRODUCTION To improve the results of treatment of patients with esoph-ageal cancer,it is important to achieve good local control.Because the esophagus is adjacent to highly radiation-sen-sitive organs such as the lungs,bone marrow,etc.,it is dif?cult to irradiate tumors with high doses.Although irra-diation techniques have improved as a result of advances in treatment planning equipment and irradiation equipment,there are still some patients in whom radical radiation therapy within doses that the bone marrow and lung can tolerate is dif?cult with external irradiation alone.On the other hand,intraluminal brachytherapy allows high-dose irradiation of esophageal cancer,with little exposure of Reprint requests to:Tomohiko Okawa,M.D.,Department of Radiology and Oncology,Tokyo Women’s Medical University,8-1Kwada-cho,Shinjuku Ku Tokyo,162-8666,Japan.Tel:03-3353-8111;Fax:035269-7355. Acknowledgments —We wish to express our sincere thanks to the medical institutions and physicians who kindly contributed to this investigation by registering cases and recording case report forms.Accepted for publication 28April 1999. Int.J.Radiation Oncology Biol.Phys.,Vol.45,No.3,pp.623–628,1999 Copyright ?1999Elsevier Science Inc.Printed in the USA.All rights reserved 0360-3016/99/$–see front matter 623
第一节实验动物的捉拿、固定和编号方法 在基础医学实验中，正确捉拿与固定动物，是实验工作的基础，也是实验顺利进行的保证。掌握正确捉拿、固定动物的目的就是防止实验者被动物咬伤抓伤，同时也是为了维持动物的正常生理活动，从而不影响实验观察结果。 一、实验动物的捉拿与固定方法 在基础医学实验中，最常用的动物有小鼠、大鼠、豚鼠、蟾蜍、蛙、家兔和犬，现分别就其捉拿与固定方法依次予以介绍。 1. 小鼠 捉拿时先用右手将鼠尾抓住提起，放在较粗糙的台面或鼠笼上，在其向前爬行时，右手向后拉尾(图5-1)，用左手拇指和食指抓住小鼠的两耳和头颈部皮肤，将其置于左手心中，拉直四肢并用左手无名指压紧尾和后肢(图5-2)，右手即可作注射或其他实验操作。取尾血及尾静脉注射时，可将小鼠固定在金属或木制的固定器上(图5-11)。 图5-1 图5-2 2. 大鼠 方法基本与捉拿小鼠相同，但实验者事先应戴帆布防护手套。用右手将鼠尾抓住提起，放在较粗糙的台面或鼠笼上，抓住鼠尾向后轻拉，左手拇指和食指抓紧两耳和头颈部皮肤，余下三指紧捏鼠背部皮肤，如果大鼠后肢挣扎厉害，可将鼠尾放在小指和无名指之间夹住，将整个鼠固定在左手中，右手进行操作。若进行手术或解剖，则应事先麻醉或处死，然后用棉线活结缚四肢，用棉线固定门齿，背卧位固定在大鼠固定板上。需取尾血及尾静脉注射时，可将其固定在大鼠固定盒里，将鼠尾留在外面供实验操作。 3. 豚鼠 豚鼠具有胆小易惊的特性，因此抓取时要求快、稳、准。一般方法是：先用右手掌迅速、轻轻地扣住豚鼠背部，抓住其肩中上方，以拇指和食指环握颈部，对于体型较大或怀孕的豚鼠，可用另一只手托住其臀部(图5-3、图5-4)。
疼痛实验动物模型 科研探索2007-04-25 23:11:36 阅读147 评论0 字号：大中小订阅 疼痛是机制非常复杂的神经活动。疼痛研究已经成为当前神经科学研究的重要课题之一。由于疼痛机制的复杂性，使得在患者身上研究与疼痛有关的神经机制成为不可能的事。因而，我们的研究需要相应的动物模型。本章介绍了在现代神经科学研究中常用的疼痛动物模型。在概要介绍了疼痛研究的意义及其现状之后，重点介绍了在生理痛研究和急性、慢性病理痛研究中所应用的动物模型。生理痛的模型即常用的动物伤害性感受阈测定法；急性病理痛的模型则主要是各种急性炎症模型模型；慢性病理痛的模型则包 括慢性炎症模型和慢性神经损伤模型。 前言 疼痛(pain)是人们一生中经常遇到的不愉快的感觉。它提供躯体受到威胁的警报信号，是生命不可缺少的一种特殊保护功能。另一方面，它又是各种疾病最常见的症状，也是当今困扰人类健康最严重的问题之一。近年来，仅在美国就有三至四千万人患有慢性痛。据估计，美国每年用于治疗慢性痛的费用约为400~600亿美元；澳大利亚每年用于治疗疼痛的费用占全部医疗费用的40%。随着医学的进步和人类生活水平的提高，烈性传染病逐渐得到控制，疼痛在人的身心痛苦和医疗费用消耗上的相对地位将越来越重要。 由于难以在人体对疼痛进行深入的机制研究，有必要建立疼痛的动物模型。但疼痛是是包括性质、强度和程度各不相同的多种感觉的复合，并往往与自主神经系统、运动反应、心理和情绪反应交织在一起，它既不是简单地与躯体某一部分的变化有关，也不是由神经系统某个单一的传导束、神经核和神经递质进行传递的，所以很难将某种客观指标与疼痛直接联系起来。因而，我们只能根据模型动物对伤害性刺激的 保护反应和保护性行为来推测它们的疼痛程度。 伤害性感受(nociception)和痛觉是两个有密切关系但又不相同的概念。前者是指中枢神经系统对由于伤害性感受器的激活而引起的传入信息的加工和反应，以提供组织损伤的信息；痛觉则是指上升到感觉水 平的疼痛感觉。两者之间有时并没有严格的相关性。 生理痛模型与常用的痛阈测定法 概述 为了能够对痛觉现象及其机制作深入细致的观察，特别是在中枢神经系统的形态学、细胞生物学和分子生物学水平研究痛觉机制，必须建立动物的痛觉模型。又由于痛觉是意识水平的感觉，我们无法确定动物是否具有痛觉，只能观察其对伤害性刺激的行为反应。因而在下文的描述中有时用伤害性感受阈 (nociceptive threshold)取代痛阈(pain threshold)。 正常情况下，疼痛是机体对外界伤害性刺激的感受，它是一种报警系统，提示实存的或潜在的组织损伤的可能性。如果这种伤害性刺激是可以回避的，那么痛觉就是一种具有完全的积极意义的感觉形式，称为生理痛。这种意义上的疼痛模型实际上就是对伤害性感受阈的测量。它是通过观察动物对伤害性温度 和机械刺激的逃避反应实现的。 如果动物遇到无法逃避的伤害性刺激，就会引起它的情绪反应，发出嘶叫声。这是需要高级神经中枢配合的反应，并且不受局部运动功能的影响。因而，在伤害性刺激下引起的嘶叫反应也可以作为伤害性 感受阈的测量指标。 热辐射-逃避法 这是最常见的伤害性感受阈测量方式。最常用的有热辐射-甩尾法、热辐射-甩头法和热辐射-抬足法。
动物实验的基本操作技术 实验动物 实验动物(experimental animals)是指经过人工饲养、繁育，对其携带的微生物及寄生虫实行控制，遗传背景明确或者来源清楚，应用于科研、教学、生产和检定以及其他科学实验的动物。这些个体具有较好的遗传均一性、对外来刺激的敏感性和实验再现性。 一、常用实验动物的种类和特点 （一）狗(dog)属于哺乳纲、食肉目、犬科动物。其嗅觉、视和听觉均很灵敏，对外界环境的适应能力强。消化、循环和神经系统均发达，且与人类很相似。适用于各类实验外科手术学的教学和临床科研工作，是复制休克、DIC、动脉粥样硬化等动物模型首选的动物之一，由于其价格较昂贵，教学实验中不如某些中小动物常用。 （二）家兔(rabbit) 属于哺乳纲、啮齿目、兔科、草食类动物。品种有：青紫蓝兔（livor blue rabbit）、中国白兔（china white rabbit）、新西兰白兔和大耳白兔（maximus ear white rabbit）等。具有性情温顺，对温度适应敏锐和便于静脉注射等特点，是教学实验中最常用的动物之一。可用于血压、呼吸、泌尿等多种实验，还可用于体温实验和热原的研究与鉴定。 （三）大白鼠(rat) 属哺乳纲、啮齿目、鼠科类动物。其性情凶猛、喜欢啃咬、繁殖周期短、抗病能力较强、心血管反应敏锐。用于水肿、休克、炎症、心功能不全、肾功能不全和应激反应等实验。大鼠不能呕吐，故不能做催吐实验。 （四）小白鼠(mouse) 属哺乳纲、啮齿目、鼠科类动物。具有繁殖周期短、产仔多、生长快、体型小、温顺易捉、易于饲养等特点。广泛应用于各种药物的毒理实验、药物筛选实验、生物药效学实验，以及癌症研究、营养学、遗传学、免疫性疾病研究等项实验。 （五）豚鼠(cavy) 属哺乳纲、啮齿目、豚鼠科类动物。又名天丝鼠、荷兰猪。其性情温顺，嗅觉和听觉较发达。对某些病毒反应敏锐，易引起变态反应。适用于药理学、营养学、各种传染病的实验研究。细菌、病毒诊断学研究、过敏、变态反应性实验研究和内耳及听神经疾病研究。也常用于离体心脏实验研究。 （六）蛙和蟾蜍(frog and toad ) 均属两栖纲、无尾目类动物。常用于教学实验。其心脏在离体后仍可有节律地跳动。常用于心脏生理、药理和病生实验。蛙舌与肠系膜是观察炎症和微循环变化的良好标本。此外，蛙类还可用于水肿和肾功能不全的实验研究。 二、常用实验动物的品系
常用学习记忆障碍动物模型 药理研究中常用学习记忆障碍动物模型的行为学分析(1) 摘要:目的本研究对老年、基底前脑损伤、东莨菪碱注射和双侧颈总动脉结扎大鼠四种药理研究中常用的学习记忆障碍动物模型的行为学表现进行了分析。方法采用水迷宫及旷场分析法对上述四种动物模型组及正常青年对照和假手术组进行了研究,数据采用多因素方差分析法处理。结果发现老年动物学习记忆能力减弱, 对新环境的紧张程度增强,但空间认知能力及兴奋性无明显改变; 基底前脑损伤动物的学习记忆及空间认知能力明显下降,但兴奋性及情绪反应正常; 东莨菪碱动物模型不但学习记忆及空间认知能力明显下降, 而且兴奋性异常增强;双侧颈总动脉结扎动物的学习记忆及空间认知能力明显下降, 但兴奋性及情绪反应正常。 结论从行为学角度将上述四种动物模型进行了比较,为药理学研究应用这些模型提供了背景资料。 关键词:老年性痴呆; 动物模型; 行为学 老年痴呆主要包括Alzheimer. s病和血管性痴呆, 是老年人致残最严重的疾病之一, 并且随着人口的老龄化, 患者日益增多, 将给家庭和社会带来沉重的负担。因此对老年性痴呆药物的寻求和研究是当前医学界的一个紧迫问题。良好的动物模型是进行该病研究的基础。目前作为老年痴呆的药理学研究常用动物模型主要有: M受体阻断剂东莨菪碱或樟柳碱致动物学习记忆障碍模型, 兴奋性氨基酸基底前脑注射致动物学习记忆障碍模型, 正常老年动物模型, 以及双侧颈总动脉结扎动物模型等。为更好地了解这四种模型动物的行为学表现,我们采用了水迷宫及旷场分析方法对这四种动物模型进行了比较观察, 水迷宫是一种较好地评定动物空间学习记忆能力的方法, 而旷场分析能对动物对新异环境的兴奋性、适应性、探究、紧张、记忆等行为进行评价。 1 材料和方法 1.1.1 动物SD大鼠, 雄性, 由北京实验动物研究中心提供( 许可证编号SCXK( 京) 2002-2003) 。 1.1.2 试剂氢溴酸东莨菪碱注射液( Scopolaminehydrobromide) 为上海禾丰制药有限公司产品( 批号:950901) , 鹅膏覃氨酸( Ibotenic acid, IBO) 为美国Sigma公司产品。 1.1.3 模型制备 1.1.3.11 老年动物组: 正常24月龄雄性SD大鼠; 青年对照组为3月龄大鼠。 1.1.3.12 基底前脑损伤大鼠: 采用3月龄SD大鼠,1%戊巴比妥钠麻醉, 脑立体定位仪固定, 平颅头位,按大鼠脑定位图谱, 在AP( 前囟后) : 018mm; ML( 中线旁) : 310mm; DV( 自脑表面深度) : 710mm; 双侧注射1LL鹅膏覃氨酸( 10LgPLL, 溶于生理盐水中) 。假手术对照组注射等量生理盐水, 手术后1个月进行实验。 1.1.3.13 东莨菪碱模型: 3月龄大鼠, 按5mgPkg体重于实验前20min腹腔注射氢溴酸东莨菪碱溶液。以上述青年对照组为对照。
动物实验基本操作一（固定、性别判定、标识） 【实验目的】在做动物试验时，为确保给药、实验顺利进行，防止被动物咬伤、准确辨别动物性别、准确标识动物，要学会用正确方法捉拿实验动物、掌握辨别动物性别的方法以及掌握标识动物的方法。 【实验对象】SD大鼠，KM小鼠，雌雄各半，体重180-250g。 【实验器材和药品】 器材：鼠笼、大小鼠固定器、方木板、美式图钉、细绳、防护手套 药品：苦味酸80%~90%酒精饱和溶液、20%乌拉坦 【实验步骤】 一、小鼠的捉拿 1、徒手固定：用右手提起尾巴中部，放在鼠笼盖或其他粗糙面上。向后上方轻拉，此时，小鼠前肢紧紧抓住粗糙表面。左手拇指和食指迅速捏住小鼠颈背部皮肤，再置小鼠于左手心，并以左手掌心和中指夹住小鼠背部皮肤，无名指压住小鼠尾根部，将其固定于手中。右手可行注射或其它操作。 2、固定器固定：尾静脉注射或给药时，将小鼠放进固定器中或者大小和重量适当的容器（如烧杯），只露出尾巴，该类容器能够压住尾部，避免其活动。勿固定过紧造成窒息死亡。进行腹腔手术或心脏采血时，先准备一个15-20cm的方木板，边缘钉入五颗钉子。将小鼠四肢分别用20-30cm的线绳捆绑，线的另一头分别绑在方木板的钉子上，并且在头部上颚切齿牵引一根线绳，也固定在钉子上，达到完全固定。 二、大鼠的捉拿
4-5周内的大鼠，方法同小鼠。周龄较大的，则：1、首先戴好防护手套。2、用右手拇指和食指抓住大鼠尾巴中部将大鼠提起，放在大鼠饲养盒的面罩上。3、左手顺势按、卡在大鼠躯干背部，稍加压力向头颈部滑行。4、以左手拇指和食指捏住大鼠两耳后部的头颈皮肤，其余三指和手掌握住大鼠背部皮肤，完成抓取保定。三、性别判定 小鼠、大鼠性别判定 （1）幼鼠外生殖器与肛门间隔短的是♀，外生殖器与肛门间隔长的是♂。 （2）成年动物可直接肉眼辨认，雄性有膨起的阴囊和阴茎，雌性动物有阴道口。四、动物的标记 小鼠的短期标记法：苦味酸80%~90%酒精饱和溶液（黄色），标出属于自己的编号【注意事项】 1、实验人员要有精神准备：掌握方法，胆大心细，做好防护。 2、动物兴奋的时候不要抓取，待其安静下来。 3、根据受试动物的给药部位或采血方法的不同，事先选择徒手固定还是固定器固定。 4、固定时把握好力度，过分用力会使小鼠颈椎脱臼或窒息死亡，若用力过轻头部能反转过来咬伤实验者的手。 【思考题】 1.在固定实验动物时如何才能快、准、稳？
呼吸系统疾病动物模型 （一）慢性支气管肺炎模型 常选用大鼠、豚鼠或猴吸入刺激性气体（如二氧化硫、氯、氨水、烟雾等）复制人类慢性气管炎。现发现猪粘膜下腺体与人类相似，且经常发生气管炎及肺炎，故认为是复制人类慢性气管炎较合适的动物。用去甲肾上腺素可以引起与人类相似的气管腺体肥大。 （二）肺气肿模型 给兔等动物气管内或静脉内注射一定量木瓜蛋白酶、菠罗蛋白酶（Bromelin）、败血酶（Alcalas）、胰蛋白酶（Trypsin）、致热溶解酶（Thermolysin），以及由脓性痰和白细胞分离出来的蛋白溶解酶等，可复制成实验性肺气肿。以木瓜蛋白酶形成的实验性肺气肿病变明显而且典型，或用瓜蛋白酶基础上再加用气管狭窄方法复制成肺气肿和肺心病模型，其优点是病因病变更接近于人。猴每天吸入一定深度的SO2和烟雾（烟草丝50g，持续2.5小时），一年后，可出现不同程度的肺气肿。这种模型比较符合人的临床发病规律，有利于进行肺气肿的病理生理及药物治疗研究。还可用1%三氯化铁水溶液1～3ml，自兔耳静脉注入，每周2～3次，可在短期内造成肺心病模型。 （三）肺水模肿型 用氧化氮吸入可造成大鼠和小鼠中毒性肺水肿，或用气管内注入50%葡萄糖液（家兔及狗分别为1及10ml）引起渗透性肺气肿。麻醉下用37～38℃生理盐水注入兔颈外静脉或股静脉使血液总量增加0.6～1倍（血液总量相当体重1/12），可形成稀血性多血症肺水肿。切断豚鼠、家兔、大鼠颈部两则迷走神经可引起肺水肿。家兔（1.5～2kg）耳静脉注入1：1000肾上腺素0.54～0.6毫克，可使动物发生肺水肿并在5～15分钟死亡，肺系数自4.1～5g/kg 增至6.3～12.5g/kg；5mg肾上腺素肌注，8分钟左右大鼠死亡，肺系数20g/kg，静脉注入10%氯仿（兔0.1ml/kg，狗0.5ml/kg）也可引起急性肺水肿。腹腔注入6%氯化铵水溶液可引
O RIGINAL A RTICLE Paclitaxel Poliglumex(PPX-Xyotax)and Concurrent Radiation for Esophageal and Gastric Cancer A Phase I Study Tom Dipetrillo,MD,*Luka Milas,MD,?Devon Evans,MD,*Paul Akerman,MD,*Thomas Ng,MD,* Tom Miner,MD,*Dennis Cruff,MD,*Bharti Chauhan,MD,*David Iannitti,MD,* David Harrington,MD,*and Howard Safran,MD* Objectives:To determine the maximal tolerated dose(MTD)and dose limiting toxicities of poly(L-glutamic acid)-paclitaxel(PPX)and con-current radiation(PPX/RT)for patients with esophageal and gastric cancer. Methods:Patients with esophageal or gastric cancer receiving chemo-radiation for loco-regional,adjuvant,or palliative intent were eligible. The initial dose of PPX was40mg/m2/wk,for6weeks with50.4Gy radiation.Dose levels were increased in increments of10mg/m2/wk of PPX. Results:Twenty-one patients were enrolled over5dose levels.Sixteen patients had esophageal cancer and5had gastric cancer.Twelve patients received PPX/RT as de?nitive loco-regional therapy,4patients had undergone resection and received adjuvant PPX/RT,and5patients had metastatic disease and received PPX/RT for palliation of dyspha-gia.Dose limiting toxicities of gastritis,esophagitis,neutropenia,and dehydration developed in3of4patients treated at the80mg/m2dose level.Four of12patients(33%)with loco-regional disease had a complete clinical response. Conclusions:The maximally tolerated dose of PPX with concurrent radiotherapy is70mg/m2/wk for patients with esophageal and gastric cancer. Key Words:paclitaxel polyglumex,concurrent chemoradiation, esophageal cancer,gastric cancer,macromolecule drug conjugate (Am J Clin Oncol2006;29:376–379) P PX is a drug conjugate that links paclitaxel to a biodegrad-able polymer,poly-L-glutamic acid.1Preclinically,poly(L-glutamic acid)-paclitaxel(PPX)has demonstrated tumor tissue radiation enhancement factors from4.0to8.0compared with1.5 to 2.0for paclitaxel.2PPXs macromolecular structure may underlie its improved radiation enhancement.PPX has a molec- ular weight of40,000compared with854for paclitaxel.3 Solid tumors are more permeable to macromolecules than normal tissue because of altered capillary endothelium.4Further- more,macromolecules persist longer in tumor tissue because of the relative decrease in lymphatics compared with non-neoplas- tic tissue.5 Radiation increases the vascular permeability of solid tumors.6,7Local tumor irradiation in tumor cell lines increases vascular permeability and increases PPX uptake.2Tumor se- creted vascular endothelial growth factor also increases vascular permeability.8The combination of PPX and radiation in the ovarian Oca-1carcinoma implanted in C3Hf/Kam mice pro- duced a signi?cantly greater tumor growth delay than treatment with radiation and paclitaxel when both drugs were given at an equivalent paclitaxel dose(enhancement factors of4.44versus 1.50).2These preclinical?ndings support the hypothesis that the supra-additive effect of combined PPX/RT is because of the modulation of the enhanced permeability and retention effect of macromolecules by radiation. Paclitaxel is an important radiation sensitizer in upper gastrointestinal malignancies.9–14We initiated a phase I trial of PPX/RT for patients with esophageal and gastric cancer.This is the?rst report of a clinical study utilizing a poly L-glutamic acid macromolecule as a radiation sensitizer in human malignancies. MATERIALS AND METHODS Eligibilty Patients with a histologically con?rmed diagnosis of esoph- ageal adenocarcinoma or squamous cell carcinoma,or gastric ade- nocarcinoma were enrolled between January2004and May2005. PPX/RT could be administered as de?nitive loco-regional or adjuvant therapy.Patients with metastatic disease with dyspha- gia were also eligible.Required laboratory parameters included absolute neutrophil count?1,500?L hemoglobin?10g/dL, platelets?100,000?L,creatinine?1.5mg/dL,bilirubin?1.5 mg/dL,alanine aminotransferase,and alkaline phosphatase ?2.5?upper limit of normal,and calcium?12mg/dL.Patients with previous irradiation,Eastern Cooperative Oncology Group (ECOG)performance status?1,life expectancy?12weeks, tracheobronchial invasion,tracheoesophageal?stula,unstable From The*Brown University Oncology Group,Providence,RI;and the ?Department of Experimental Radiation Oncology,The University of Texas M.D.Anderson Cancer Center,Houston TX. Reprints:Devon Evans,MD,Brown University,Department of Hematology/ Oncology,164Summit Avenue,Providence,RI02906.E-mail:devans@ https://www.doczj.com/doc/484325575.html,. Copyright?2006by Lippincott Williams&Wilkins ISSN:0277-3732/06/2904-0376 DOI:10.1097/01.coc.0000224494.07907.4e
006111 7 栏目中药药物评价>>非临床安全性和有效性评价 标题银屑病药效学动物模型及其在中药新药研究中应用的思考 作者韩玲 部门 正文内容 审评一部韩玲 摘要：本文综述了银屑病药效学动物模型的进展，对治疗银屑病中药新药 药效学研究方面存在的问题进行了分析，提出了应结合银屑病发病机制的 研究进展完善药效学方法学研究的建议。 银屑病是一种慢性复发性皮肤病，其发病机制复杂，至今尚未明了。可能是一种具有基因遗传特性的疾病，也与感染、内分泌机能障碍、免疫功 能紊乱等有关。中医学认为以风湿毒邪内侵为标，血虚、血燥为根源，血 热、血虚、血瘀是本病的基本病机，故清热解毒、凉血、活血化瘀、祛风 除湿成为治疗本病的基本法。 银屑病的组织病理学主要表现为角质形成细胞过度增生、炎症细胞聚集和真皮乳头部血管增生扩张三大特征，从而出现角化过度、角化不全、颗 粒层消失、棘细胞层增厚等。尽管许多不同的动物模型能复制银屑病的某 个方面的病理特征，并能解释部分病因，但长期以来仍缺乏一种能够研究 所有有关的潜在因素的动物模型。即-缺乏理想的动物模型。 一、银屑病药效学动物模型 银屑病动物模型是根据银屑病的特征，人为建立的一种与之相似的模型。 有人总结了银屑病动物模型的建立过程，包括以下4个不断完善的阶段： 1．诱发性动物模型 该模型是通过工方法在动物，如豚鼠、大鼠等皮肤上诱发银屑病样增生。
其方法包括紫外线照射、化学刺激剂外涂或药物外涂、缺乏必须脂肪酸的饲料喂养等。这些方法均为过度增殖模型，可引起表皮增生加快。但这些方法仅是针对表皮损伤的一种迟发反应，持续时间较短。 如缺乏必须脂肪酸饲料喂养的大鼠模型，因缺乏必须脂肪酸，故可使大鼠皮肤逐渐出现角化过度、棘层肥厚、基底细胞有丝分裂加快，DNA合成增加等变化，表皮细胞呈慢性增生状态，与药物诱发的急性表皮增生相比，其与银屑病更为相似。 如心得安诱导银屑病动物模型，是利用心得安特有的药理作用，即心得安可阻断角阮细胞的β-肾上腺能受体而降低了细胞内cAMP水平所致。应用后可出现动物表皮角化过度、角化不全和棘层肥厚等类似银屑病的组织病理改变，其皮损并非是刺激性皮炎。黄畋[1]和Gaylarde[2]等分别用心得安成功地在豚鼠背部和耳部皮肤上制备了银屑病样皮肤模型，并发现药物可抑制其所致的表皮异常角化与增殖、角化不全等。 已有研究[3-4]发现IL-20是与银屑病密切相关的细胞因子，在体外可明显促进角质形成细胞增殖；IL-20的转基因鼠可出现皮损，其组织病理学改变与人类银屑病有相似之处；此外，IL-20受体在银屑病皮损处有高表达。有人在心得安造模的基础上，利用原位杂交等方法检测了动物皮损中IL-20 及角蛋白的K14,K16，K17表达水平的变化情况，发现正常皮肤IL-20的表达仅限于基底层，且为低表达，但模型动物皮损中基底层及棘层均有高表达，且显著高于正常组。应用IL-20反义寡核苷酸外涂皮肤后，可使其明显好转。也观察到皮损中角蛋白K14,K16表达水平明显增高。提示心得安所致的豚鼠银屑病样动物模型不仅在组织病理学，而且在分子水平上与人类银屑病皮损一致。从而在分子水平上证实了此动物模型的可行性。 上述这些方法的优点是可在短时间内复制出某些方面与银屑病相似的动物模型，方法简便、易行、重复性较好，可用于表皮增殖动力学、药物筛选等研究，但这些模型只是模拟了银屑病中的部分组织病理特征，在许多方面与诱发或自然产生的疾病毕竟存在差异。 2．裸鼠-皮损移植模型 该模型是将银屑病人皮损组织移植到裸鼠背部。其具体方法是，选择经组织学证实，2周内未经任何治疗的寻常型银屑病患者的典型皮损，麻醉后取全层皮肤，裸鼠背部麻醉后切除部分圆形皮肤至肌肉筋膜，将皮损间段缝合到移植点。因裸鼠是先天性无胸腺小鼠，其免疫缺陷，在一定情况
常用实验动物的实验基本操作技术 第一节常用实验动物的生物学特征 1．蛙（或蟾蜍）的生物学特点是什么？主要用于哪些实验？ 属于两栖变温动物，皮肤光滑湿润，有腺体无外鳞。蛙的心脏有两个心房，一个心室，心房与心室区分不明显，动静脉血液混合，有冬眠习性。生存环境比哺乳动物简单，在机能学实验中有多种实验选择该动物。如：①离体蛙心实验，常用来研究心脏的生理功能及药物对心脏活动的影响。②蛙的腓肠肌和坐骨神经可用于观察外周神经及其肌肉的功能，以及药物对周围神经、骨骼肌或神经肌肉接头的影响。③缝匠肌可用于记录终板电位。脊休克、脊髓反射、反射弧分析、肠系膜微循环等。在临床检验中，可用雄蛙作妊娠反应实验。 2．小白鼠的生物学特征是什么？主要用于哪些实验？ 小白鼠性情温顺，易于捕捉，胆小怕惊，对外来刺激敏感。它胃容量小，不耐饥渴，随时采食。在机能学实验中常选用该动物。故适用于大量的实验动物，如：某些药物的筛选实验、半数致死量（LD50）测定、药效比较、毒性实验、妊娠期20天左右，常用于避孕药实验及抗癌药实验。 3．大白鼠的生物学特征是什么？主要用于哪些实验？ 大白鼠性情温顺，行动迟缓，易于捕捉，但受惊吓或粗暴操作时，会紧张不安甚至攻击人。大鼠嗅觉发达，对外界刺激敏感，抵抗力较强。大鼠无胆囊，肾单位表浅，肝再生能力强。大鼠的血压反应比兔稳定，可用它作血压实验，也可用于慢性实验、抗炎、降脂、利胆、子宫实验及心血管系统的实验。药典规定该动物为催产素效价测定及药品指控中升压物质检查指定动物。 4．豚鼠生物学特征是什么？主要用于哪些实验？ 豚鼠性情温和，胆小，饲养管理方便，可群养。豚鼠耳蜗管发达，听觉灵敏，存在可见的普赖厄反射（听觉耳动反射），乳突部骨质薄弱。豚鼠对组织胺、人型结核杆菌很敏感。能耐受腹腔手术，使用于肾上腺机能的研究。其自身不能制造维生素C，是研究实验性坏血症的唯一动物。 5．家兔生物学特征是什么？主要用于哪些实验？ 家兔属于草食性动物，性情温顺但群居性差，听觉、嗅觉十分灵敏，胆小易惊，具夜行
药理学实验动物的基本操 作实验心得 Revised by Jack on December 14,2020
这次是第一次药理学实验，我们学习了很多实验动物的基本操作方法。在做药理学实验之前我们就有做过人体解剖生理学实验，解剖过蟾蜍，小白鼠和家兔，这次的药理学实验更是一次对所学知识的巩固和深化，教给了我们很多在生理学实验中并没有学过的知识点。在刚做实验时，黄老师就向我们介绍了3R原则，即减少，优化，替代。动物是我们人类的朋友，首先我们应该尊重动物。它们用生命来换取人类的健康，推动着医学的进步，人类医学的发展离不开动物实验，动物为我们人类的健康做出了牺牲，我们应遵循“3R”原则。黄老师还通过视频给我们重点介绍了常用麻醉药及用法，实验动物的捉拿、麻醉、固定、给药、取血和处死方法。让我学到了很多实用性很强的东西。 之后便是分组自己做实验了，首先陈老师向我们讲解了小白鼠的标记方法，用中性品红表示十位数，苦味酸表示个位数，加上空白对照，一共可以标记一百支小白鼠。标记顺序为先左后右，从上至下。用苦味酸作为标记物的一个原因是它不容易被分解和弄掉，不会因为小鼠的活动而消失。其次是因为其有苦味，避免了被其他老鼠舔掉。之后讲解了小鼠的性别鉴定方法，除了书上说的方法外还可通过观察小鼠的乳房辨别。关于大鼠和家兔的捉持方法，大鼠在捉持前最好对其进行安抚，避免其急躁而咬人，而家兔则不可用手扯其双耳将其拉起。在给药方法方面，灌胃法要注意从口角插入口腔，用灌胃针抵住舌头，插入不可过深，一般入喉即可。腹腔注射时最好将其倒转，头部朝下，这样不容易刺入内脏。是否插入腹腔的判断方法：推注完后，轻微回抽，若有负压将注射器的推杆拉回，则已入腹腔。皮下注射时是否的入皮下的判断方法上同，皮下无负压，回抽不拉回。尾静脉注射时注意静脉在尾的两侧，不在上下，注射时用手捏住尾巴前段有利于暴露血管。 家兔的灌胃用木质开口器，使用时要想办法将其舌头压在开口器下，因为舌头会阻碍导尿管插入口腔，可以另外使用棉签配合，一边用棉签压住舌头，一边将开口器插入口腔。耳缘静脉注射时要注意选择小号针头，因家兔耳静脉较小，插入时应仔细谨慎。