Fluorescence spectroscopy for in vivo characterization of ovarian tissue

BACKGROUND AND OBJECTIVE: The objective of this study was to explore whether fluorescence spectroscopy signatures differed between normal variations within the ovary, benign neoplasms, and ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Ovarian tissue fluorescence emission spectra were collected sequentially at 18 excitation wavelengths ranging from 330 to 500 nm from 11 patients undergoing oophorectomy and assembled into fluorescence excitation emission matrices (EEMs); biopsies corresponded to the area interrogated. Spectral areas that could differentiate normal ovary, benign neoplasms, and cancers were evaluated, using histopathology as the reference standard. RESULTS: The most promising measurements are (1) the integrated fluorescence intensity from 400 to 430 nm excitation at 460 nm emission, and (2) the ratios of fluorescence intensities at 330 nm excitation, 385 and 500 nm emission, and at 375 and 415 nm excitation, 460 nm emission. Simple systems to visualize these optical signatures at laparoscopy could be designed. CONCLUSION: Fluorescence spectroscopy may have the ability to distinguish ovarian cancers from normal ovarian structures and benign neoplasms, as well as differentiate between normal variations and metaplastic structures and should be further explored as a device for the early detection of ovarian cancers.     

Biochemical, biomechanical, and physical changes in the skin in an experimental animal model of therapeutic tissue expansion

Biochemical, biomechanical, and physical changes occurring in the skin during tissue expansion have been studied using an animal model in which a Silastic expander was inserted into the peritoneal cavity. Forty-eight Sprague-Dawley rats were divided into four groups to be studied at 4, 8, 16 and 32 days after expansion. In the experimental animals (6 per group) the expander was inflated by a single injection of 120 ml of saline. Sixteen hours prior to sacrifice each animal received a single injection of tritiated proline. Sixteen days after expansion both the specific activity and the total content of hydroxyproline in the skin were significantly elevated in experimental animals. Intrinsic skin tension increased dramatically at the time of inflation but fell almost to control values at the end of 32 days. Skin thickness, initially decreased, returned to normal by the end of the experiment. There were no significant differences in breaking strengths between skin from experimental and control animals. Skin surface area, initially increased by stretching at the time of inflation, increased further between Days 0 and 8, possibly as a result of stress relaxation combined with enhanced remodelling of connective tissue macromolecules, and again from Days 24 to 32. We conclude that, during tissue expansion, there is a net accumulation of collagen in the skin and that this allows the local cellular environment to return to normal with respect to pressure and/or tension.     

Characterization of the immunosuppressive effect of burned tissue in an animal model

The immunosuppressive effect of burned tissue was studied using a mouse burn model. To evaluate the immunologic status an in vivo measure of cell-mediated immunity (CMI) involving contact sensitization of mice by painting the skin with dinitrofluorobenzene was used; mice were challenged 5 days later by painting the ear with the same antigen. Ear swelling in response to antigenic challenge was used as a quantitative measure of CMI; diminution in ear swelling in treatment mice compared to sensitized, unburned control mice indicated the degree of immunosuppression. A full-thickness steam burn covering 20% body surface ares (BSA) was profoundly immunosuppressive as reflected by ear swelling of 45 to 60% of that found in normal mice; partial thickness burns and burns of 10% BSA extent were not significantly immunosuppressive. Transfer into unburned mice of burned skin equivalent in size to a 20% BSA burn eschar resulted in marked immunosuppression, but transfer of smaller amounts of burned skin, or of larger amounts of unburned skin and normal and burned liver tissue, did not produce immunosuppression. Mice receiving a very high-temperature (300°C), dry burn were only slightly more suppressed than mice receiving a standard steam burn. Normal immunity was preserved in burned mice which received daily application of cerium nitrate to the wound for 7 days, but application of other topical agents commonly used in burn treatment did not preserve immunity. Postburn immunosuppression thus appears related quantitatively to toxic factors in burned skin, and these toxic factors can be abrogated in burned mice by the topical application of cerium nitrate.     

Evaluation of tissue oxygen measurements for flap monitoring in an animal model

Tissue oxygen tension (p (ti)O (2)) measurements are common in neurosurgery but uncommon in plastic surgery. We examined this technique as a monitoring method with probe placement in the subcutaneous tissue and addressed the importance of probe placement. Myocutaneous flaps were raised in an animal model and p (ti)O (2) measurements performed at different levels in the subcutaneous fat. Flap artery and vein were occluded until a 50% p (ti)O (2) reduction had occurred (T (1/2)). We found no significant effect of depth ( P > 0.10) on the level of p (ti)O (2). T (1/2)(arterial) was 7.2 minutes and T (1/2)(venous) was 18 minutes. We found no significant relation between intitial levels of p (ti)O (2) and T (1/2). Location of the probe and absolute p (ti)O (2) value is of little relevance for flap monitoring. It is the relative change in p (ti)O (2) that is important. The p (ti)O (2) technique is well suited for monitoring in the subcutaneous tissue and is highly sensitive to changes in both arterial and venous blood flow.     

Hemodynamic changes during reperfusion of the graft in an animal model of liver xenotransplantation

Our goal was to determine the hemodynamic changes that are witnessed during the initial minutes of reperfusion of the graft in liver xenotransplantation from pig to baboon. METHOD: We studied a group of 12 baboons undergoing transplantation of a pig liver via the classic technique with arterial anastomosis to the aorta. The anesthesia technique was similar to that used in humans. Hemodynamic monitoring, due to the size of the recipient, consisted of heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP) recorded at the beginning and end of each of the three phases: preanhepatic (A1, A2), anhepatic (B1, B2), and neohepatic (C1 and C2). We aimed to maintain the following values by means of crystalloids, colloids, and blood derivates: HR >50 beats/minute; MAP >60 mm Hg; and CVP >10 mm Hg. RESULTS: Both HR and CVP remained unchanged throughout the procedure. MAP droped briefly after vascular clamping (B1) but not on reperfusion (C1). CONCLUSION: In cirrhotic patients there is an autonomic dysfunction, demonstrated as cardiovascular instability at times like the clamping of major vessels and reperfusion of the graft. On the other hand, the intact baboon has an intact nervous system. After vascular clamping, the sharp decrease in venous return lead to an adequate vasopressor response. Likewise, the extreme vasodilatation involved with reperfusion managed to maintain MAP above 70 mm Hg.     

Standardized qualitative evaluation of scar tissue properties in an animal wound healing model

There is a great need to establish reproducible methods for evaluative studies of wound treatment and wound healing. Validation of the healing process through optical techniques, as well as histologic and immunohistochemical methodologies, have been improved and to some extent have become well-established assays. Data relating to biomechanical properties, e.g., evaluation of the tensile strength of scar tissue that forms in experimental wound treatment strategies, are less widely available. We chose the domestic pig as an animal model in which to examine epidermal wound healing. We implanted specially made chambers that served to isolate the wounds and prevent epidermal migration from the edges. We performed histologic and immunohistochemical analyses as well as evaluation of biomechanical qualities of scar tissue using laser tensiometry. Pig skin is well suited for wound healing studies, and wound creation, implantation of the chambers, and the regular changing of dressings could all be carried out in the operating theater. In addition to established macroscopic evaluation and microscopic documentation, the need for objective biomechanical assessment of scar tissue by measuring tensile strength has been met using laser tensiometry. By optimizing methods for measuring tensile strength, it is possible to evaluate the biomechanical quality of scar tissue formed following different courses of wound treatment, as well as histologic assessment.     

Effect of optical clearing agents on the in vivo optical properties of squamous epithelial tissue

BACKGROUND AND OBJECTIVES: Optical clearing agents (OCAs) have previously been shown to increase depth penetration within turbid tissue ex vivo. This paper quantifies tissue optical properties of the hamster cheek pouch model in order to provide a means to assess the effect of OCAs quantitatively in vivo. STUDY DESIGN/MATERIALS AND METHODS: Diffuse reflectance spectra were obtained from both cheeks of 12 hamsters before and after immersion in dimethyl sulfoxide (DMSO), glycerol or a phosphate buffer saline (PBS) control for 20 minutes. A Monte Carlo model was then utilized to derive the wavelength dependent reduced scattering and absorption coefficients. RESULTS: DMSO caused a statistically significant decrease in the absorption and reduced scattering coefficients derived by the model. Glycerol caused a statistically significant increase in the wavelength dependent absorption coefficient, but no statistically significant changes in the reduced scattering coefficient. CONCLUSIONS: DMSO and glycerol act upon tissues differently as reflected by the tissue optical properties, implying that not all OCAs are equally effective in optically clearing tissues.     

AsRed2示踪前列腺癌肺转移模型的建立

目的:使用携带红色荧光蛋白(AsRed2)的前列腺癌PC-3细胞(PR7细胞)建立可用活体荧光成像技术监测的前列腺癌肺转移模型。方法:分别采用MTT及Transwell实验比较前列腺癌PC-3与PR7细胞体外增殖、迁移及侵袭能力;将20只BALB/c裸鼠随机平均分为4组,分别尾静脉注射不同浓度PR7细胞悬液200μl,A组:1×107/ml,B组:2.5×107/ml,C组:5×107/ml,D组:2.5×107/ml,并于1周后重复注射2.5×107/ml细胞悬液200μl。第2、4、6、8周分别用活体荧光成像技术检测各组肺转移灶形成情况。结果:前列腺癌PC-3细胞与PR7细胞体外增殖、迁移、侵袭能力均无明显差异(P>0.05);第4周D组40%裸鼠用活体荧光成像检测到肺转移灶;第8周,A组20%、B组60%、C组100%、D组100%裸鼠可检测到肺转移灶;通过病理检查证实肺转移灶形成。结论:通过尾静脉注射携带红色荧光蛋白的前列腺癌PR7细胞可成功建立用活体荧光成像技术监测的前列腺癌肺转移模型。     

"穿刺抽取髓核"诱导腰椎间盘退变的时间相关性评估

目的对穿刺纤维环抽取髓核诱导的腰椎间盘退变模型，进行时间相关的放射学和组织学评估，明确椎间盘退变程度的时间相关性。方法1岁山羊12只，以粗针穿刺纤维环抽取髓核（L1，2-15，6）建立腰椎间盘退变模型。术后第2、4、8、16周分别行放射学观察、髓核蛋白多糖（GAG）定量、组织学及微观结构评估。结果影像学示术后16周椎间隙高度显著降低（P〈0．01），但各椎间盘间差异无统计学意义（P〉0．05）。GAG定量示所有节段髓核内GAG含量随时间持续下降（P〈0．01），但与椎间盘序列间无相关性。大体标本及组织学观察显示，椎间盘退变组织学表现与抽取髓核后时间显著相关：术后2周组织学未见明显异常；4周始出现退变表现；16周时髓核已近完全纤维化。电镜观察示术后2—16周，髓核细胞从基本正常至大量凋亡，髓核基质逐渐纤维化。结论针刺抽取髓核法是较理想的腰椎间盘退变模型诱导方法。本研究观察16周，椎间盘退变未见缓解及自行修复，诱发的退变严重度与术后时间因素显著相关，而与椎间盘节段序列间无相关性。该模型在术后2周尚未出现明显组织学改变，或许是进行干预的良好时机。     

Cardiopulmonary changes during laparoscopy and vessel injury: comparison of CO2 and helium in an animal model

BACKGROUND: Injury of venous vessels during elevated intraperitoneal pressure is thought to cause possible fatal gas embolism, and helium may be dangerous because of its low solubility. METHODS: Twenty pigs underwent laparoscopy with either CO2 (n=10) or helium (n=10) with a pressure of 15 mm Hg and standardized laceration (1 cm) of the vena cava inferior. After 30 s, the vena cava was clamped, closed endoscopically by a running suture and unclamped again. During the procedure changes of cardiac output (CO), heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), pulmonary artery pressure (PAP), pulmonary artery wedge pressure (PAWP), end tidal CO2 pressure (PETCO2), and arterial blood gas analyses (pH, pO2 and pCO2) were investigated. RESULTS: No animal died during the experimental course (mean blood loss during laceration: CO2, 157+/-50 ml; helium, 173+/-83 ml). MAP and CO values showed a decrease after laceration of the vena cava in both groups that had already been completely compensated for before suturing. PETCO2 increased significantly after CO2 insufflation (P<0.01), while helium showed no effect. Laceration of the vena cava caused no significant changes in PETCO2 values in either group. Significant acidosis and an increase of pCO2 were only found in the CO2 group. CONCLUSIONS: The incidence of gas embolism during laparoscopy and accidental vessel injury seems to be very low. With the exception of acidosis and an increase of PETCO2 in the CO2 group, there were no differences in cardiopulmonary function between insufflation of CO2 and helium.     

Proprioception of the cruciate ligaments: receptor mapping in an animal model

Ten anterior and posterior cruciate ligaments (ACL and PCL) harvested from adult sheep were investigated under light microscopy for data on the frequency and localisation of neural structures. Serial sections of 25 μm thickness were stained with a modified gold chloride technique. Receptors were classified according to their histological structure. Topographic distribution and frequency within the ligament texture were determined with the help of computerized image analysis. Three distinct neural structures could be identified: Ruffini endings, Ruffini corpuscles of the Golgi tendon organ-like type and Pacinian corpuscles. Golgi tendon organs were not found. In total, 274 and 238 neural structures were present in the 10 ACL and 10 PCL, respectively. Pacinian receptors were the most common structures, with a mean frequency of 13.6 ± 5.3 (ACL) and 12.4 ± 5.1 (PCL), followed by Ruffini endings with 8.9 ± 3.2 (ACL) and 7.8 ± 2.9 (PCL), whereas Ruffini corpuscles had the lowest frequency with a mean value of 4.9 ± 2.1 (ACL) and 3.4 ± 1.1 (PCL). The majority of the neural structures were located in the subsynovial sheath or closely associated with endotenon structures. The tibial and femoral insertion areas had a significantly increased receptor density compared with the midpart of the ACL and PCL (P < 0.001), where only 19.3% and 23.7% of the receptors were located. These results emphasise the complex sensory structure of the cruciate ligaments and provide a valid morphological basis for further neurophysiological investigations. Received: 3 June 1997     

Numerical simulation of hemodynamic changes during beating-heart surgery: analysis of the effects of cardiac position alteration in an animal model

Hemodynamic instability, mostly due to vertical lifting of the heart, is usually observed during beating-heart surgical procedures. However, some hemodynamic parameters, such as coronary blood flow, are not routinely measured. A digital computer model of the circulation able to simulate and analyze the effects of heart lifting and the Trendelenburg maneuver, and thus supply detailed hemodynamic information to the clinicians would provide a useful analytical tool. A lumped parameters model of the circulation was applied to both beta-blocked and not beta-blocked pigs. The results confirmed a drop of cardiac output and coronary flow during heart lifting and a rise of both variables after the Trendelenburg maneuver for beta-blocked animals. In not beta-blocked pigs, the analysis was more complex but the model reproduced experimental data and permitted coronary flow to be estimated. These results showed the feasibility of numerical simulation for specific circulatory conditions encountered during beating-heart surgery.     

A cell-based approach to study changes in the pancreas following nicotine exposure in an animal model of injury

Background Cigarette smoking is a recognized risk factor for the induction of pancreatic diseases and is suspected to play a major role in the development of pancreatic cancer in smokers. Materials and methods This study was designed to characterize the mechanisms of nicotine-induced injury to the pancreas. AR42Jcells, a stable mutant pancreatic tumor cell line, was chosen for the study because of its stability in culture media and also because of its known secretory capacity, which is like that of a normal pancreatic acinar cell. It is hypothesized that nicotine-induced effects on the pancreas are triggered by oxidative stress induced in pancreatic acinar cell via oxidative stress signaling pathways. Results The results from our study showed that, in vitro, nicotine induced generation of oxygen free radicals measured as malondialdehyde, an end product of lipid peroxidation. Treatment of AR42J cells with nicotine induced p-ERK 1/2 activation as confirmed by Western blot and immunofluorescence imaging of cytoplasmic localization of mitogen-activated protein kinase (MAPK) signals. Nicotine enhanced AR42J cell proliferation and cholecystokinin-stimulated amylase release in AR42J cells. These effects of nicotine were confirmed by simultaneous studies conducted on the same cells by hydrogen peroxide, a known oxidative biomarker. Allopurinol, a XOD inhibitor, suppressed these effects induced by nicotine and H₂O₂ with the exception that cholecystokinin-stimulated amylase release by H₂O₂ remained unaltered when AR42J cells were preincubated with allopurinol. These results suggest that nicotine-induced effects on pancreatic acinar cells were associated with generation of oxyradical mediated via the XOD pathway. The results have a direct impact on cell proliferation, MAPK signaling, and acinar cell function. Conclusion We conclude that nicotine induces oxidative stress in pancreatic acinar cells and that these events trigger pathophysiological changes in the pancreas, leading to increased cell proliferation and injury. A part of this communication was presented in the International Pancreatic Cancer meeting held on September 5–9, 2006, in Schloss Reisensburg, Ulm, Germany     

一种可用活体荧光成像技术检测的前列腺原位肿瘤模型的建立

目的建立可用活体荧光成像技术动态观察前列腺原位肿瘤发生及生长的裸鼠模型。方法 10只BALB/c裸鼠前列腺背侧包膜内注入携带红色荧光蛋白（RFP）的前列腺癌PC-3细胞（PR7细胞）悬液20μL,第2、4、6、8周分别用非侵入式活体分子影像系统观察前列腺原位肿瘤发生及其转移情况。尸检取前列腺原位肿瘤、肺、肝、肾、股骨、盆腔淋巴结等组织,制成冰冻切片,通过荧光显微镜下观察及HE染色等方法分别检测肿瘤发生及有无转移等情况。结果第2周开始可用非侵入式活体分子影像系统检测到前列腺原位肿瘤形成,第4周开始可于裸鼠下腹部触及原位肿瘤,第6～8周裸鼠逐渐出现恶液质。荧光信号随原位肿瘤长大而增强,但并未见明显转移灶。尸检也未发现转移灶。结论前列腺包膜内注入携带RFP的前列腺癌PR7细胞可成功建立可用活体荧光成像技术监测的前列腺原位肿瘤模型,该模型可作为研究前列腺肿瘤发生发展及治疗方式的较理想的动物模型。     

Psychological stress accelerates the onset of tumour formation and alters the type and location of tumours in a DMBA mouse carcinogenesis model

Psychological stress is recognized as a factor that contributes to increased susceptibility to a number of diseases, including cancer. Psychological stress, via release of chemical mediators, can induce long‐term changes in the organism resulting in an altered responsiveness of the organism to external carcinogens. The present investigation sought to evaluate the impact of stress on polycyclic aromatic hydrocarbon (PAH)‐induced carcinogenesis. We utilized a repetitive restraint stress mouse model and the model PAH, 7,12‐dimethylbenz[a]anthracene (DMBA). Restraint stress was applied three times a week for 6 weeks and DMBA was administered intra‐gastrically once a week for 6 weeks and formation of skin, mammary and ovarian tumours was examined at 26 weeks by pathological analyses. The results indicate that stress accelerates the onset of tumour formation produced in response to DMBA and significantly influences the type and location of tumours. Taken together, these results demonstrate that stress enhances the carcinogenicity of DMBA. These results clearly indicate the need for further characterization of the impact of stress on carcinogen metabolism in oncology. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of sodium hydroxide exposure on esophageal epithelial cells in an in vitro ovine model: implications for esophagus tissue engineering

BackgroundEsophagus tissue engineering holds promises for esophageal replacement after severe caustic injuries. The aim of this study was to determine whether viable esophageal epithelial cells could be isolated from an esophagus exposed to varying concentrations of alkali with regard to number, viability, and morphology during in vitro culture.MethodsOvine esophagi were exposed to phosphate-buffered saline 2.5%, 15%, or 25% sodium hydroxide (NaOH). The effect of NaOH concentrations on epithelial damage was assessed histologically. Esophageal epithelial cells were then isolated, and cell count and viability were investigated. Finally, cell number, viability, and morphology of esophageal epithelial cells were determined for 24 days of in vitro culture.ResultsHistologic analysis showed a progressive destruction of the epithelium proportional to increasing NaOH concentrations. Esophagi treated with phosphate-buffered saline and 2.5% NaOH showed significantly higher viable cell counts after isolation and culture in comparison with those treated with 15% to 5% NaOH.ConclusionThe evidence presented in this study indicates that epithelial biopsies from an esophagus exposed to low concentrations (2.5%) of NaOH will still yield large numbers of viable cells suitable for tissue engineering applications. In cases of exposure to higher concentrations (15%-25%), alternative cell sources for epithelial regeneration, such as stem cells, will be necessary for tissue engineering applications.     

Collagen gel contraction as a measure of fibroblast function in an animal model of subsynovial connective tissue fibrosis

Carpal tunnel syndrome (CTS) is a peripheral neuropathy characterized by non‐inflammatory fibrosis of the subsynovial connective tissues (SSCT). A rabbit model of CTS was developed to test the hypothesis that SSCT fibrosis causes the neuropathy. We used a cell‐seeded collagen‐gel contraction model to characterize the fibrosis in this model in terms of cellular mechanics, specifically to compare the ability of SSCT cells from the rabbit model and normal rabbits to contract the gel, and to assess the effect of transforming growth factor‐β1,which is upregulated in CTS, on these cells. SSCT fibrosis was induced in six retired breeder female rabbits which were sacrificed at 6 weeks (N = 3) and 12 weeks (n = 3). An additional two rabbits served as controls. SSCT was harvested according to a standard protocol. Gels seeded with SSCT cells from rabbits sacrificed at 6 weeks had significantly higher tensile strength (p < 0.001) and Young's modulus (p < 0.001) than gels seeded with cells from rabbits sacrificed at 12 weeks or control animals. TGF‐β1 significantly increased the decay time constant (p < 0.001), tensile strength (p < 0.001), and Young's modulus (p < 0.001) regardless of the cell source. This model may be useful in screening therapeutic agents that may block SSCT fibrosis, identifying possible candidates for CTS treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:668–674, 2015.