Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

PURPOSE: We aimed to assess the effects of a cocktail comprising three specific antiHER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. METHODS: Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of the three specific anti-HER2 antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phagescFv cocktail/kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. RESULTS: The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). CONCLUSION: Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Microplastics- and copper-induced changes in neurogenesis and DNA methyltransferases in the early life stages of zebrafish.

In this study, zebrafish embryos were exposed to microplastics (MPs, 2 mg/L) and copper (Cu, 60 and 125 µg/L), alone or combined, for 14 days, and the development of motor neurons was assessed through gene expression and immunohistochemistry. DNA methyltransferases (DNMTs) genes expression was also evaluated. The results showed a downregulation of neuronal proliferation (sox2, pcna), neurogenesis (neuroD, olig2), and motor neurons development (islet) related genes, implying potential deficits in the neurogenesis of the exposed zebrafish early life stages. Downregulation of the maintenance and de novo DNMTs expression was also found, indicating that the DNA methylation patterns could be modulated by MPs and Cu. A high relative volume of proliferating cell nuclear antigen (PCNA)-positive cells was found in the fish retina from the MPs exposed group, suggesting that MPs increased the rate of cellular division. In contrast, a significant decrease of PCNA-positive cells, and therefore a lower cell proliferation, was found in the retina and brain of zebrafish exposed to Cu and Cu + MPs, which could lead to cognitive and behavioral functions impairment. No alterations were found in the relative volume of ISL1&2-positive cells. This study contributes to the knowledge of the mechanisms by which MPs and Cu cause neurotoxicity, fundamental for a comprehensive and realistic ecological risk assessment in aquatic populations.

Possible role of antioxidative capacity of (-)-epigallocatechin-3-gallate treatment in morphological and neurobehavioral recovery after sciatic nerve crush injury.

OBJECTIVE This study examined the capacity of the major polyphenolic green tea extract (-)-epigallocatechin-3-gallate (EGCG) to suppress oxidative stress and stimulate the recovery and prompt the regeneration of sciatic nerve after crush injury. METHODS Adult male Wistar rats were randomly assigned to one of 4 groups: 1) Naïve, 2) Sham (sham injury, surgical control group), 3) Crush (sciatic nerve crush injury treated with saline), and 4) Crush+EGCG (sciatic nerve crush injury treated with intraperitoneally administered EGCG, 50 mg/kg). All animals were tested for motor and sensory neurobehavioral parameters throughout the study. Sciatic nerve and spinal cord tissues were harvested and processed for morphometric and stereological analysis. For the biochemical assays, the time points were Day 1, Day 7, Day 14, and Day 28 after nerve injury. RESULTS After sciatic nerve crush injury, the EGCG-treated animals (Crush+EGCG group) showed significantly better recovery of foot position and toe spread and 50% greater improvement in motor recovery than the saline-treated animals (Crush group). The Crush+EGCG group displayed an early hopping response at the beginning of the 3rd week postinjury. Animals in the Crush+EGCG group also showed a significant reduction in mechanical allodynia and hyperalgesia latencies and significant improvement in recovery from nociception deficits in both heat withdrawal and tail flick withdrawal latencies compared with the Crush group. In both the Crush+EGCG and Crush groups, quantitative evaluation revealed significant morphological evidence of neuroregeneration according to the following parameters: mean cross-sectional area of axons, myelin thickness in the sciatic nerve (from Week 4 to Week 8), increase of myelin basic protein concentration and gene expression in both the injured sciatic nerve and spinal cord, and fiber diameter to axon diameter ratio and myelin thickness to axon diameter ratio at Week 2 after sciatic nerve injury. However, the axon area remained much smaller in both the Crush+EGCG and Crush groups compared with the Sham and Naïve groups. The number of axons per unit area was significantly decreased in the Crush+EGCG and Crush groups compared with controls. Sciatic nerve injury produced generalized oxidative stress manifested as a significant increase of isoprostanes in the urine and decrease of the total antioxidant capacity (TAC) of the blood from Day 7 until Day 14. EGCG-treated rats showed significantly less increase of isoprostanes than saline-treated animals and also showed full recovery of TAC levels by Day 14 after nerve injury. In spinal cord tissue analysis, EGCG-treated animals showed induced glutathione reductase and suppressed induction of heme oxygenase 1 gene expression compared with nontreated animals. CONCLUSIONS EGCG treatment suppressed the crush-induced production of isoprostanes and stimulated the recovery of the TAC and was associated with remarkable alleviation of motor and sensory impairment and significant histomorphological evidence of neuronal regeneration following sciatic nerve crush injury in rats. The findings of this study suggest that EGCG can be used as an adjunctive therapeutic remedy for nerve injury. However, further investigations are needed to establish the antioxidative mechanism involved in the regenerative process after nerve injury. Only upregulation of glutathione reductase supports the idea that EGCG is acting indirectly via induction of enzymes or transcription factors.

Quantitative stereological analysis of the highly porous hydroxyapatite scaffolds using X-ray CM and SEM.

BACKGROUND: Material properties of the scaffolds as well as their microstructure are vital in determining in vivo cellular response. Three-dimensional (3D), highly porous scaffolds are used in tissue engineering to provide a suitable microenvironment and to support regeneration of bone. Both pore sizes and their architecture, in particular interconnection density, impact functionality of scaffold during its biomedical applications. OBJECTIVE: In this paper a comparative study of the microstructure of highly porous hydroxyapatite scaffolds produced via gelcasting of foamed slurries and replication of polyurethane sponge were carried out. METHODS: Quantitative stereological analysis of the microstructure was conducted using transmission X-ray computed microtomography (µCT) and scanning electron microscopy (SEM). Application of the X-ray microtomography allowed obtaining the 2D cross-sectional images of examined samples, and then the 3D reflection of individual samples. RESULTS: In our studies we proved that the distribution of pores in HAp bioceramics can be controlled by selection of the manufacturing method. In the case of material produced by the gelcasting method, the porosity of the samples was about ∼78 vol.%, while for the method of replication of the porous organic matrix it was higher ∼84 vol.%. Application of gelcasting method resulted in bioceramics with the macropores ranging from 95 µm to 158 µm (the modal value of 120 µm). Furthermore, micropores of size 34 µm-60 µm - so called "windows", were observed on spherical macropores surfaces. In the case of replication of polyurethane sponge only macropores from 295 µm to 337 µm (the modal value of 300 µm) were obtained. Application of µCT and SEM give more information than classical mercury intrusion porosimetry in studies of porous bioceramics. Developed materials met the criteria for porous bone substitutes. CONCLUSIONS: The results of quantitative description of microstructure allowed determining the differences between porous hydroxyapatite bioceramics obtained via replication of porous organic matrix and gelcasting of foamed slurry. The stereological analysis demonstrated, that bioceramics prepared via gelling of foamed slurry has a lower pore size and grains (1.1-1.9 µm) than the material obtained by the method of replication of polyurethane sponge (2.1-2.3 µm). Based on morphological analysis the porosity of tested materials was determined. In the case of material produce by the gelcasting, porosity of the samples was about ∼78 vol.%, while for method of replication of the porous organic matrix the porosity was higher and constituted ∼84 vol.%. Furthermore, evaluated materials varied in porosity and the pore size distribution. It was stated that the method of gelcasting resulted in hydroxyapatite bioceramics with the macropores diameter (95-158 µm), micropores so called "windows" (34-60 µm) - observed on spherical macropores walls and micropores of size 0.6 µm-1.3 µm, which were visible in sintered areas. When the method of replication of polyurethane sponge was applied only macropores from 295 µm to 337 µm were obtained. The comparable values of shape factors such as elongation, curvature of pours boundary and convexity, confirmed that macropores in both studied series had similar shape.

A clinically relevant in vivo model for the assessment of scaffold efficacy in abdominal wall reconstruction.

An animal model that allows for assessment of the degree of stretching or contraction of the implant area and the in vivo degradation properties of biological meshes is required to evaluate their performance in vivo. Adult New Zealand rabbits underwent full thickness subtotal unilateral rectus abdominis muscle excision and were reconstructed with the non-biodegradable Peri-Guard®, Prolene® or biodegradable Surgisis® meshes. Following 8 weeks of recovery, the anterior abdominal wall tissue samples were collected for measurement of the implant dimensions. The Peri-Guard and Prolene meshes showed a slight and obvious shrinkage, respectively, whereas the Surgisis mesh showed stretching, resulting in hernia formation. Surgisis meshes showed in vivo biodegradation and increased collagen formation. This surgical rabbit model for abdominal wall defects is advantageous for evaluating the in vivo behaviour of surgical meshes. Implant area stretching and shrinkage were detected corresponding to mesh properties, and histological analysis and stereological methods supported these findings.

5α-Dihydrotestosterone enhances wound healing in diabetic rats.

UNLABELLED: Wound healing involves a complex interaction between the cells, extracellular matrix and oxidative response. AIMS: Analyze the effects of 5α-Dihydrotestosterone (5α-DTH) ointment in cutaneous wound healing by secondary intention in diabetic Wistar rats. MAIN METHODS: Rats (302.23±26.23g, n=48) were maintained in cages with food and water ad libitum in accordance with the Guiding Principles in the Use of Animal Ethics Committee. Diabetes was induced by intraperitoneal injection of streptozotocin (60mg/kg). Three skin wounds (12mm diameter) were created on the animals' back, which were randomized into 6 groups according to the application received: VT group: Vehicle (lanolin), SA group: 0.9% saline solution, NC group: Non-diabetic, CP group: positive control (silver sulfadiazine 0001%), T1 group: Testosterone (10%), T2 group: Testosterone (20%) emulsified in lanolin. The applications were made daily within 21days, and tissues from different wounds were removed every 7days. KEY FINDINGS: Both groups treated with testosterone (T1 and T2) showed a significantly higher proportion of type I and type III collagen fibers. Superoxide dismutase levels were significantly higher on days 7 and 14 in testosterone treated groups. Protein carbonyls and MDA were lower in both groups. SIGNIFICANCE: We conclude that groups treated with 5α-DTH showed a better healing pattern with complete wound closure, and proved to have a positive effect on the morphology of the scar tissue as well as an antioxidant stimulating effect during secondhand intention skin wounds repair in diabetic rats.

Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods.

BACKGROUND: NeuN and Nissl staining (toluidine blue, cresyl violet staining) are routinely used methods in unbiased stereological estimation of the total number of hippocampal neurons. NEW METHOD: In the present study, we stained serial frozen coronal sections from 5 normal adult male Sprague-Dawley rat brains with different methods, measured the deformation of hippocampal area in brain sections and estimated the total number of hippocampal neurons using the optical fractionator. RESULTS: The deformation in x, y-axis was not obviously different with different staining protocols, but shrinkage in z-axis was significant after staining (p < 0.001). NeuN staining produced significant higher estimate number than cresyl violet staining by 24% (p = 0.002), however, NeuN and Cresyl Violet staining showed a high degree of correlation in quantification of total neuronal numbers and both methods are suitable for unbiased stereological estimation. COMPARISON WITH EXISTING METHOD (S): NeuN is more reliable but if time is limited or the number of animals used in experiments is high, cresyl violet staining may be a feasible method. CONCLUSIONS: Compared with previous estimates of the neurons number in rat hippocampus, our present data is reliable and the stereological analysis based on our system is a cost-effective unbiased method for estimation of neuron number.

Efficient estimation of the total number of acini in adult rat lung.

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) µL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model.

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each: Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250-1 500 human mesenchymal stem cells (total volume of 50 µL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may represent a very promising clinical tool in peripheral nerve reconstructive surgery. Yet, umbilical cord human mesenchymal stem cells, that can be expanded in culture and induced to form several different types of cells, may prove, in future experiments, to be a new source of cells for cell therapy, including targets such as peripheral nerve and muscle.

Effects of intrahepatic injection with hepatocyte growth factor and dexamethason on stereological quantitative changes of hepatic sinusoidal disease and expression of IV-collagen on hepatic sinusoidal walls / 中国基层医药

Objective To observe the effects of intrabepatic injection with bepatocyte growth factor and dexamethason on stereological quantitative changes of hepatic sinusoidal tissues and expression of IV-collagen on hepatic sinusoidal walls of patients with hepatic cirrhosis.Methods Under the guide of hypersound,98 cases of hepatic cirrhosis were intrahepatic injected with 80mg hepatocyte growth factor and 1mg dexamethason at one time,twice a week,12 times a course,pathological changes of hepatic sinusoidal tissues and the expression of IV-coliagen on hepatic sinusoidal walls were detected by liver biopsy after one course.Results Pathological changes of hepatic sinusoidal tissues were improved obviously(P＜0.01 ) and the expression of IV-collagen was alleviated significantly(P＜0.01 ) on 98cases of hepatic cirrhosis after one course.Conclusion Pathological changes of hepatic sinusoidal tissues and the expression of IV-collagen on hepatic sinusoidal wails can be improved obviously on patients with hepatic cirrhosis who had been intrahepatic injected with hepatocyte growth factor and dexamethason under the guide of hypersound.

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 ± 0.7 percent in 2-month-old Cx (N = 5) vs 13.4 ± 0.3 percent (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 ± 0.9 percent in 2-month-old Cx (N = 5) vs 15.5 ± 0.3 percent (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 ± 1.2 percent in 10-month-old vs 9.8 ± 0.9 percent in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 ± 0.2 percent in 10-month-old vs 18.6 ± 0.7 percent in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.

Protective Effects of VE on Renal Proximal Tubular Cells of Mice With Chronical Cadmium Poisoning / 环境与健康杂志

Objective To study protective effects of vitamin E(VE) on renal proximal tubular cells of mice treated with cadmium chronically. Methods 75 Kunming mice were divided into 3 groups randomly, cadmium group, VE group, control group. The mice in cadmium group were treated with cadmium in 2 mg/kg by subcutaneously injected, twice per week, in VE group they were treated with VE in 10 mg/(kg?d) additionally, in control group the animals were treated with saline only. 3 months later the finestuctural changes of the renal proximal tubule cells were observed by electron microscopy and immunohistochemistry(Tunel method). The ultrastuctures of nuclei were revealed with stereological analysis. The apoptotic cells were counted with image analysis. Results The structure of the renal proximal tubule cells of mice in the VE group was similar to those in the control group, but it had significant changed compared with those of the cadmium group. Compared with the control group, the nuclear important morphological parameters of VE group increased significantly (P

A STEREOLOGICAL STUDY ON THE ULTRASTRUCTURE OF CHROMAFFIN CELLS IN GUINEA PIG ADRENAL MEDULLA / 解剖学报

Stereological methods were applied in the qantitative ultrastructural analysis of the chromaffin cells in adrenal medulla of normal adult guinea pigs.The main result includes:(1)The average volume(V)of each cell(714?m~3)and its nucleus (167?m~3);(2)volume density(Vv)of mitochondria(0.082?m~3/?m~3),lysosomes (0.0045?m~3/?m~3),rough-endoplasmic reticulium(0.013?m~3/?m~3),smooth-endopas- mic reticulium including Golgi apparatus(0.024?m~3/?m~3)and granule vesicles (0.23?m~3/?m~3);(3)surface density(Sv)of cell membrane(0.87/?m~2/?m~3)and mitochondrial outer membrane(0.90?m~2/?m~3);(4)numerical density(Nv)of mitochondria(0.89/?m~3),lysosomes(0.11/?m~3)and granule vesicles(59.98/?m~3); (5)the mean diameter of granule vesicles(144 nm),In addition,several small- granule chromaffin cells were quantified separately from the general chromaffin cells.They contain granule vesicles with an average diameter of 97 nm and show a significant difference in surface density of cell membrane(1.54?m~2/?m~3)from that relevant value of general chromaffin cells(P