Use of electron microscopy to study megakaryocytes

Abstract

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.     

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.     

Use of electron microscopy to study platelets and thrombi

ABSTRACT

Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and scanning electron microscopy (EM) provided a foundation for our initial understanding of platelet structure and how it changes upon platelet activation. EM approaches have since been utilized to study platelets and thrombi in the context of basic, translational and clinical research, and they are instrumental in the diagnosis of multiple platelet function disorders. In this brief review, we provide a sampling of the many contributions EM based studies have made to the field, including both historical highlights and contemporary applications. We will also discuss exciting new imaging modalities based on EM and their utility for the study of platelets, hemostasis and thrombosis into the future.     

Biomechanics of DNA structures visualized by 4D electron microscopy

We present a technique for in situ visualization of the biomechanics of DNA structural networks using 4D electron microscopy. Vibrational oscillations of the DNA structure are excited mechanically through a short burst of substrate vibrations triggered by a laser pulse. Subsequently, the motion is probed with electron pulses to observe the impulse response of the specimen in space and time. From the frequency and amplitude of the observed oscillations, we determine the normal modes and eigenfrequencies of the structures involved. Moreover, by selective “nano-cutting” at a given point in the network, it was possible to obtain Young’s modulus, and hence the stiffness, of the DNA filament at that position. This experimental approach enables nanoscale mechanics studies of macromolecules and should find applications in other domains of biological networks such as origamis.     

巴尔通体Ⅰ、Ⅱ代扫描透射电镜观察

目的探寻巴尔通体培养中子代不如原代长得好、甚至不长的原因。方法收集培养中的Ⅰ代、Ⅱ代巴尔通体菌用扫描、透射电镜观察其超微结构。结果扫描电镜见多个菌缠绕成团，菌体密集。用超声波冲击后有少量孤立的巴尔通体菌似圆球形，外围呈云雾状，中间凹陷。透射电镜见Ⅰ代菌细胞数量多、体积大、饱满，胞膜完整，Ⅱ代数量少，菌细胞出现畸形、衰退，胞膜不完整。结论菌体之间有相互缠绕、聚集、不易分开现象，这是否与它的侵袭、致病性有关，需进一步探讨;传代后原来的生长条件不适合其生长，进一步研究相适宜的培养基很有必要。     

From the Cover: Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by “blinking” with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This “continuous switching mode” is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.     

On the dynamical nature of the active center in a single-site photocatalyst visualized by 4D ultrafast electron microscopy

Understanding the dynamical nature of the catalytic active site embedded in complex systems at the atomic level is critical to developing efficient photocatalytic materials. Here, we report, using 4D ultrafast electron microscopy, the spatiotemporal behaviors of titanium and oxygen in a titanosilicate catalytic material. The observed changes in Bragg diffraction intensity with time at the specific lattice planes, and with a tilted geometry, provide the relaxation pathway: the Ti⁴⁺=O²⁻ double bond transformation to a Ti³⁺−O¹⁻ single bond via the individual atomic displacements of the titanium and the apical oxygen. The dilation of the double bond is up to 0.8 Å and occurs on the femtosecond time scale. These findings suggest the direct catalytic involvement of the Ti³⁺−O¹⁻ local structure, the significance of nonthermal processes at the reactive site, and the efficient photo-induced electron transfer that plays a pivotal role in many photocatalytic reactions.Single-site catalysts of both the thermally and photoactivated kind now occupy a prominent place in industrial- and laboratory-scale heterogeneous catalysis (1–8). Among the most versatile of these are the ones consisting of coordinatively unsaturated transition metal ions (Ti, Cr, Fe, Mn…) that occupy substitutional sites in well-defined, three-dimensionally extended, open-structure silicates of the zeolite type. The well-known and most widely used are the 4- or 5-coordinated Ti(IV) ions accommodated within the crystalline phase of silica, silicalite (9–14).Titanosilicates, especially, are used extensively both industrially and in the laboratory for a wide range of chemo-, regio-, and shape-selective oxidations of organic compounds (15–18). These single-site heterogeneous photocatalysts are quite distinct from those typified by TiO₂, SrTiO₃, and other titaniferous photocatalysts where the Ti(IV) ions are in 6-coordination; and where, in interpreting the processes involved in harnessing solar radiation, electronic band structure considerations hold sway in preference to the localized states (see, e.g., refs. 19, 20). It has been demonstrated (16–18, 21, 22) that single-site, coordinatively unsaturated Ti(IV)-centered photocatalysts are especially useful in the aerial oxidation of environmental pollutants in the photodegradation of NO (to N₂ and O₂), of H₂O (to H₂ and O₂), and in the photocatalytic reduction of CO₂ to yield methanol. There is an exigent need to explore the precise nature of the electronic, temporal, and spatial changes accompanying the initial act of photoabsorption that sets in train the ensuing elementary chemical processes that are of vital environmental significance in, for example, the utilization of anthropogenic CO₂ as a chemical feedstock (23).Here, we report the use of 4D ultrafast electron microscopy (UEM) (24–26) to trace the spatiotemporal behavior of the Ti(IV) and O²⁻ ions at the photocatalytic active center in the structurally well-characterized titanosilicate Na₄Ti₂Si₈O₂₂·4H₂O, known as JDF-L1 (27–29). JDF stands for Jilin–Davy–Faraday, as the crystalline solid described here was discovered and characterized in joint work involving Jilin University (P. R. China) and the Davy–Faraday Laboratory at the Royal Institution of Great Britain. L1 stands for the first layered catalyst formed during that collaboration; 5-coordinated solids containing Ti(IV) ions are rare among the hundred or so titaniferous minerals, the prime example being fresnoite, Ba₂Ti₂Si₂O₈. We choose this photocatalyst with 5-coordinated Ti because of its unique bonding structure. Our approach entails monitoring, at femtosecond resolution, the changes in intensities and anisotropies of Bragg (electron) diffraction reflections in such a manner as to retrieve the change in valency and the time scales involved in both the formation of Ti³⁺−O¹⁻ bond and the relaxation of the energy back to the local structure of the Ti = O bond in JDF-L1. Through these diffraction studies, and the associated Debye–Waller effect and structural factors anisotropies, it is found that a Ti³⁺−O¹⁻ bond is formed on the femtosecond time scale; whereas, the back relaxation from the site to the structure occurs on a much longer time scale, permitting ample time for reactivity involving Ti³⁺−O¹⁻, and indicating the potential significance of nonthermal processes in the photocatalytic activity at the reactive site.     

花鲈感染简单异尖线虫及扫描电镜观察

目的简单异尖线虫(Anisakis simplex)是一类寄生于海水鱼类及哺乳类动物,可引起人类异尖线虫病(anisaki-osis)的食源性人兽共患寄生虫。作者从浙江宁波患病花鲈(Latealabrax japonicus)体内检出简单异尖线虫,检出率为100%(11/11)。患病鲈鱼体表无异常症状,解剖鱼体在肝脏、胃壁、肠壁、肠系膜等部位均有简单异尖线虫寄生,线虫盘成螺旋状包于包囊内,病鱼感染线虫强度为20～30条/尾。感染严重病鱼出现胃穿孔、胃壁出血、肝脏具白色结节等病症。光学显微镜观察线虫虫体为长圆筒形,两端较细,肌质食道,腺体的胃位于食管后面,黑色不透明,和肠连接处有一斜的分界线。扫描电镜观察虫体体表具致密环纹,前端钝圆,具1个钻齿和4个钝角状乳突,排泄口位于腹侧两个乳突之间,尾部短而圆,末端具一明显尾突。     

Analysis of microvessels in pancreatic cancer: by light microscopy,confocal laser scan microscopy,and electron microscopy

Background/Purpose

The microvessel density (MVD) of most malignant tumors is considered to be strongly related to metastasis and prognosis. Weidner’s “hot spot method” for determining MVD is in general use, but it is possible that cells other than endothelial cells will also be stained. In our previous study, no correlations were observed between MVD determined by the “hot spot method” and prognosis/metastasis. But, using the “lumen method,” we found a correlation with the number of vessel structures only. In the present study, we analyzed the staining of microvessels in pancreatic cancer, using light microscopy, confocal laser scan microscopy (CLSM), and transmission electron microscopy (TEM).

Methods

Microvessel staining of pancreatic cancer with CD34, factor VIII, and CD45 antibodies was examined in consecutive slices by light microscopy. For CLSM, freshly resected specimens were immunostained with factor VIII and fluorescein isothiocynate. For TEM, specimens were fixed with 2.5% glutaraldehyde, treated with 1% osmium tetroxide, and embedded in epoxy resin.

Results

Staining of vessels with CD34 and factor VIII antibodies appeared similar under light microscopy. However, CD34-stained consecutive slices were judged not to reveal vessel structures, and some cells stained with CD45 antibody were similar in appearance to CD34-stained cells. Under CLSM, irregular arrangements of neovascularization, consisting of many branches, were observed, but many positively stained cells not identified as vessels were also seen. Microvessels were distinctly identified under TEM, but the types of individual cells could not be determined.

Conclusions

An integrated, reproducible method for the measurement of MVD is vital. For pancreatic cancer, the “lumen method” is recommended.

     

Fatty acids and the initial events of endothelial damage seen by scanning and transmission electron microscopy

A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endothelium was produced by arachidonic, linoleic, γ-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin.,It..appears that damage produced by the fatty acids is non-specific.     

Genetics study and transmission electron microscopy of pili in susceptible and resistant clinical isolates of Mycobacterium tuberculosis

ObjectiveTo study genetic bases and morphology of pili in Mycobacterium tuberculosis (M. tuberculosis).MethodsPCR and sequencing were used to investigate two related pili, Mtp and Flp genes in clinical isolates of M. tuberculosis. The primers were designed and PCR program were set for whole genes amplification. PCR products of the two genes from all isolates were sequenced by an applied biosystems apparatus and the results were analysed by online software. In the other hands, harvested cells from fresh cultures of isolates were undergoing specific sample preparation for sectional and negative staining for transmission electron microscopy.ResultsElectrophoresis revealed two specific bonds of 361 bp for Mtp and 150 bp for Flp genes and confirmed primer and PCR conditions designing. There were not any mutations in sequencing results of Mtp and Flp in comparison with reference sequence. Transmission electron microscopy examination revealed two distinct types of pili in the isolates as a bundle-forming pilus and rope-like pilus. From total investigated cells, 10% harbored pili in their structure.ConclusionsTwo genes of pili in all clinical isolates of M. tuberculosis were conserved and two morphological types of pili were detected. We proposed that by targeting pili proteins by a suitable inhibitor, it could affect the pathogenesis especially in resistant forms.     

心肌mtDNA4977缺失与扩张型心肌病猝死的关系

目的探讨扩张型心肌病(DCM)猝死者心肌线粒体DNA(mtDNA)4977缺失情况及其与猝死的关系.方法从近7年尸检案例中挑选11例DCM猝死和14例对照组病例及心肌组织蜡块,按常规方法提取心肌mtDNA,用PCR、琼脂糖紫外凝胶成像技术确定扩增产物激光密度,初步定量检测mtDNA4977缺失率.结果DCM猝死11例中,未成年人2例;成人9例,均为男性,年龄22～49(平均38)岁.对照组14例中,未成年人1例;成人13例,其中男11例,女2例,年龄19～47(平均37.23)岁,死因为机械性损伤和窒息各2例,电击死2例,中毒2例,非心肌病猝死6例.DCM11例(占100%)、对照组2例(占14.28%)检见不同程度的mtDNA4977缺失;两组病例mtDNA4977缺失率均值分别为0.92%和0.09%,差异有统计学意义.结论DCM猝死者心肌可检见mtDNA49 77缺失;其心肌mtDNA4977缺失突变与DCM猝死有关.     

心力衰竭患者心肌线粒体DNA缺失突变的定量分析及与心功能损害程度的关系

目的为探讨心肌线粒体ＤＮＡ（ｍｔＤＮＡ）获得性损伤在衰竭心肌发病中的意义。方法对２８例慢性心力衰竭患者活检心肌，用定量ＰＣＲ方法，以常发缺失型突变ｍｔＤＮＡ４９７７ｂｐ（ｍｔ－ＤＮＡ４９７７）和７４３６ｂｐ（ｍｔＤＮＡ７４３６）缺失率为指标，观察心力衰竭不同发展阶段ｍｔＤＮＡ损伤程度与患者临床心功能受损之间的关系。结果慢性心力衰竭患者活检心肌中ｍｔＤＮＡ４９７７缺失频率为１００％，缺失率在０．１０３％～１．０２８％之间；７０％的扩张型心肌病（ＤＣＭ）和３８．９％的风湿性心脏病（ＲＨＤ）患者存在ｍｔＤＮＡ７４３６缺失，缺失率在０．００９％～０．４８８％之间。１０例健康对照者中，仅２例４５岁以上者有低水平的ｍｔＤＮＡ缺失。心功能愈差，左房扩大愈明显者，ｍｔＤＮＡ缺失率愈高；扩张型心肌病缺失程度明显高于风湿性心脏病。结论心肌ｍｔＤＮＡ损伤与心脏功能的受损程度密切相关，特别是ＤＣＭ患者。     

Filming the formation and fluctuation of skyrmion domains by cryo-Lorentz transmission electron microscopy

Magnetic skyrmions are promising candidates as information carriers in logic or storage devices thanks to their robustness, guaranteed by the topological protection, and their nanometric size. Currently, little is known about the influence of parameters such as disorder, defects, or external stimuli on the long-range spatial distribution and temporal evolution of the skyrmion lattice. Here, using a large (7.3 × 7.3?μm²) single-crystal nanoslice (150 nm thick) of Cu₂OSeO₃, we image up to 70,000 skyrmions by means of cryo-Lorentz transmission electron microscopy as a function of the applied magnetic field. The emergence of the skyrmion lattice from the helimagnetic phase is monitored, revealing the existence of a glassy skyrmion phase at the phase transition field, where patches of an octagonally distorted skyrmion lattice are also discovered. In the skyrmion phase, dislocations are shown to cause the emergence and switching between domains with different lattice orientations, and the temporal fluctuation of these domains is filmed. These results demonstrate the importance of direct-space and real-time imaging of skyrmion domains for addressing both their long-range topology and stability.In a noncentrosymmetric chiral lattice, the competition between the symmetric ferromagnetic exchange, the antisymmetric Dzyaloshinskii–Moriya interaction, and an applied magnetic field can stabilize a highly ordered spin texture, presenting as a hexagonal lattice of spin vortices called skyrmions (1–4).Magnetic skyrmions have been experimentally detected in materials having the B20 crystal structure such as MnSi (5), Fe_1?xCo_xSi (6, 7), FeGe (8), and Cu₂OSeO₃ (9) and, recently, also on systems like GaV₄S₈ (10) and beta-Mn-type alloys (11). Small-angle neutron scattering studies of bulk solids evidenced the formation of a hexagonal skyrmion lattice confined in a very narrow region of temperature and magnetic field (T-B) in the phase diagram (5, 6). In thin films and thinly cut slices of the same compounds, instead, skyrmions can be stabilized over a wider T-B range as revealed by experiments using cryo-Lorentz transmission electron microscopy (LTEM) (12, 13). Furthermore, it was proposed and recently observed that skyrmions can also exist as isolated objects before the formation of the ordered skyrmion lattice (14, 15). A recent resonant X-ray diffraction experiment also suggested the formation of two skyrmion sublattices giving rise to regular superstructures (16).In a 2D landscape, long-range ordering can be significantly altered by the presence of defects and disorder. Indeed, the competition between order and disorder within the context of lattice formation continues to be an issue of fundamental importance. Condensed matter systems are well known to provide important test beds for exploring theories of structural order in solids and glasses. An archetypal and conceptually relevant example is the superconducting vortex lattice, where real-space imaging studies allow direct access to the positional correlations and local coordination numbers (17–19). Up until now, however, analogous studies of skyrmion lattices have not been reported even though (as for superconducting vortices) it is well known that defects and dislocations present in a sample can pin the motion of skyrmions induced by external perturbations such as an electric field (20) or a magnetic field (16). This competition between disorder and elasticity will clearly give rise to a complex energy landscape promoting diverse metastable states (21) and superstructures (22, 23). Furthermore, previous imaging studies of skyrmion lattices could probe only the short-range order due to limitations in the size of the imaged area and its homogeneity.In this paper, by systematic observations using cryo-LTEM, we reveal the magnetic field-dependent evolution of the skyrmion-related spin textures in a Cu₂OSeO₃ thin plate and study their long-range ordering properties imaging up to ∼1,000 lattice constants. The different phases of the spin textures are analyzed with state-of-the-art methods to unravel their spatial properties. At low magnetic fields, the coexistence of two helical domains is observed, in contrast to previous studies (9); the angle between the two helices’ axis is retrieved via a reciprocal space analysis. At the magnetic field close to the helical–skyrmion phase transition, evidence for a glassy skyrmion phase is found via cross-correlation analysis, a method that has recently been applied to the analysis of both X-rays and electron diffraction patterns to retrieve information on the local order and symmetry of colloidal systems (24–26). In this phase, we reveal also patches of octagonally distorted skyrmion lattice crystallites. In the skyrmion phase, by locating the position of each skyrmion and generating an angle map of the hexagonal unit cell they formed, we obtain a direct-space distortion map of the skyrmion lattice. This distortion map evidences the presence of orientation-disordered skyrmion lattice domains present within the single-crystalline sample. Each domain boundary coincides with a dislocation formed by a seven–five or a five–eight–five Frenkel-type defect. The number of such dislocations decreases with increasing magnetic field, and large single-domain regions are formed. The formation of these mesoscopic domains was also filmed with camera-rate (millisecond) time resolution. The presence of differently oriented skyrmion lattice domains was observed in spatially separated regions, or in the same area of the sample but at a different moment in time. Based on our observation, we propose an alternative scenario for the appearance of split magnetic Bragg peaks reported in ref. 16. Instead of the formation of regular superstructures of coexisting misoriented skyrmion lattices in real space, we suggest that the splitting is caused by a spatial or temporal integration of an orientation-fluctuating skyrmion lattice. This result highlights the importance of a direct-space, real-time probe for assessing the dynamical topological properties of a large number of skyrmions.     

Comparative studies on the ultrastructure of malignant melanoma in fish and human by freeze-etching and transmission electron microscopy

Summary Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes (compound melanosomes) occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing local differences in frequency and distribution patterns in the tumor. Intercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable avantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.     

线粒体DNA ND1基因nt3394 T→C突变与老年人2型糖尿病

目的：了解线粒体烟酰胺腺嘌呤脱氧核苷酸（NADH）脱氢酶第一亚单位（ND1）基因中339位点T→C突变与我国老年人2型糖尿病的关系。方法：收集无血缘关系的208例老年2型糖尿病患者，同时以180例无糖尿病家族史的老年糖耐量正常者作为对照，用聚合酶链反应扩增。限糖尿病患者，同时以180例无糖尿病家族史的老年糖耐量正常者作为对照，用聚合酶链反应扩增、限制性内切酶HaeⅢ消化进行点突变筛选。结果：发现9例患者存在线粒体DNA ND1基因3394位点T→C突变（4．3％），非糖尿病者中仅有1例突变（0．6％），两组比较差异有显著性（P＜0．05）。结论：线粒体DNA ND1基因中3394位点T→C突变可能与我国老年人2型糖尿病的发病有关。     

From the Cover: Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo

Mitochondrial dysfunction is heavily implicated in Parkinson disease (PD) as exemplified by the finding of an increased frequency of respiratory chain-deficient dopamine (DA) neurons in affected patients. An inherited form of PD is caused by impaired function of Parkin, an E3 ubiquitin ligase reported to translocate to defective mitochondria in vitro to facilitate their clearance. We have developed a reporter mouse to assess mitochondrial morphology in DA neurons in vivo and show here that respiratory chain deficiency leads to fragmentation of the mitochondrial network and to the formation of large cytoplasmic bodies derived from mitochondria. Surprisingly, the dysfunctional mitochondria do not recruit Parkin in vivo, and neither the clearance of defective mitochondria nor the neurodegeneration phenotype is affected by the absence of Parkin. We also show that anterograde axonal transport of mitochondria is impaired in respiratory chain-deficient DA neurons, leading to a decreased supply of mitochondria to the axonal terminals.     

Variation in articular cartilage in rats between 3 and 32 months old. A histomorphometric and scanning electron microscopy study

In this study we assess the thickness, the cellular density, the cell sizes and the collagen of the three superficial cartilage zones and the morphology of the articular surface of the femoral trochlea in rats with 3, 12 and 32 months of age. The cartilage was studied using light microscopy and scanning electron microscopy. The quantitative results are expressed as means ± SEM. The data were compared statistically (P < 0.05). Both the thickness and the cellular density significantly diminish with age, in the three cartilage zones studied. The reduction of cellular density is more pronounced in the superficial and intermediate zones of the cartilage (zones I and II, respectively). In zone III (deep zone), the cellular density declines only as from 12 months of age. The area of the chondrocytes diminishes in the superficial and deep zones, but only as from 12 months old. In the intermediate zone, there is no chondrocyte hypotrophy with age. The types of collagen in the zones of the cartilage change with age. In the superficial zone, the collagen type I predominates at 3 months of age while the collagen type II predominates at 12 and 32 months of age. In the intermediate and deep zones, the collagen type I that predominates at 3 months of age is substituted by the collagen type III at 12 and 32 months of age. The articular surface in the 3-month-old rats is relatively smooth, presenting few undulations. In 12-month-old animal cartilages, few fissures and craters are found. In the 32-month-old animals, it was observed a higher number of this kind of degenerative changes and with a more severe look.