Predicting the mechanical response of the vaginal wall in ball burst tests based on histology.

A histologically motivated (HM) coefficient that establishes a link between tissue's microstructure and material model through histological data is used in the prediction of the mechanical properties of vaginal tissue that is subjected to multiaxial loading conditions. Therefore, the material parameters were based on an HM coefficient obtained from tensile testing and histological data of comparable tissues. Uniaxial tensile test data and histological data were collected from three groups of sheep at different time points in their life cycle, including virgins, pregnant, and parous ewes. From this data, a correlation between material parameters and histological data was obtained. Spherical indentation (ball burst [BB]) tests were then performed in specimens with similar tissue structure. The histological data of these samples were used in conjunction with the correlations already established for the uniaxial samples data, to define the material parameters of the BB samples. Mechanical properties of the BB specimens were predicted through basic histology and using finite element modeling (FEM) simulations, without direct mechanical measurements. The predicted force and displacement values of the FEM simulation displayed a good correlation with the experimental (BB) testing results. No fitting of the BB results was performed. In this way, the use of uniaxial tests coupled with useful histological information offers a promising approach to predicting macroscopic material behavior under multiaxial loading conditions in biomechanics.

The effect of consecutive pregnancies on the ovine pelvic soft tissues: Link between biomechanical and histological components.

BACKGROUND: Pelvic organ prolapse, various types of incontinence (urinary incontinence, defecatory dysfunction), chronic cystourethritis, and sexual dysfunctions remain between the most common disorders in urogynecology. Currently, it is believed that the nature and number of births plays a major role in their development. Moreover, after these events, pelvic floor tissues may not recover to their original statuses. The close anatomical relationship among the vaginal wall, bladder and rectum often contribute to the emergence of anatomical-functional failure of adjacent organs and systems. BASIC PROCEDURES: The aim of this study was to investigate the effect of consecutive pregnancies on pelvic floor soft tissues, conducting biomechanical and histological analysis. Fifteen Swifter ewes: virgins, parous and pregnant were used. Samples, for uniaxial tension tests and histological analysis, were cut out from fresh tissue. A description of the mechanical properties of native tissue was obtained from the stress-strain curve. Histological samples were stained with Miller's Elastica staining and analyzed using ImageJ software. Collagen, elastin, and smooth muscle contents (%) were analyzed along the full wall thickness of the selected organs. The links between mechanical properties of the soft tissues and histological parameters were analyzed. MAIN FINDINGS: Mechanically, vaginal wall tissue and cervix of pregnant sheep were more compliant. In contrast, bladder and rectum became stiffer and had the highest total collagen content. Parous sheep rectum and bladder were stiffer, compared to virgin sheep. PRINCIPAL CONCLUSIONS: Tensile strength appears to be linked to total collagen content. Elastin and smooth muscle show a direct influence on tissue compliance.

Linking hyperelastic theoretical models and experimental data of vaginal tissue through histological data.

Mechanical characterization of living tissues and computer-based simulations related to medical issues, has become increasingly important to improve diagnostic processes and treatments evaluation. This work proposes a link between the mechanical testing and the material model predictions through histological data of vaginal tissue. Histological data was used to link tensile testing experiments with material-dependent parameters; the approach was adequate to capture the nonlinear response of ovine vaginal tissue over a large strain range. The experimental data obtained on a previous study, has two main components: tensile testing and histological analysis of the ovine vaginal tissue. Uniaxial tensile test data and histological data were collected from three sheep groups: virgins, pregnant and parous. The distal part of vaginal wall was selected since it is prone to tears induced by vaginal delivery. The HGO (Holzapfel-Gasser-Ogden) model parameters were fitted using a stochastic approach, namely the Simple Genetic Algorithm (SGA). The SGA was able to fit the experimental data successfully (R2 > 0.986). The dimensionless coefficient ξ, was highly correlated with histological data. The ratio was seen to increase linearly with increasing collagen content. Coefficient ξ brings a new way of interpreting and understanding experimental data; it connects the nonlinear mechanical behaviour (tensile test) with tissue's morphology (histology). It can be used as an 'inverse' (approximate) method to estimate the mechanical properties without direct experimental measurements, through basic histology. In this context, the proposed methodology appears very promising in estimating the response of the tissue via histological information.

In vitro study of the mechanical performance of hernia mesh under cyclic loading.

The use of prostheses for hernia surgery, made from synthetic polymers may lead to development of postoperative complications. The reason for this can be the mismatch of the mechanical properties of meshes and the loads acting on them. The aim of this work was to investigate the behavior of 3 different hernia meshes under in vitro simulated physiological conditions followed by cyclic loadings. Meshes, Ultrapro (poliglecaprone and polypropylene), Dynamesh (polyvinylidenefluoride) and Surgipro (polypropylene) were selected. For in vitro degradation test, samples were kept in alkaline and acid mediums at 37 °C during 42 and 90 days and analyzed in terms of their weight loss and thickness changes. This was followed by cyclic loading in three increasing load stages. The greatest weight loss and thickness reduction were suffered by Ultrapro mesh. The mesh showed pH independent characteristics. Surgipro mesh had pH independent behavior due to the degradation process, with slight weight loss and thickness reduction. The degradation mechanism of Dynamesh is highly dependent on the pH, with acid surrounding medium acting as a degradation catalyst. Mechanical hysteresis was observed in all three meshes. The larger deformations occurred in Surgipro (25%); necking phenomenon was also observed. The deformation of Dynamesh was 22%, the mesh unweaves under applied load and was unable to withstand the third period of cyclic loads. Ultrapro mesh exhibits the lowest level of deformation (10%). Despite the different compositions and architectures of the meshes, all three underwent permanent plastic deformation, which will induce decreased mesh flexibility over time.

In vitro degradation of polydimethylsiloxanes in breast implant applications.

BACKGROUND: The durability of breast implant material is associated with failure probability, increasing with time from implantation. The current study avoided the bias introduced by biological factors, to systematically investigate the degradation over time of shell materials. The same fundamental physical and chemical conditions were maintained (temperature and pH) throughout the study, to decouple biological aspects from the degradation process. METHODS: Six virgin implants of 2 brands were submitted to the in vitro degradation process, mechanical testing of shell materials, surface change analysis (via scanning electron microscopy [SEM]) and chemical composition analysis by Fourier transform infrared (FTIR) spectroscopy. RESULTS: FTIR results showed that the principal chemical bonds of the material remained intact after 12 weeks of degradation. Apparently the implants' shell structures remained unchanged. Despite this observation, there were statistically significant differences between strain at failure at different time points for the shells of both brands, translated into a stiffening of the material over time. CONCLUSIONS: Material stiffening is reported as an indicator of material degradation. This altered mechanical behavior, added to the mechanical friction from tissue-tissue and tissue-implant contact and to the external mechanical loading (physical activity), may alter the material performance in women's bodies. Ultimately these changes may affect the implants' durability. Further work is needed to understand the biological aspects of the degradation process and their impact on implant durability.

Biomechanical and morphological properties of the multiparous ovine vagina and effect of subsequent pregnancy.

Pelvic floor soft tissues undergo changes during the pregnancy. However, the degree and nature of this process is not completely characterized. This study investigates the effect of subsequent pregnancy on biomechanical and structural properties of ovine vagina. Vaginal wall from virgin, pregnant (in their third pregnancy) and parous (one year after third vaginal delivery) Swifter sheep (n=5 each) was harvested. Samples for biomechanics and histology, were cut in longitudinal axis (proximal and distal regions). Outcome measurements describing Young's modulus, ultimate stress and elongation were obtained from stress-strain curves. For histology samples were stained with Miller's Elastica staining. Collagen, elastin and muscle cells and myofibroblasts contents were estimated, using image processing techniques. Statistical analyses were performed in order to determine significant differences among experimental groups. Significant regional differences were identified. The proximal vagina was stiffer than distal, irrespective the reproductive status. During the pregnancy proximal vagina become more compliant than in parous (+47.45%) or virgin sheep (+64.35%). This coincided with lower collagen (-15 to -21%), higher elastin (+30 to +60%), and more smooth muscle cells (+17 to +37%). Vaginal tissue from parous ewes was weaker than of virgins, coinciding with lower collagen (-10%), higher elastin (+50%), more smooth muscle cells (+20%). It could be proposed that after pregnancy biomechanical properties of vagina do not recover to those of virgins. Since elastin has a significant influence on the compliance of soft tissues and collagen is the main "actor" regarding strength, histological analysis performed in this study justifies the mechanical behavior observed.

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures.

Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50µg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole).

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50µg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Biomechanical properties of breast tissue, a state-of-the-art review.

This paper reviews the existing literature on the tests used to determine the mechanical properties of women breast tissues (fat, glandular and tumour tissue) as well as the different values of these properties. The knowledge of the mechanical properties of breast tissue is important for cancer detection, study and planning of surgical procedures such as surgical breast reconstruction using pre-surgical methods and improving the interpretation of clinical tests. Based on the data collected from the analysed studies, some important conclusions were achieved: (1) the Young's modulus of breast tissues is highly dependent on the tissue preload compression level, and (2) the results of these studies clearly indicate a wide variation in moduli not only among different types of tissue but also within each type of tissue. These differences were most evident in normal fat and fibroglandular tissues.

Effects of 17α-ethinylestradiol at different water temperatures on zebrafish sex differentiation and gonad development.

In the current climate change scenario, studies combining effects of water contaminants with environmental parameters, such as temperature, are essential to predict potentially harmful impacts on aquatic organisms. In zebrafish (Danio rerio), sex determination seems to have a polygenic genetic basis, which can be secondarily influenced by environmental factors, such as temperature and endocrine disrupting chemicals (EDCs). The present study aimed to evaluate the effects of the EDC 17α-ethinylestradiol (EE2), a potent synthetic estrogen, on zebrafish sex differentiation and gonad development at different water temperatures. Therefore, zebrafish raised at three distinct water temperatures (23, 28 or 33±0.5°C), were exposed to 4ng/L of EE2, from 2hours to 60days post-fertilization (dpf). Subsequently, a quantitative (stereological) assessment of zebrafish gonads was performed, at 35 and 60dpf, to identify alterations on gonadal development and differentiation. The results show that low temperature delayed general growth of zebrafish, as well as gonad differentiation and maturation, while high temperature induced an opposite effect. Moreover, sex ratio was skewed toward males when zebrafish were exposed to the high temperature. In general, EE2 exposure promoted gonad maturation in both genders, independently of the temperature. However, at the high temperature condition, exposure to EE2 induced a delay in the male gonad development, with some individuals still showing differentiating gonads at 60dpf. The findings of this study support the notion that zebrafish has a genetic sex determination mechanism highly sensitive to environmental factors and show that it is essential to study the effects of water contaminants at different climate scenarios in order to understand potential future impacts on organisms.

Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study.

Current knowledge on zebrafish (Danio rerio) sex determination suggests that this trait has a polygenic genetic basis, although environmental factors, such as endocrine disrupting chemicals (EDC), may also be involved in modeling or disturbing the species sex differentiation and development. This study aimed to assess how sex steroids imbalance triggers impact on sex differentiation and gonad development in zebrafish. Fish where exposed to an estrogen (EE2, i.e. 17α-ethinylestradiol, 4ng/L), to an inhibitor of estrogen synthesis (Fad, i.e. fadrozole, 50µg/L) or to their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L), from 2h to 60 days post-fertilization (dpf). Afterwards, a quantitative (stereological) analysis using light microscopy, based on systematic sampling, was made at 35 and 60dpf, to identify alterations on gonad differentiation and development. During the sex differentiation period, our histological data showed that not all zebrafish males develop a "juvenile ovary", contrarily to what is currently taken for granted. Furthermore, the stereological analysis suggests that EE2 alone enhanced both zebrafish growth and gonad development. On the other hand, exposure to Fad affected the sexual development in zebrafish, inducing masculinization of the specimens, with some degree of intersex observed in males. In addition, the binary mixture allowed identifying sex-dependent roles of steroid hormones in the general growth and gonad development of zebrafish, with estrogens acting as growth promoters in females and being essential for ovary development. Data further support that sex-specific and single EDC impact studies are important, but clearly not sufficient to understand what may occur in the environment.

Screening and identification of potential sex-associated sequences in Danio rerio.

Current knowledge on zebrafish (Danio rerio) suggests that sex determination has a polygenic genetic basis in this species, although environmental factors may also be involved. This study aimed to identify sex-associated genomic regions using two different marker systems: inter-simple sequence repeats (ISSRs) and random-amplified polymorphic DNA (RAPDs). Two bulks were constructed: one with DNA from zebrafish females and the other from males; then, a total of 100 ISSR and 280 RAPD primers were tested. Three DNA fragments presenting sexual dimorphism (female-linked: OPA17436 and OPQ191027 ; male-linked: OPQ19951 ) were determined from sequential analysis of the bulks followed by assessment in individuals. These fragments were cloned and convert into the following sequenced characterized amplified regions (SCAR): DrSM_F1, DrSM_F2, and DrSM_M, which share identities with sequences located in chromosomes 2, 3, and 11 (Zv9), respectively. Using these potential markers in zebrafish samples it was possible to correctly identify 80% of the males (DrSM_M) and 100% of the females (DrSM_F1 + DrSM_F2) in the analyzed population.

A current overview of the Papaya meleira virus, an unusual plant virus.

Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease, which is characterized by a spontaneous exudation of fluid and aqueous latex from the papaya fruit and leaves. The latex oxidizes after atmospheric exposure, resulting in a sticky feature on the fruit from which the name of the disease originates. PMeV is an isometric virus particle with a double-stranded RNA (dsRNA) genome of approximately 12 Kb. Unusual for a plant virus, PMeV particles are localized on and linked to the polymers present in the latex. The ability of the PMeV to inhabit such a hostile environment demonstrates an intriguing interaction of the virus with the papaya. A hypersensitivity response is triggered against PMeV infection, and there is a reduction in the proteolytic activity of papaya latex during sticky disease. In papaya leaf tissues, stress responsive proteins, mostly calreticulin and proteasome-related proteins, are up regulated and proteins related to metabolism are down-regulated. Additionally, PMeV modifies the transcription of several miRNAs involved in the modulation of genes related to the ubiquitin-proteasome system. Until now, no PMeV resistant papaya genotype has been identified and roguing is the only viral control strategy available. However, a single inoculation of papaya plants with PMeV dsRNA delayed the progress of viral infection.

Copper induced upregulation of apoptosis related genes in zebrafish (Danio rerio) gill.

Copper (Cu) is an essential micronutrient that, when present in high concentrations, becomes toxic to aquatic organisms. It is known that Cu toxicity may induce apoptotic cell death. However, the precise mechanism and the pathways that are activated, in fish, are still unclear. Thus, this study aimed to assess which apoptotic pathways are triggered by Cu, in zebrafish (Danio rerio) gill, the main target of waterborne pollutants. Fish where exposed to 12.5 and 100 µg/L of Cu during 6, 12, 24 and 48 h. Fish gills were collected to TUNEL assay and mRNA expression analysis of selected genes by real time PCR. An approach to different apoptosis pathways was done selecting p53, caspase-8, caspase-9 and apoptosis inducing factor (AIF) genes. The higher incidence of TUNEL-positive cells, in gill epithelia of the exposed fish, proved that Cu induced apoptosis. The results suggest that different apoptosis pathways are triggered by Cu at different time points of the exposure period, as the increase in transcripts was sequential, instead of simultaneous. Apoptosis seems to be initiated via intrinsic pathway (caspase-9), through p53 activation; then followed by the extrinsic pathway (caspase-8) and finally by the caspase-independent pathway (AIF). A possible model for Cu-induce apoptosis pathways is proposed.

Computational modeling approach to study the effects of fetal head flexion during vaginal delivery.

OBJECTIVE: The purpose of this study was to investigate the influence of fetal head flexion during vaginal delivery with a 3-dimensional computational finite element model. STUDY DESIGN: A finite element model of the pelvic skeletal structure, pelvic floor, and fetus was developed. The movements of the fetus during birth were simulated in engagement, descent, flexion, internal rotation, and extension of the fetal head. The opposite forces against the fetal descendent and the stress of the pelvic floor muscles were obtained on simulations with different degrees of head flexion. RESULTS: The simulated increase in fetal head flexion is associated with lower values of opposite forces against the fetal descent. The descending fetus with abnormal head flexion also meets resistance in later stations. Lower stress on the pelvic floor was demonstrated with simulated increase in fetal head flexion during vaginal delivery. CONCLUSION: This analytic evidence suggests that the fetal head flexion during vaginal delivery may facilitate birth and protect the pelvic floor.

Spatial relationships of the intrahepatic vascular-biliary tracts and associated pancreatic acini of Nile tilapia, Oreochromis niloticus (Teleostei, Cichlidae): a serial section study by light microscopy.

Reports on teleost liver morphology reflect both controversial and confirmed interspecies variations. Choosing Nile tilapia as a model, we described the histology and 3D organization of all types of vascular-biliary tracts and their spatial relationships from the organ hilum toward the hepatic vein opening(s). The portal tracts entering the hilum, termed pancreatic-venous-biliary-arteriolar tracts (P-VBAT), are associated with pancreocytes and have an afferent axially located vein, plus biliary duct(s) and small artery(ies). The P-VBAT gradually disappears toward the anterior (efferent) end of the liver; those tracts ramify and originate new types of tracts, which may carry one type of element (vascular or biliary) or groups of two, in all possible combinations. Most tracts carrying afferent veins had pancreocytes, thus forming (pancreatic-venous tracts (P-VT), pancreatic-venous-biliary tracts (P-VBT), and pancreatic-venous-arteriolar tracts (P-VAT). There were terminal (and smaller) afferent isolated veins that had no associated pancreocytes. Also, the pancreatic sleeve of a vein could end abruptly or attenuate and disappear, reappearing in distal portions of the same vein. Thus, veins without pancreatic covering as seen in sections are not always efferent. Small arterioles can enter the liver retrogradely, via the adventitia of efferent hepatic veins, thus forming venous-arteriolar tracts (VAT). In comparison with the salmonid-liver type, there were no VBAT without associated pancreocytes and there was a smaller degree of ambiguity in identification of the afferent vs. efferent veins. Thus, the tilapine-liver type is proposed to be a more promising model for studying hepatic metabolic zonation in fish, defined not as in mammals, but eventually considering a gradient radiating from the hilum. Our data and differences from mammals supported the adequacy of the previously proposed nomenclature for the vascular-biliary tracts of fish livers, extending it to those that contain the exocrine pancreas.

Temperature and gender influences on the hepatic stroma (and associated pancreatic acini) of Nile tilapia, Oreochromis niloticus (Teleostei, Cichlidae): a stereological analysis by light microscopy.

The normal organ morphology and function in fishes varies according to several natural factors, and such variability is found in liver. Knowledge about the normal liver microanatomy is fundamental to pathological evaluation. Even though gender and temperature are important factors for modulating morphophysiological processes in fishes, their influences on liver stroma are virtually unknown. Because temperature- and gender-related changes exist in liver parenchyma, we predict both factors should also influence the normal stromal structure. Using Nile tilapia as a model, we undertook a study to: 1) establish baseline quantitative structural data on the hepatic stroma (and intimately associated pancreatic acini); 2) compare data with those available from other species, namely, salmonids that do not have a liver with pancreatic acini; and 3) test our hypothesis that, within normal healthy limits, the stroma and its structural components may vary significantly with temperature and gender. We used 1-year-old male and female specimens acclimated to 17 degrees C (breeding noncompatible) and 27 degrees C (breeding compatible) for 45 days. Basic morphometric fish parameters were recorded. After estimation of liver volume, the organ was sliced and pieces systematically sampled for light microscopy. Stereology allowed estimation of the relative volumes of organ components. The total volumes were computed by combining the relative volumes with the total liver volumes. Nile tilapia of both genders, held at 17 vs. 27 degrees C, showed structural quantitative differences in the relative volumes of stroma and most of its components, and in the total volumes of certain stromal elements. The total volume of the stroma and of associated pancreatic acini did not differ. We first established that, in fishes, the total amount (volume) of liver biliary ducts and of eosinophilic granule cells might significantly change (increase and decrease, respectively) with a higher acclimation temperature. Indeed, virtually all the stereological changes were, essentially, temperature- and not gender-related. At 27 degrees C, parallel changes in the parenchyma caused a decreased liver volume and hepatic-somatic index (HSI). The relative volumetric proportion of stroma vs. parenchyma in tilapia is higher than in salmonids. The differences found in this study could not be detected with a qualitative approach, thus stressing the importance of using stereology for analyzing histological patterns and for establishing reliable baseline values in healthy conditions. It was also anticipated that in experimental settings with fish the baseline liver stromal architecture may be different according to temperature and breeding status; in consequence, the effects of the tested variable may also diverge. Our data do not fully explain the lower liver volume and HSI at 27 degrees C, thus justifying studies on the parenchyma, particularly on cell size and number.