Electron Microscopic Stereology Research Articles

Obesity and hypoxia have differential effects on myocardial innervation in the right ventricle of the male mouse heart.

Obesity, along with hypoxia, is known to be a risk factor for pulmonary hypertension (PH), which can lead to right ventricular hypertrophy and eventually heart failure. Both obesity and PH influence the autonomic nervous system (ANS), potentially aggravating changes in the right ventricle (RV). This study investigates the combined effects of obesity and hypoxia on the autonomic innervation of the RV in a mouse model. Male C57BL/6N mice were subjected to a control diet (CD) or a high-fat diet (HFD) for 30 weeks, with subsets of the mice exposed to chronic normobaric hypoxia (13% O2) during the final 3 weeks. Light and electron microscopic stereology was used to quantify various parameters of nerve fibres innervating the RV myocardium. HFD-induced obesity significantly increased the total length of nerve fibres and axons in the RV under normoxic conditions, indicating hyperinnervation. Quantitatively, the length density of nerve fibres per unit volume of RV (unit: x10-3 µm-2) was similar in CD (0.158 ± 0.04), CD-Hyp (0.176 ± 0.06) and HFD-Hyp (0.147 ± 0.05). In contrast, in HFD the length density of nerve fibres showed higher values 0.206 ± 0.054. The total length of nerve fibres increased by 67% from 2.61 m ± 0.77 m in CD to 4.37 m ± 1.51 m in HFD. The total length of axons increased by 80% from 8.87 m ± 2.75 m to 15.95 m ± 4.62 m. However, when obesity was combined with hypoxia, the total axon length was significantly reduced by 27% in HFD-Hyp compared with HFD. In addition, the mean number of axon profiles per nerve fibre profile decreased from 3.44 ± 0.68 in HFD to 2.95 ± 0.43 in HFD-Hyp. Interestingly, chronic hypoxia alone did not significantly alter RV innervation but led to RV hypertrophy, independent of the diet. The attenuation of obesity-induced hyperinnervation by hypoxia suggests a complex and potentially antagonistic interaction between these conditions. In conclusion, obesity induced by a HFD caused hyperinnervation of the RV, whereas chronic hypoxia alone did not significantly alter RV innervation. Surprisingly, chronic hypoxia attenuated the obesity-induced changes in RV innervation. These findings indicate that the effects of obesity and hypoxia-induced PH on RV innervation are distinct and potentially antagonistic.

Stress and remodeling of hippocampal spine synapses.

By nature's original design, stress is a protective mechanism, signaling danger to homeostasis and, as a result, stimulating the brain to initiate coping and adaptive responses, which ultimately increases the chances of survival. On the other hand, stress may become a danger to homeostasis itself, when it is so severe or so prolonged that it overpowers cellular reserves. One of the consequences of traumatic stress is loss of hippocampal spine synapses, which is the main topic of this chapter summarizing research findings from the last 10+ years. Loss of spine synapses is thought to be a neuronal defense mechanism against excitotoxic damage, so the subcellular mechanisms of synapse loss are reviewed in the context of glutamatergic insults. One of the main conceptual derivates of stress-induced synaptic alterations is the "synaptogenic" hypothesis of major depressive disorder. The synaptogenic hypothesis postulates an inverse correlation between the number of limbic, mainly prefrontal cortical and hippocampal, spine synapses and the severity of depressive behavior/symptoms. The synaptogenic hypothesis implies that synaptoprotective interventions, that are capable of countering the stress-induced loss of limbic spine synapses, are probably also capable of promoting stress resilience, which may provide the conceptual basis for a preventive approach in antidepressant therapy. Finally, we discuss why electron microscopic stereology is a reliable and highly accurate technique for the quantitative assessment of ultrastructural particulate objects, such as spines and synapses.

Cellular and acellular ex vivo lung perfusion preserve functional lung ultrastructure in a large animal model: a stereological study

BackgroundEx vivo lung perfusion (EVLP) is used by an increasing number of transplant centres. It is still controversial whether an acellular or cellular (erythrocyte enriched) perfusate is preferable. The aim of this paper was to evaluate whether acellular (aEVLP) or cellular EVLP (cEVLP) preserves functional lung ultrastructure better and to generate a hypothesis regarding possible underlying mechanisms.MethodsLungs of 20 pigs were assigned to 4 groups: control, ischaemia (24 h), aEVLP and cEVLP (both EVLP groups: 24 h ischaemia + 12 h EVLP). After experimental procedures, whole lungs were perfusion fixed, samples for light and electron microscopic stereology were taken, and ventilation, diffusion and perfusion related parameters were estimated.ResultsLung structure was well preserved in all groups. Lungs had less atelectasis and higher air content after EVLP. No significant group differences were found in alveolar septum composition or blood-air barrier thickness. Small amounts of intraalveolar oedema were detected in both EVLP groups but significantly more in aEVLP than in cEVLP.ConclusionsBoth EVLP protocols supported lungs well for up to 12 h and could largely prevent ischaemia ex vivo reperfusion associated lung injury. In both EVLP groups, oedema volume remained below the level of functional relevance. The group difference in oedema formation was possibly due to inferior septal perfusion in aEVLP.

Lipid-body containing interstitial cells (lipofibroblasts) in the lungs of various mouse strains.

Pulmonary alveolar septa are thought to contain at least two types of fibroblasts that are termed myofibroblasts and lipofibroblasts based on their morphological characteristics. Lipofibroblasts possess cytoplasmic lipid inclusions (lipid bodies or droplets) and are involved in several important functions, such as surfactant synthesis, development, vitamin A storage and presumably regeneration. As vitamin A was shown to reduce pulmonary emphysema in several but not all mouse and rat strains, we hypothesized that these strain differences might be explained by a differential occurrence of lipofibroblasts and their lipid bodies in various mouse strains. Therefore, mouse lungs of six strains (NMRI, BALB/c, C3H/HeJ, C57BL/6J, C57BL/6N and FVB/N) were investigated by light and electron microscopic stereology to quantify the amount of lipid bodies and the composition of alveolar septa. Lipofibroblasts were observed qualitatively by transmission electron microscopy in every investigated mouse strain. The total volume and the volume-weighted mean volume of lipid bodies were similar in all mouse strains. The results on the composition of the interalveolar septa did not show major differences between the groups. The only mouse strain that differed significantly from the other strains was the NMRI strain because the lungs had a higher volume and consequently many of the morphological parameters were also larger than in the other groups. In conclusion, the present study showed that lipofibroblasts are a common cell type in the mouse lung across various strains. Therefore, the mere presence or absence of lipofibroblasts does not explain differences in the pulmonary regenerative potential among mouse strains.

P2X7 Receptors Drive Spine Synapse Plasticity in the Learned Helplessness Model of Depression.

BackgroundMajor depressive disorder is characterized by structural and functional abnormalities of cortical and limbic brain areas, including a decrease in spine synapse number in the dentate gyrus of the hippocampus. Recent studies highlighted that both genetic and pharmacological invalidation of the purinergic P2X7 receptor (P2rx7) leads to antidepressant-like phenotype in animal experiments; however, the impact of P2rx7 on depression-related structural changes in the hippocampus is not clarified yet.MethodsEffects of genetic deletion of P2rx7s on depressive-like behavior and spine synapse density in the dentate gyrus were investigated using the learned helplessness mouse model of depression.ResultsWe demonstrate that in wild-type animals, inescapable footshocks lead to learned helplessness behavior reflected in increased latency and number of escape failures to subsequent escapable footshocks. This behavior is accompanied with downregulation of mRNA encoding P2rx7 and decrease of spine synapse density in the dentate gyrus as determined by electron microscopic stereology. In addition, a decrease in synaptopodin but not in PSD95 and NR2B/GluN2B protein level was also observed under these conditions. Whereas the absence of P2rx7 was characterized by escape deficit, no learned helpless behavior is observed in these animals. Likewise, no decrease in spine synapse number and synaptopodin protein levels was detected in response to inescapable footshocks in P2rx7-deficient animals.ConclusionOur findings suggest the endogenous activation of P2rx7s in the learned helplessness model of depression and decreased plasticity of spine synapses in P2rx7-deficient mice might explain the resistance of these animals to repeated stressful stimuli.

How common is the lipid body-containing interstitial cell in the mammalian lung?

Pulmonary lipofibroblasts are thought to be involved in lung development, regeneration, vitamin A storage, and surfactant synthesis. Most of the evidence for these important functions relies on mouse or rat studies. Therefore, the present study was designed to investigate the presence of lipofibroblasts in a variety of early postnatal and adult mammalian species (including humans) to evaluate the ability to generalize functions of this cell type for other species. For this purpose, lung samples from 14 adult mammalian species as well as from postnatal mice, rats, and humans were investigated using light and electron microscopic stereology to obtain the volume fraction and the total volume of lipid bodies. In adult animals, lipid bodies were observed only, but not in all rodents. In all other species, no lipofibroblasts were observed. In rodents, lipid body volume scaled with body mass with an exponent b = 0.73 in the power law equation. Lipid bodies were not observed in postnatal human lungs but showed a characteristic postnatal increase in mice and rats and persisted at a lower level in the adult animals. Among 14 mammalian species, lipofibroblasts were only observed in rodents. The great increase in lipid body volume during early postnatal development of the mouse lung confirms the special role of lipofibroblasts during rodent lung development. It is evident that the cellular functions of pulmonary lipofibroblasts cannot be transferred easily from rodents to other species, in particular humans.

Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea

Cancer patients often suffer from dyspnea the pathogenesis of which is incompletely understood. Both dyspnea and pulmonary diseases are closely linked to airway innervation. Recently, it was shown that Lewis lung carcinoma induces cardiac hypoinnervation in the mouse. We hypothesized that airway innervation undergoes similar changes as myocardial innervation and that this effect occurs in different mouse models of cancer. C57Bl6 mice were randomly assigned to subcutaneous injection of Lewis lung carcinoma cells (LLC, n=6), B16 melanoma cells (B16, n=6), or saline (control group, C, n=10). After 16 or 21 days, respectively, the trachea was processed for light and electron microscopic design-based stereology and the volume, surface area and length of axons ramifying in the tracheal wall were estimated. Body weight was reduced both in LLC and B16 vs. C. Hypoinnervation was present in both tumor groups compared to controls as volume and surface area of axons were significantly reduced in LLC and B16. However, the total length of tracheal axons and the mean number of axons per nerve fiber were reduced only in LLC but not in B16 compared to C indicating a differentially pronounced effect of cancer on tracheal innervation. In conclusion, reduced innervation of the trachea was observed in two different murine tumor models. These findings add to the pathophysiological concepts explaining cancer-related dyspnea and open new perspectives of treating this symptom.

Rebuilding the lung: signals for a complex architectural task

WHAT A REMARKABLE ORGAN THE human lung is! At the end of a deep breath, the vast majority of its volume (80%) is air. More than half of the remaining volume (10%) is blood. These two phases have to be separated strictly but also brought into close contact over a large surface to allow efficient gas exchange. However, what about those remaining 10% that serves this function: the “real” lung tissue? Those few hundred grams of tissue consist of more than 40 cell types, originating from all 3 germ layers, and a sophisticated connective tissue network. Together they form an organ with a complex architecture optimized to serve its main function. The branching airway and vascular trees come in close proximity in the parenchymal region where gas exchange is facilitated by a minimized barrier thickness over a maximized surface area. This delicate parenchymal structure is stabilized by two factors: the surface tension modifying properties of the pulmonary surfactant system and the lung’s connective tissue “backbone.” A continuous network of connective tissue, consisting of collagenous and elastic fibers and the fibroblasts that produce them, forms a tensegrity structure in the lung (15). This network, however, appears as “focal” (as it is often described in cases of proliferation, e.g., in idiopathic pulmonary fibrosis, although it is actually a highly interconnected reticulum; Ref. 3) in thin histological sections, which underscores the importance of qualitative and quantitative threedimensional information for proper characterization of lung structure in health and disease. Mechanical signals that act within and on the lung are translated into biochemical signals that lead to effects at the cellular level, e.g., inducing secretion, tissue growth, or remodeling, which, in turn, affect lung mechanics (for review, see e.g., Ref. 16). One example specific to the lung is the stimulation of surfactant secretion by mechanical distension of surfactant-producing type II alveolar epithelial cells or their neighboring type I cells (reviewed in Ref. 5). An interesting and relevant model to study the effects of mechanical forces on lung structure and function is compensatory lung growth following pneumonectomy. Moreover, compensatory lung growth is a paradigmatic example for the structural plasticity of the adult lung. Can the mechanical stimuli that induce compensatory lung growth after pneumonectomy, tissue expansion vs. microvascular perfusion, be dissected? A study by Ravikumar et al. (12) in this issue of the Journal of Applied Physiology elegantly demonstrates that this is possible. In adult dogs, the right lung was replaced by an inflated silicone prosthesis for 4 mo. Then, the prosthesis was either kept inflated or deflated for another 8 mo (long-term cohort). This study design was complemented by a short-term cohort with right pneumonectomy, prosthesis inflation for 1 mo, and immediate analysis either with or without deflation. This setting allowed the temporal separation of expansion- and perfusion-related stimuli for compensatory lung growth. A comprehensive quantitative analysis of lung structure was undertaken. Noninvasive chest imaging by high-resolution computed tomography was performed before and after pneumonectomy in the inflation and deflation groups. This was followed by post mortem morphometric analysis by light and electron microscopic stereology. The prosthesis prevented mediastinal shift and lateral expansion of the left lung but allowed some caudal elongation. Capillary blood volume increased and microvascular congestion was noted at low inflation. This was accompanied by an increase in alveolar septal volume but not surface area. Subsequent deflation led to an immediate mediastinal shift and lateral expansion of the remaining left lung which, in the long-term cohort, further increased lung and alveolar septal tissue volume and surface area in comparison to the inflation group. These quantitative data demonstrate that lung tissue expansion and microvascular perfusion contribute nearly

Loss of synapse-related genes in depression

A reduced number and function of synapses may underlie the smaller size and density of neurons in the dorsolateral prefrontal cortex (dlPFC) of patients with major depressive disorder (MDD). However, a direct causal relationship has not been demonstrated. Using microarray gene profiling and electron microscopic stereology, this study showed that patients with MDD have a reduced expression of various synapse-related genes and fewer synapses. Moreover, expression of the transcriptional repressor GATA1 is increased in dlPFC neurons of patients with MDD. In rats, Gata1 expression caused repression of these synaptic genes and loss of dendritic spines and dendrites, and induced depression-like behaviour. These data suggest that reversing dlPFC atrophy could be a strategy for treating depression.

Decreased expression of synapse-related genes and loss of synapses in major depressive disorder

Previous imaging and postmortem studies have reported a reduction in brain volume and a decrease in the size and density of neurons in the dorsolateral prefrontal cortex (dlPFC, area 9) of subjects with major depressive disorder (MDD).1,2 These findings suggest that synapse number and function are decreased in dlPFC of depressed patients. However, there has been no direct evidence for synapse loss in MDD and the gene expression alterations underlying these effects have not been identified. Here we use microarray gene profiling and electron microscopic stereology to reveal decreased expression of synaptic function-related genes in dlPFC of MDD subjects and a corresponding reduction in the number of synapses. We also identify a transcriptional repressor that is increased in MDD, and that when expressed in PFC neurons is sufficient to decrease expression of synapse-related genes, cause loss of spines and dendrites, and produce depressive behavior in rodent models of depression.

Glomerulopathy in spontaneously obese rhesus monkeys with type 2 diabetes: a stereological study.

Animal models could provide insights into the diabetic nephropathy pathogenesis; however, available rodent models do not mirror the heterogeneity of lesions in type 2 diabetic patients, and do not progress to end-stage renal disease. Previous studies showed that spontaneously obese type 2 diabetic rhesus monkeys develop many of the features of human diabetic glomerulopathy, and may progress to end-stage renal disease. Here, in order to further characterize diabetic glomerulopathy in this model, we used electron microscopic stereology. Renal biopsies from 17 diabetic, 17 pre-diabetic/metabolic syndrome and 11 non-diabetic monkeys were studied. Fractional volumes of mesangium [Vv(Mes/glom)], mesangial matrix [Vv(MM/glom)] and mesangial cells [Vv(MC/glom)], glomerular basement membrane width and peripheral glomerular basement membrane surface density per glomerulus [Sv(PGBM/glom)] were estimated. Glomerular filtration and albumin excretion rates were measured in a limited number of animals. Glomerular structural and biochemical/metabolic data were compared among the groups. Compared to non-diabetic monkeys, diabetic rhesus monkeys showed classic diabetic nephropathy changes, including glomerular basement membrane thickening (p = 0.001), increased fractional volumes of mesangium (p = 0.02), and reduced peripheral glomerular basement membrane surface density per glomerulus (p = 0.03) compared to non-diabetic monkeys. Increased fractional volumes of mesangium was primarily due to increased mesangial matrix (p = 0.03). Glomerular structural parameter inter-relationships in diabetic monkeys mirrored those of human diabetic glomerulopathy. Albumin excretion rate was greater (p = 0.03) in diabetic vs. non-diabetic monkeys. There was trend for a positive correlation between albumin excretion rate and fractional volumes of mesangium. This rhesus primate model shares many features of human diabetic glomerulopathy. Mesangial expansion in this model, similar to human diabetic nephropathy and different from available rodent models of the disease, is primarily due to increased mesangial matrix.

Allometry of the mammalian intracellular pulmonary surfactant system

Alveolar epithelial (AE) surface area is closely correlated with body mass (BM) in mammals. The AE is covered by a surfactant layer produced by alveolar epithelial type II (AE2) cells. We hypothesized that the total number of AE2 cells and the volume of intracellular surfactant-storing lamellar bodies (Lb) are correlated with BM with a similar slope as AE surface area. We used light and electron microscopic stereology to estimate the number and mean volume of AE2 cells and the total volume of Lb in 12 mammalian species ranging from 2 to 3 g (Etruscan shrew) to 400-500 kg (horse) BM. The mean size of Lb was evaluated using the volume-weighted mean volume and the volume-to-surface ratio of Lb. The mean volume of AE2 cells was 500-600 μm(3) in most species, but was higher in Etruscan shrew, guinea pig, and human lung. The mean volume of Lb per AE2 cell was 80-100 μm(3) in most species, with the same exceptions as above. However, the total number of AE2 cells and the total volume of Lb were closely correlated with BM and exhibited an allometric relationship similar to the slope of AE surface area. The mean size of Lb was similar in all investigated species. In conclusion, the mean volume of AE2 cells and their Lb are independent of BM but show some interspecific variations. The adaptation of the intracellular surfactant pool size to BM is obtained by the variation of the number of AE2 cells in the lung.

Quantitative Analysis of Collagen Network Structure and Fibril Dimensions in Cartilage Repair with Autologous Chondrocyte Transplantation

Objective: The aim of this study was to undertake a stereological analysis to quantify the dimensions of the collagen network in the repair tissue of porcine joints after they had been subjected to autologous chondrocyte transplantation (ACT). Method: ACT was used to repair cartilage lesions in knee joints of pigs. Electron-microscopic stereology, immunostaining for type II collagen, and quantitative polarized-light microscopy were utilized to study the collagen fibrils in the repair tissue 3 and 12 months after the operation. Results: The collagen volume density (V_V) was lower in the repair tissue than in normal cartilage at 3 months (20.4 vs. 23.7%) after the operation. The collagen surface density (S_V, 1.5·10^–2 vs. 3.1·10^–2 nm²/nm³) and V_V increased with time in the repair tissue (20.4 vs. 44.7%). Quantitative polarized-light microscopy detected a higher degree of collagen parallelism in the repair tissue at 3 months after the operation (55.7 vs. 49.7%). In contrast, 1 year after the operation, fibril parallelism was lower in the repair tissue than in the control cartilage (47.5 vs. 69.8%). Conclusion: Following ACT, V_V and S_V increased in the repair tissue with time, reflecting maturation of the tissue. One year after the operation, there was a lower level of fibril organization in the repair tissue than in the control cartilage. Thus, the newly synthesized collagen fibrils in the repair tissue appeared to form a denser network than in the control cartilage, but the fibrils remained more randomly oriented.

Effects of Estradiol on Learned Helplessness and Associated Remodeling of Hippocampal Spine Synapses in Female Rats

Despite the fact that women are twice as likely to develop depression as men, our understanding of depression neurobiology in female subjects is limited. We have recently reported in male rats that development of helpless behavior is associated with a severe loss of hippocampal spine synapses, which is reversed by treatment with the antidepressant desipramine. Considering that estradiol has a hippocampal synaptogenic effect similar to those of antidepressants, the presence of estradiol during the female reproductive life might influence behavioral and synaptic responses to stress and depression. With electron microscopic stereology, we analyzed hippocampal spine synapses in association with helpless behavior in ovariectomized female rats (n = 70), under different conditions of estradiol exposure. Stress induced an acute and persistent loss of hippocampal spine synapses, whereas subchronic treatment with desipramine reversed the stress-induced synaptic loss. Estradiol supplementation given either before stress or before escape testing of nonstressed animals increased the number of hippocampal spine synapses. Correlation analysis demonstrated a statistically significant negative correlation between the severity of helpless behavior and hippocampal spine synapse numbers. These findings suggest that hippocampal spine synapse remodeling might be a critical factor underlying learned helplessness and, possibly, the neurobiology of depression.

Cartilage Collagen Fibril Network in Newborn Transgenic Mice Analyzed by Electron Microscopic Stereology

The objective was to investigate by electron microscopic stereology the properties of the cartilage collagen fibril network in newborn transgenic mice. The mice harbored transgenes targeted to affect the structure or assembly of the collagen fibrils. The mouse lines investigated here harbored either (i) one or (ii) two human COL2A1 genes with Arg519Cys mutation in addition to one or (iii) no active allele(s) of the murine Col2a1 gene, (iv) two inactive alleles of the procollagen N-proteinase genes, or (v) a human COL2A1 gene with deleted exons 16–27. In all newborn mice carrying the COL2A1 transgene with Arg519Cys mutation, the growth plate collagen fibrils were thinner than in the wild-type (wt) mice and showed clearly reduced volume fraction of the fibril network. In mice with the inactive procollagen N-proteinase genes, the fibril thickness and the volume fraction of collagen did not differ from the wt mice. In mice harboring the transgene of human COL2A1 gene with internally deleted exons 16–27, the collagen fibril diameter remained the same, but the volume density of collagen network was reduced. Using the indirect stereology, the differences in the collagen fibril stereological estimates could be reliably detected in newborn mice harboring mutations that affect the structure and assembly of collagen fibrils. The EM stereology permitted early detection of altered phenotype of the collagen fibril network in newborn transgenic mice. It is recommended that the indirect model-based stereological technique is utilized instead of the direct design-based technique for the estimation of collagen volume, surface, and length densities.

Pre-ischaemic exogenous surfactant reduces pulmonary injury in rat ischaemia/reperfusion

The optimal timing of exogenous surfactant application to reduce pulmonary injury and dysfunction was investigated in a rat lung ischaemia and reperfusion injury model. Lungs were subjected to flush perfusion, surfactant instillation, cold ischaemia (4 degrees C, 4 h) and reperfusion (60 min). Animals received surfactant before (group 1) or at the end (2) of ischaemia, or during reperfusion (3) or not at all (4). Control groups included "worst case" without Perfadex and surfactant (5), "no injury" without (6) or with surfactant (7), and ischaemia with pre-ischaemic surfactant (8). Intra-alveolar oedema and blood-air barrier injury were estimated by light and electron microscopic stereology. Perfusate oxygenation and pulmonary arterial pressure (P(pa)) were determined during reperfusion in groups 1 to 4. Intra-alveolar oedema was almost absent in groups 1, 6, 7 and 8, pronounced in 2, 3 and 4, and severe in 5. Blood-air barrier injury was moderate in groups 1 and 8, slightly pronounced in 2, 3 and 4, extensive in 5 and almost absent in 6 and 7. Perfusate oxygenation was significantly higher in group 1 compared with groups 2 to 4. P(pa) did not differ between the groups. In conclusion, exogenous surfactant attenuates intra-alveolar oedema formation and blood-air barrier damage and improves perfusate oxygenation in the rat lung, especially when applied before ischaemic storage.

Remodeling of Hippocampal Spine Synapses in the Rat Learned Helplessness Model of Depression

Although it has been postulated for many years that depression is associated with loss of synapses, primarily in the hippocampus, and that antidepressants facilitate synapse growth, we still lack ultrastructural evidence that changes in depressive behavior are indeed correlated with structural synaptic modifications. We analyzed hippocampal spine synapses of male rats (n=127) with electron microscopic stereology in association with performance in the learned helplessness paradigm. Inescapable footshock (IES) caused an acute and persistent loss of spine synapses in each of CA1, CA3, and dentate gyrus, which was associated with a severe escape deficit in learned helplessness. On the other hand, IES elicited no significant synaptic alterations in motor cortex. A single injection of corticosterone reproduced both the hippocampal synaptic changes and the behavioral responses induced by IES. Treatment of IES-exposed animals for 6 days with desipramine reversed both the hippocampal spine synapse loss and the escape deficit in learned helplessness. We noted, however, that desipramine failed to restore the number of CA1 spine synapses to nonstressed levels, which was associated with a minor escape deficit compared with nonstressed control rats. Shorter, 1-day or 3-day desipramine treatments, however, had neither synaptic nor behavioral effects. These results indicate that changes in depressive behavior are associated with remarkable remodeling of hippocampal spine synapses at the ultrastructural level. Because spine synapse loss contributes to hippocampal dysfunction, this cellular mechanism may be an important component in the neurobiology of stress-related disorders such as depression.

Bisphenol A Prevents the Synaptogenic Response to Testosterone in the Brain of Adult Male Rats

Exposure measurement data from several developed countries indicate that human beings are widely exposed to low levels of the synthetic xenoestrogen, bisphenol A. We reported previously that bisphenol A, even at doses below the reference safe daily limit for human exposure, recommended by the U.S. Environmental Protection Agency, impairs the synaptogenic response to 17beta-estradiol in the hippocampus of ovariectomized rats. Recent experiments revealed that bisphenol A also interferes with androgen receptor-mediated transcriptional activities. Thus, to investigate whether bisphenol A impairs synaptogenesis in the medial prefrontal cortex (mPFC) and hippocampus of adult male rats, castrated and sham-operated animals were treated with different combinations of bisphenol A (300 microg/kg), testosterone propionate (1.5 mg/kg), and sesame oil vehicle. The brains were processed for electron microscopic stereology, and the number of asymmetric spine synapses in the mPFC and CA1 hippocampal area was estimated. In both regions analyzed, bisphenol A reduced the number of spine synapses in sham-operated, gonadally intact animals, which was accompanied by a compensatory increase in astroglia process density. In addition, bisphenol A prevented both the prefrontal and hippocampal synaptogenic response to testosterone supplementation in castrated males. These results demonstrate that bisphenol A interferes with the synaptogenic response to testosterone in the mPFC and hippocampus of adult male rats. Because the hippocampal synaptogenic action of androgens seems to be independent of androgen and estrogen receptors in males, the potential mechanisms that underlie these negative effects of bisphenol A remain the subject of further investigation.

Collagenase-Induced Changes in Articular Cartilage as Detected by Electron-Microscopic Stereology, Quantitative Polarized Light Microscopy and Biochemical Assays

The purpose of this study was to compare the ability of electron-microscopic (EM) stereology with quantitative polarized light microscopy (PLM) and biochemical collagen (hydroxyproline) and crosslink (pyridinoline) analyses to detect changes in the superficial collagen network of bovine articular cartilage after digestion in vitro with purified bacterial (Clostridium histolyticum) collagenase (30 U/ml) for 24 and 48 h. Collagen volume (V_V) and surface (S_V) densities of the uppermost third of the superficial zone were estimated indirectly from zonal isotropic uniform random sections using collagen length density (L_V) and average collagen fibril diameter, or its average second power. Collagenase digestion caused a significant decrease in fibril diameter (64 to 62%), V_V (89 to 95%) and S_V (64 to 86%) after incubation for 24 and 48 h. Collagen L_V remained unchanged after 24 h incubation but decreased 63% after 48 h. Collagen concentration per dry weight, assayed biochemically from the whole superficial zone, decreased also significantly (29 to 60%) after 24 and 48 h digestions, respectively. The pyridinoline concentration per dry weight of the superficial zone decreased (31 to 57%) whereas the pyridinoline concentration per collagen remained unchanged. PLM revealed that the birefringence of the uppermost third of the superficial zone was decreased by 36% after digestion for 24 h though the total birefringence of the whole zone was not reduced. However, after 48 h, the birefringence of the whole superficial zone was significantly reduced (76%). All of the techniques compared in this study could detect collagen network degradation in bovine articular cartilage but the EM stereological technique was more sensitive at detecting the changes than PLM or biochemical assays.

Electron microscopic stereology of capillary endothelial cells and cardiomyocytes in artificially arrested canine hearts.

In open heart surgery and transplantation, sufficient structural preservation of the myocardium immediately following cardioplegic arrest is a precondition for overcoming ischemia and for resumption of postischemic function. Therefore, we compared the protective effect of three clinically applied cardioplegic solutions with fibrillating and beating hearts using structural criteria. Left ventricular samples were taken from (1) beating, or (2) fibrillating or arrested hearts following coronary perfu-sion with (3) St. Thomas' Hospital solution, (4) histidine tryptophane ketoglutalate (HTK) (Custodiol), or (5) University of Wisconsin (UW) solution and fixed by immersion. Ultrastructural differences in the swelling of capillary endothelial cells and myocytes were quantitatively evaluated using stereological methods. Endothelial cells were somewhat more swollen after St. Thomas perfusion than those in beating and fibrillating hearts. HTK-arrested hearts showed significantly lower values for cellular edema than beating hearts. UW perfusion resulted in the (significantly) lowest degree of endothelial cell edema. Edematous changes in myocytes were significantly greater in St. Thomas-arrested hearts than in UW- or HTK-arrested hearts. Cardiomyocyte edema in beating and fibrillating hearts was comparable to that in St. Thomas-perfused hearts. Thus, the stereol-ogical analysis revealed significant differences between cardioplegic solutions in structural preservation of myocardial ultrastructure.