Cavalieri Estimator Research Articles

Applying the area fraction fractionator (AFF) probe for total volume estimations of somatic, dendritic and axonal domains of the nigrostriatal dopaminergic system in a murine model

BackgroundThe Cavalieri estimator is used for volume measurement of brain and brain regions. Derived from this estimator is the Area Fraction Fractionator (AFF), used for efficient area and number estimations of small 2D elements, such as axons in cross-sectioned nerves. However, to our knowledge, the AFF has not been combined with serial sectioning analysis to measure the volume of small-size nervous structures. New methodUsing the nigrostriatal dopaminergic system as an illustrative case, we describe a protocol based on Cavalieri's principle and AFF to estimate the volume of its somatic, nuclear, dendritic, axonal and axon terminal cellular compartments in the adult mouse. The protocol consists of (1) systematic random sampling of sites within and across sections in regions of interest (substantia nigra, the nigrostriatal tract, caudate-putamen), (2) confocal image acquisition of sites, (3) marking of cellular domains using Cavalieri's 2D point-counting grids, and 4) determination of compartments’ total volume using the estimated area of each compartment, and between-sections distance. ResultsThe volume of the nigrostriatal system per hemisphere is ∼0.38 mm3, with ∼5 % corresponding to perikarya and cell nuclei, ∼10 % to neuropil/dendrites, and ∼85 % to axons and varicosities. Comparison with existing methodsIn contrast to other methods to measure volume of discrete objects, such as the optical nucleator or 3D reconstructions, it stands out for its versatility and ease of use. ConclusionsThe use of a simple quantitative, unbiased approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.

Volumetric study on sheep brain using stereology technique

AbstractThree‐dimensional morphometric data better show the structural and functional characteristics of the brain. The objective of this study was to estimate the volume of the cerebral structures of the sheep using design‐based stereology. The brains of five sheep were used, fixed in formalin 10% and embedded in agar 6%. An average of 10–12 slab was obtained from each brain. All slabs were stained using Mulligan's method and photographs were recorded. The volume of the brain and its structures were estimated using the Cavalieri's estimator and the point counting system. The total volume was 70604.8 ± 132.45 mm3. The volume fractions of the grey and white matters were calculated as 42.55 ± 0.21% and 24.23 ± 0.51% of the whole brain, respectively. The fractional volume of the caudate nucleus and claustrum were estimated at 2.39 ± 0.08% and at 1.008 ± 0.057% of total brain volume. The volumes of corpus callosum, internal capsule and external capsule were 1.24 ± 0.053%, 3.63 ± 0.22% and 0.698 ± 0.049% of total cerebral volume, respectively. These data could help improve the veterinary comparative neuroanatomy knowledge and development of experimental studies in the field.

RIBM3DU‐Net: Glioma tumour substructures segmentation in magnetic resonance images using residual‐inception block with modified 3D U‐Net architecture

AbstractGlioma brain tumour is one of the life‐threatening diseases in the world. Tumour substructure segmentation by physicians is a time‐consuming task with the magnetic resonance imaging (MRI) technique due to the size of clinical data. An automatic and well‐trained method is essential to detect and segment the tumour which increase the survival of the patients. The proposed work aims to produce high accuracy on glioma substructures segmentation with less computation time using deep learning. From the literature survey, the following challenges are found: (i) computing complex spatial boundaries between normal and tumour tissues, (ii) feature reduction and (iii) overfitting problems. Hence, we proposed a fully automatic glioma tumour segmentation using a residual‐inception block (RIB) with a modified 3D U‐Net (RIBM3DU‐Net). It includes three phases: pre‐processing, modified 3D U‐Net segmentation and post‐processing. From the results, RIB with U‐Net enhances the segmentation accuracy. GPU parallel architecture reduces the computation time while training and testing. For quantitative analysis, comprehensive experiment results were computed and compared with state‐of‐the‐art methods. It achieves better Dice scores on enhancing tumour, tumour core and complete tumour of 87%, 87% and 94%, respectively. GPU speedup folds yield up to 48× when compared with CPU. Quantitatively, 3D glioma volume is rendered from the obtained segmented results and estimated using the Cavalieri estimator.

Three-dimensional morphometry of the testis in dog using design-unbiased stereology.

Testis is considered the main organ of the male reproductive system. Dogs are used as a suitable experimental model of testicular diseases in humans. From the veterinary aspect, several disorders have been reported to affect the testis in dogs. Thus, the objective of the present study was to investigate the morphometrical features of the dog testis using design-based stereology. The testes of six male dogs were used. Isotropic, uniform random sections were obtained and processed for light microscopy. Testicular total volume and the fractional volume of the seminiferous tubules, interstitial tissue and germinal epithelium were measured using the Cavalieri's estimator and the point counting system. Germinal epithelial surface area was estimated using test lines, and total length of seminiferous tubules was analysed using the counting frames. The total volume of testis was calculated 13.64 ± 1.94 cm3 . The relative volume fractions of the seminiferous tubules, interstitial tissue and germinal layer expressed as a percentage of total testicular volume were found to be 75.87 ± 6.11%, 23.68 ± 5.15% and 64.15 ± 4.82%, respectively. The surface area of the germinal layer was 915.25 ± 150.48 cm2 . The thickness of germinal layer was estimated to be 96.18 ± 10.72 μm. The total length of seminiferous tubules measured 290.8 ± 35.86 m. No statistical difference in investigated parameters was found between the left and right testes (p > 0.05). Our data might contribute to the male reproductive knowledge, help develop experimental studies in this field and possibly lead to advancement in the diagnosis and treatment of testicular diseases in the dog.

A stereological study on determination of volume of the heart ventricles in female and male quails

In this study, ventricular wall volume of female and male quails was investigated stereologically. Six females and six males were used in this study. All of the animals were perfused. After the perfusion, the quails were kept in 10% formaldehyde solution. Afterwards, chests of quails were cut and their hearts were resected. Ventricles of the hearts were separated. Specific ratio of tissue samples was obtained from each ventricle. The 5-µm thick samples were cut by using a microtome. Sequentially, 10 sections were obtained. These sections were stained by hematoxylin eosin and photographed. Volumes of all tissues of the ventricles were estimated by using the Cavalieri Estimator. In this study, the volume values of female and male quails were compared. Some differences were found between these values. The volume values of six female quails were compared with each other. While the lowest volume value was 0.398 cm³, the highest volume value was 0.612 cm³. The male quails’ volume values were between 0.438 cm³-0.817 cm³. It was found that volume values of male quails were higher than volume values of female quails. As a result, although there was a specific distinction between volume values of female and male quails’ ventricles, there was no difference between statistic values (P>0.05). It was thought that this study will be guiding for other related studies.

Improving Cavalieri volume estimation based on non-equidistant planar sections: The trapezoidal estimator.

The Cavalieri estimator allows one to infer the volume of an object from area measurements in equidistant planar sections. It is known that applying this estimator in the non-equidistant case may inflate the coefficient of error considerably. We therefore consider a newly introduced variant, the trapezoidal estimator, and make it available to practitioners. Its typical variance behaviour for natural objects is comparable to the equidistant case. We state this unbiased estimator, describe variance estimates and explain how the latter can be simplified under rather general but realistic models for the gaps between sections. Simulations and an application to a synthetic area function based on parietal lobes of 18 monkeys illustrate the newmethods.

Stereological study of the kidney during prenatal development in sheep.

The development of metanephros is a complex and gradual process. The number, size and distribution of nephrons provide important information about the organization of the kidney. Stereology is the current gold-standard technique for the morphometrical evaluation of kidney structures. This study describes morphometric features of the kidney development in sheep using design-based stereological techniques aimed to introduce the sheep as a translational model in human nephrogenesis. Left kidneys of 16 sheep fetuses in four groups at 9-11, 12-14, 15-17, and 18-20 weeks of gestation were used in the present study. Systematic uniform random sections were obtained. The kidney volume, volume fraction of nephrogenic zone, cortex and medulla, and glomerular volume were estimated using point counting and Cavalieri's estimator. The total glomerular number was estimated using a physical disector/fractionator technique. The kidney and its compartments presented gradual changes with aging, with differences found in the last fetal ages. The kidney volume increased from 0.94 ± 0.22 cm3 to 8.6 ± 0.88 cm3 during development. The volume of cortex increased from 406 ± 85 mm3 to 5,151 ± 309 mm3 and the volume of medulla showed increase from 301 ± 91 mm3 to 3,426 ± 599 mm3 . The total glomerular volume increased from 13.8 ± 1.6 mm3 to 235 ± 44 mm3 . The total glomerular number increased from 4,683 ± 757 to 639 × 103 ± 11.6 × 103 . Our data might contribute to the knowledge of embryological urology and promote future experimental investigations in this field.

Morphometry of cervical spinal cord in cat using design-based stereology.

The spinal cord harbours nerve fibres that facilitate reflex actions and that transmit impulses to and from the brain. The cervical spinal cord is an area of particular interest in medicine and veterinary due to frequent pathologic alterations in this region. This study describes the morphometric features of the cervical spinal cord in cat using design-unbiased stereological methods. The cervical spinal cords of four male cats were dissected and samples were taken according to systematic uniform random sampling. Each sample was embedded in agar and cut into 60-µm thick sections and stained with cresyl violet 0.1% for stereological estimations. The total cervical spinal cord volume obtained by the Cavalieri estimator was 2,321.21±285.5mm3 . The relative volume of grey matter and white matter was 23.8±1.3% and 76.1±1.3%. The dorsal horn and ventral horn volume were 12.3±1.2% and 11.4±0.7% of the whole cervical spinal cord. The volume of central canal was estimated to 3.8±1mm3 . The total number of neurons was accounted 3,405,366.2±267,469.4 using the optical disector/fractionator method. The number of motoneurons and interneurons was estimated to be 1,120,433.2±174,796.7 and 2,284,932.9±127,261.5, respectively. The average volume of the motoneurons and interneurons was estimated to 1980µm3 and 680µm3 , respectively, using the spatial rotator method. This knowledge of cat spinal cord findings may serve as a foundation as a translational model in spinal cord experimental research and provide basic findings for diagnosis and treatment of spinal cord disorders.

Asymptotic variance of Newton–Cotes quadratures based on randomized sampling points

AbstractWe consider the problem of numerical integration when the sampling nodes form a stationary point process on the real line. In previous papers it was argued that a naïve Riemann sum approach can cause a severe variance inflation when the sampling points are not equidistant. We show that this inflation can be avoided using a higher-order Newton–Cotes quadrature rule which exploits smoothness properties of the integrand. Under mild assumptions, the resulting estimator is unbiased and its variance asymptotically obeys a power law as a function of the mean point distance. If the Newton–Cotes rule is of sufficiently high order, the exponent of this law turns out to only depend on the point process through its mean point distance. We illustrate our findings with the stereological estimation of the volume of a compact object, suggesting alternatives to the well-established Cavalieri estimator.

Improving the Cavalieri estimator under non-equidistant sampling and dropouts

Motivated by the stereological problem of volume estimation from parallel section profiles, the so-called Newton-Cotes integral estimators based on random sampling nodes are analyzed. These estimators generalize the classical Cavalieri estimator and its variant for non-equidistant sampling nodes, the generalized Cavalieri estimator, and have typically a substantially smaller variance than the latter. The present paper focuses on the following points in relation to Newton-Cotes estimators: the treatment of dropouts, the construction of variance estimators, and, finally, their application in volume estimation of convex bodies.Dropouts are eliminated points in the initial stationary point process of sampling nodes, modeled by independent thinning. Among other things, exact representations of the variance are given in terms of the thinning probability and increments of the initial points under two practically relevant sampling models. The paper presents a general estimation procedure for the variance of Newton-Cotes estimators based on the sampling nodes in a bounded interval. Finally, the findings are illustrated in an application of volume estimation for three-dimensional convex bodies with sufficiently smooth boundaries.

Human kidney graft survival correlates with structural parameters in baseline biopsies: a quantitative observational cohort study with more than 14 years' follow-up.

This prospective cohort study evaluates associations between structural and ultrastructural parameters in baseline biopsies from human kidney transplants and long-term graft survival after more than 14 years' follow-up. Baseline kidney graft biopsies were obtained prospectively from 54 consecutive patients receiving a kidney transplant at a single institution. Quantitative measurements were performed on the baseline biopsies by computer-assisted light microscopy and electron microscopy. Stereology-based techniques estimated the fraction of interstitial tissue, the volume of glomeruli, mesangial fraction, and basement membrane thickness of glomerular capillaries. The fraction of occluded glomeruli and scores according to the Banff classification were achieved. Kidney graft survival was analyzed by Kaplan-Meier estimates and Cox regression. Association to long-term kidney function was also analyzed. The long-term surviving kidney transplants were characterized at implantation by less arteriolar hyaline thickening (P < 0.001) and less interstitial fibrosis (P = 0.001), as well as a lower fraction of occluded glomeruli (P = 0.004) and lower glomerular volume (P = 0.03). At the latest follow-up, eGFR was decreased by 12 ml/min/1.73 m2 per unit increase in the score for arteriolar hyalinosis at implantation (P = 0.02), and eGFR was decreased by 19 ml/min/1.73 m2 per 106 μm3 increase in glomerular volume at baseline (P = 0.03). The unbiased Cavalieri estimate of glomerular volume and the ultrastructural parameters are the first to be evaluated in a cohort study with prospective follow-up for more than 14 years. The study shows that baseline biopsies from human kidney grafts contain extraordinary long-term prognostic information, and it highlights the importance of these intrinsic graft factors.

Morphometrical study of the cat cerebellum using unbiased design-based stereology.

The objective of the present study was to investigate the morphometrical features of the cat cerebellum using design-based stereology. Cerebellar hemispheres from four male cats were examined. Isotropic, uniform random sections were obtained and processed for light microscopy. Cerebellar total volume (V), white (WM) and grey matter (GM) volume fractions, and the volumes of the molecular and granular layers were measured using the Cavalieri's estimator and the point counting system. Cerebellar surface area was estimated using test lines, and Purkinje cellular and nuclear volumes were analysed using the nucleator technique. The volume of the cat cerebellar hemispheres was 2.06±0.29cm3 . The relative volume fractions of the GM and WM were 70.6±2.6% and 29.3±2.6%, respectively. The surface area of the cerebellar hemisphere was 68.2±17.8cm2 . The volumes of the molecular and granular layers were estimated at 0.89±0.16cm3 and 0.56±0.1cm3 , respectively. The Purkinje cell volumes were found to be ranging from 1,717 to 28,489µm3 , of which cells with a perikaryon volume of 6,994µm3 had a higher incidence. The Purkinje nuclear volume was estimated at 440-3,561µm3 , and nuclei with a volume of 1,252µm3 were the most frequently occurring ones. Our data might contribute to the veterinary comparative neuroanatomy knowledge, help develop experimental studies in this field, and possibly lead to advancement in the diagnosis and treatment of nervous diseases in the cat.

P1620PROGNOSTIC VALUE OF RENAL BIOPSY QUANTITATIVE HISTOLOGY IN LIVING KIDNEY DONORS AND RECIPIENTS OF KIDNEYS FROM LIVING DONORS

Abstract Background and Aims Living kidney donors (LKD) have an increased risk of chronic kidney disease. Likewise, renal transplant recipients may experience early or unexplained loss of graft function. We examined the predictive value of stereology based on a CT-scan and a biopsy from the transplanted kidney in relation to renal function outcome in both LKD and recipients of kidneys from living donors. Method A pre-transplantation contrast-enhanced CT-scan was used to obtain cortical volume of both kidneys using the Cavalieri estimator. Glomerular number density in a baseline biopsy from the transplanted kidney (taken at the time of transplantation) was estimated by a model-based method and multiplied with cortex volume to estimate the total number of glomeruli in the donated kidney. Glomerular volume was estimated by the 2D nucleator (1). In addition, kidney fibrosis (point-counting), glomerular sclerosis, and arteriole dimensions (2D nucleator) were also calculated. Pre-donation single kidney glomerular filtration rate (GFR) was determined using 51chrome EDTA clearance together with a renography and the GFR measurement was repeated one year after transplantation in both donors and recipients. We examined the association between GFR at one-year follow-up and cortex volume and various histology parameters from the baseline biopsy after adjustment of age, sex, body mass index, smoking status, 24-hour mean blood pressure and single-kidney GFR before transplantation. Results CT-scans, GFR measurements and kidney histology from 49 LKD and 38 recipients of kidneys from living donors were available for analysis. Mean age of donors was 50±12 years (55% women) and of recipients 46±13 years (26% women). In donors, GFR decreased from 113±24 to 71±16 ml/min while in recipients GFR was 61±18 ml/min at one-year follow-up (all with a functioning graft). In donors, GFR at follow-up of the remaining kidney had increased by 28% as compared to baseline. Follow-up GFR correlated to cortex volume (P&amp;lt;0.001), but not to any of the histological parameters (total glomerular number, glomerular volume, % sclerosed glomeruli, % kidney fibrosis or arteriolar dimensions). In recipients, GFR at follow-up had increased by 8% as compared to single-kidney GFR before donation. Follow-up GFR correlated to % kidney fibrosis (P=0.02) and % sclerosed glomeruli (P=0.05), but not any of the other histological parameters or cortex volume. Conclusion When adjusted for relevant clinical confounders the estimation of cortex volume had predictive value for renal function outcome in LKD while % kidney fibrosis and % sclerosed glomeruli had predictive value in recipients of kidneys from living donors.

How repetitive traumatic injury alters long-term brain function.

How recurrent traumatic brain injury (rTBI) alters brain function years after insult is largely unknown. This study aims to characterize the mechanistic cause for long-term brain deterioration following rTBI using a rat model. Eighteen Sprague-Dawley wild-type rats underwent bilateral rTBI using a direct skull impact device or sham treatment, once per week for 5 weeks, and were euthanized 56 weeks after the first injury. Weekly rotarod performance measured motor deficits. Beam walk and grip strength were also assessed. Brain tissue were stained and volume was computed using Stereo Investigator's Cavalieri Estimator. The L5 cortical layer proximal to the injury site was microdissected and submitted for sequencing with count analyzed using R "DESeq2" and "GOStats." Brain-derived neurotrophic factor (BDNF) levels were determined using enzyme-linked immunosorbent assay. Rotarod data demonstrated permanent deficits 1 year after rTBI. Decreased beam walk performance and grip strength was noted among rTBI rodents. Recurrent traumatic brain injury led to thinner cortex and thinner corpus callosum, enlarged ventricles, and differential expression of 72 genes (25 upregulated, 47 downregulated) including dysregulation of those associated with TBI (BDNF, NR4A1/2/3, Arc, and Egr) and downregulation in pathways associated with neuroprotection and neuroplasticity. Over the course of the study, BDNF levels decreased in both rTBI and sham rodents, and at each time point, the decrease in BDNF was more pronounced after rTBI. Recurrent traumatic brain injury causes significant long-term alteration in brain health leading to permanent motor deficits, cortical and corpus callosum thinning, and expansion of the lateral ventricles. Gene expression and BDNF analysis suggest a significant drop in pathways associated with neuroplasticity and neuroprotection. Although rTBI may not cause immediate neurological abnormalities, continued brain deterioration occurs after the initial trauma in part due to a decline in genes associated with neuroplasticity and neuroprotection.

Kidney structural characteristics based on a kidney biopsy and contrast-enhanced computed tomography in healthy living kidney donors.

The demands for kidney transplantations are increasing, and so is the number of live kidney donors (LKDs). Recent studies show that LKDs have an increased risk of developing end-stage renal disease compared with healthy non-donors. However, the knowledge about factors predicting renal disease in kidney donors is sparse. Some evidence points to increased glomerular sclerosis and kidney fibrosis, as well as a low number of glomeruli as associated with a worse renal outcome. This methodological study investigated that which estimates are obtainable with a standard kidney biopsy taken from the donated kidney during the transplantation, and a standard contrast-enhanced computed tomography (CT) in kidney donors. CT-scans were used to obtain total volume of the kidney and kidney cortex using the Cavalieri estimator and 2D-nucleator. Glomerular number density in the biopsies was estimated by a model-based method, and was multiplied by total cortex volume in order to estimate the total number of glomeruli in the kidney. Glomerular volume was estimated by the 2D-nucleator and a model-based stereological technique. Kidney fibrosis (point-counting), glomerular sclerosis (evaluation of glomerular profiles), and arteriole dimensions (2D-nucleator) were also estimated in the biopsy sections from the donated kidney. Various studies have attempted to identify predictors of renal outcome in LKDs. There is no consensus yet, and further studies are needed to elucidate if and how the estimates described in this study are associated with renal outcome in LKDs.

Rapid effects of S-ketamine on the morphology of hippocampal astrocytes and BDNF serum levels in a sex-dependent manner

The prevalence of major depressive disorder (MDD) is higher in women than men. Importantly, a differential behavioral response by sex to the antidepressant response to ketamine in rodents has been reported. Mechanistically, male depressed-like animals showed an increased spine density after ketamine treatment via restoration of synaptic protein levels while those proteins were not altered in female rats. In addition, preclinical studies indicate that the impairment of astrocytic plasticity is one of the contributing mechanisms in the pathophysiology of MDD. Accordingly, in this study, we determined the effect of sex on the rapid morphological alteration of hippocampal astrocytes and the serum level of BDNF one hour after S-ketamine injection. A single intraperitoneal dose of S-ketamine (15 mg/kg) or saline was injected to the male and female Flinders Sensitive Line (FSL) rats, a genetic animal model of depression and their brains were perfused one hour after treatment. The size of the GFAP positive astrocytes in the hippocampal subregions was measured. The volume of different hippocampal subregions was assessed using the Cavalieri estimator. Moreover, serum levels of BDNF were measured with enzyme-linked immunosorbent assay (ELISA) kits. The volume of hippocampal subregions significantly increased one hour after S-ketamine in both male and female FSL animals. However, a substantial alteration in the morphology of the hippocampal astrocytes was observed only in the female rats. Additionally, significantly increased serum BDNF levels in the female depressed rats were observed one hour after S-ketamine treatment. Our results indicate that the rapid effects of S-ketamine on the morphology of the hippocampal astrocytes and the serum level of BDNF are sex-dependent.

T193. CHRONIC HALOPERIDOL TREATMENT INDUCES SIGNIFICANT CHANGES IN MICROGLIA AND IN THE EXPRESSION OF THE 18 KDA TRANSLOCATOR PROTEIN TSPO IN NAIVE ADULT RAT BRAINS

BackgroundPositron emission tomography (PET) using radiolabeled ligands selective for the 18 kDa translocator protein (TSPO) is the most widely used technique to assess putative neuroimmune abnormalities in vivo. However, the results of TSPO PET in schizophrenia patients are ambiguous. In particular, the influence of antipsychotic medication exposure has not been definitively tested. We have previously established that chronic exposure to two different antipsychotics resulted in increased density and amoeboid morphology of brain microglia, particularly in the cortex (ACC) and corpus striatum (STR) (Cotel et al., 2015). Here we explore the consequences of such treatment on TSPO, in relation to changes in microglia. We hypothesize that chronic haloperidol treatment would increase the expression of TSPO in microglia and correlate with increased levels of circulating proinflammatory cytokines.MethodsAdult male Sprague-Dawley rats (12 week-old) were implanted with subcutaneous osmotic mini-pumps (ALZET® 2ML4) delivering a continuous dose of either common vehicle (20% β-cyclodextrin + 5% ascorbic acid) or haloperidol (2mg/kg/d) for 28 days (Kapur et al., 2003). Plasma was collected before perfusing the animals with 4% buffered PFA. Brains were carefully removed and processed for immunostaining against Iba1 and TSPO. Levels of plasma cytokines were measured using the pro-inflammatory 9-plex panel from Meso Scale Discovery (MSD®). TSPO expression was measured by its area fraction using FIJI software (10 snaps per animal per brain region, 20x magnification) in the ACC, STR, hippocampus (HPC), secondary somatosensory cortex (S2), primary motor cortex (M1). Numbers of resting and amoeboid microglia were determined by unbiased stereology (Gundersen, 1987) in the ACC, STR, and HPC. The Cavalieri estimator (StereoInvestigator 12.0,MBF Bioscience) was used to determine the volume of these regions. Data were analyzed by t-tests corrected for multiple comparisons, using GraphPad Prism 6.0.ResultsOnly 5 cytokines from the panel were above the limit of detection. Of these, IL-6 is significantly increased in haloperidol-treated animals (+186%, p<.01). IL-10, IL-4, KC/GRO, and TNFα plasma levels were not significantly affected. In parallel, TSPO expression was significantly increased in the ACC (+56%, p<.01), the STR (+77%, p<.01) and the S2 (+158%, p<.001). In the STR, the density of both resting and amoeboid microglia were significantly elevated in the treatment group (+22%, p<.05 and +45%, p<.001, respectively). A 2-way ANOVA revealed a significant interaction between treatment and brain region for amoeboid but not resting microglia. Peripheral IL-6 levels correlated negatively with TSPO levels in the STR in saline and haloperidol-treated groups (r=-0.82 and r=-0.62, respectively, p<.05). In addition, KC/GRO, although not different between groups, correlated positively with TSPO levels in STR in controls (r=0.77, p<.05), but negatively in HPC, M1, and S2 after haloperidol treatment (r=-0.66, r=-0.62, r=-0.6, respectively, p<.05).DiscussionAs hypothesized, TSPO expression is significantly affected by chronic haloperidol, with augmented levels in the ACC, STR and S2. These data are in line with an increased TSPO PET signal in medicated schizophrenia patients as compared to matched antipsychotic-naïve patients (Holmes et al., 2016). Furthermore, these data confirm and extend our prior findings demonstrating that microglial activation is present already after 28 days of exposure to haloperidol and this effect is most prominent in the STR. Further investigations are needed to determine which cell types drive changes in TSPO (glia, endothelium, astrocytes) and how this relates to their functions at a molecular level.

OP 24 Nrf2 deficiency induces placental TNF- α expression and influences the fetal growth in mice

Introduction and objectives Increased oxidative stress and intrauterine inflammation are etiological factors for intrauterine growth restriction (IUGR). The transcription of many antioxidative genes is mediated mainly through the transcription factor Nrf2. A link between Nrf2 and the vascular haemeostasis in preeclampsia has been discussed. The influence of Nrf2 deficiency on fetal growth has not been well described. Here, we studied the effect of Nrf2-deletion on fetal and placental development in the knockout mice model. Materials and methods We paired Nrf2 heterozygote mice to conditionally induce gene deletion in the coming embryos. Placentas from Nrf2 −/− and Nrf2 +/+ fetuses were collected on days 13.5, 15.5 and 18.5 post coitum . One fixed half was processed into paraffin wax, exhaustively sectioned and then stained with Periodic Acid Schiff (PAS).The total volume of each placenta was estimated using the Cavalieri estimator of volume. The other half was used for molecular biology analyses. Expression of the pro-inflammatory cytokines IL-6, IL-1, TNF- α , the anti-inflammatory enzyme Heme oxygenase-1 and VEGF were analyzed. Results The Nrf2-deficient pups presented reduced birth weight (0.7 ± 0.06 g) when compared to the wild type pups (0.95 ± 0.03 g). In addition the placental volume of Nrf2 deleted embryos was significantly reduced in comparison to the wild type placentas (Nrf2 +/+ 121 ± 4 vs. Nrf2 −/− 92 ± 5 mm 3 ). Placentas from preterm Nrf2-deleted pups showed increased levels of the pro-inflammatory cytokines (IL-6 and TNF- α ), and decreased expression of HO-1 and VEGF. Conclusion Our results show that a disturbed Nrf2 signaling leads to a significant reduction in the growth of the fetuses. The increased expression of the pro-inflammatory cytokines IL-6 and TNF- α as well as the reduced expression of the anti-inflammatory enzyme HO-1 and VEGF in Nrf2 deleted placentas suggest that intrauterine inflammation may be a cause for the IUGR in this model.

THE CAVALIERI ESTIMATOR WITH UNEQUAL SECTION SPACING REVISITED

The Cavalieri method allows to estimate the volume of a compact object from area measurements in equidistant parallel planar sections. However, the spacing and thickness of sections can be quite irregular in applications. Recent publications have thus focused on the effect of random variability in section spacing, showing that the classical Cavalieri estimator is still unbiased when the stack of parallel planes is stationary, but that the existing variance approximations must be adjusted. The present paper considers the special situation, where the distances between consecutive section planes can be measured and thus where Cavalieri’s estimator can be replaced by a quadrature rule with randomized sampling points. We show that, under mild conditions, the trapezoid rule and Simpson’s rule lead to unbiased volume estimators and give simulation results that indicate that a considerable variance reduction compared to the generalized Cavalieri estimator can be achieved.

Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.

Neuronal injury and neurodegeneration are the hallmark pathologies in a variety of neurological conditions such as epilepsy, stroke, traumatic brain injury, Parkinson’s disease and Alzheimer’s disease. Quantification of absolute neuron and interneuron counts in various brain regions is essential to understand the impact of neurological insults or neurodegenerative disease progression in animal models. However, conventional qualitative scoring-based protocols are superficial and less reliable for use in studies of neuroprotection evaluations. Here, we describe an optimized stereology protocol for quantification of neuronal injury and neurodegeneration by unbiased counting of neurons and interneurons. Every 20th section in each series of 20 sections was processed for NeuN(+) total neuron and parvalbumin(+) interneuron immunostaining. The sections that contain the hippocampus were then delineated into five reliably predefined subregions. Each region was separately analyzed with a microscope driven by the stereology software. Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator. This protocol yielded an estimate of 1.5 million total neurons and 0.05 million PV(+) interneurons within the rat hippocampus. The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images. The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system. This unbiased stereology protocol allows for sensitive, medium-throughput counting of total neurons in any brain region, and thus provides a quantitative tool for studies of neuronal injury and neurodegeneration in a variety of acute brain injury and chronic neurological models.