Biomedical subjects

Jens R Nyengaard

Publications and source records attributed to Jens R Nyengaard.

At least 19 recordsLinked to original sources

PubMed2006

Parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion: a divided treatment response?

BACKGROUND: Anti-inflammatory treatment affects ischemic damage and neurogenesis in rodent models of cerebral ischemia. We investigated the potential benefit of COX-2 inhibition with parecoxib in spontaneously hypertensive rats (SHRs) subjected to transient middle cerebral artery occlusion (tMCAo). METHODS: Sixty-four male SHRs were randomized to 90 min of intraluminal tMCAo or sham surgery. Parecoxib (10 mg/kg) or isotonic saline was administered intraperitoneally (IP) during the procedure, and twice daily thereafter. Nineteen animals were euthanized after 24 hours, and each hemisphere was examined for mRNA expression of pro-inflammatory cytokines and COX enzymes by quantitative RT-PCR. Twenty-three tMCAo animals were studied with diffusion and T2 weighted MRI within the first 24 hours, and ten of the SHRs underwent follow-up MRI six days later. Thirty-three SHRs were given 5-bromo-2'-deoxy-uridine (BrdU) twice daily on Day 4 to 7 after tMCAo. Animals were euthanized on Day 8 and the brains were studied with free-floating immunohistochemistry for activated microglia (ED-1), hippocampal granule cell BrdU incorporation, and neuronal nuclei (NeuN). Infarct volume estimation was done using the 2D nucleator and Cavalieri principle on NeuN-stained coronal brain sections. The total number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus was estimated using the optical fractionator. RESULTS: We found a significant reduction in infarct volume in parecoxib treated animals one week after tMCAo (p < 0.03). Cortical ADC values in the parecoxib group were markedly less increased on Day 8 (p < 0.01). Interestingly, the parecoxib treated rats were segregated into two subgroups, suggesting a responder vs. non-responder phenomenon. We found indications of mRNA up-regulation of IL-1beta, IL-6, TNF-alpha and COX-2, whereas COX-1 remained unaffected. Hippocampal granule cell BrdU incorporation was not affected by parecoxib treatment. Presence of ED-1+ activated microglia in the hippocampus was related to an increase in BrdU uptake in the DG. CONCLUSION: IP parecoxib administration during tMCAo was neuroprotective, as evidenced by a large reduction in mean infarct volume and a lower cortical ADC increment. Increased pro-inflammatory cytokine mRNA levels and hippocampal granule cell BrdU incorporation remained unaffected.

Journal Article↗

PubMed2006

MMP-2 in the left rat ventricle is increased by growth hormone.

In cardiac hypertrophy induced by GH, the turn-over of collagen seems to be increased. Matrix metalloproteinases (MMP) are enzymes suggested to contribute to the remodelling of the extracellular matrix in the myocardium. The aim of the present experiment was to investigate how GH influenced MMP concentration, collagen concentration, and structure of the connective tissue of the LV in young rats in relation to time. Three-month-old female rats were injected with GH (5mg/kg/day) or vehicle for 5, 10, 20, 40, or 80 days. MMPs and structural changes of the connective tissue were analysed using zymography and stereology, respectively. Wet weight of the LV was increased time-dependently by GH (r = 0.89, P < 0.001). Furthermore, GH increased the MMP-2 concentration (P < 0.01, two-way ANOVA), whereas no collagenases (MMP-1, MMP-8, or MMP-13) could be demonstrated. The increase in MMP-2 was accompanied by a time-dependent decrease in the collagen concentration (r = -0.46, P < 0.05), whereas the total collagen content (r = 0.85, P < 0.01) and total number of non-myocyte nuclei (GH: r = 0.89, P < 0.001) were time-dependently increased. These results indicate that MMP-2 may be involved in the remodelling process of the extracellular matrix in GH-induced cardiac hypertrophy.

Animals↗

PubMed2006

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Animals↗

PubMed2006

Postnatal food restriction in the rat as a model for a low nephron endowment.

A low nephron endowment may be associated with hypertension. Nephrogenesis is the process that leads to the formation of nephrons until week 36 of gestation in humans and may be inhibited by many factors like intrauterine growth restriction and premature birth. To study the consequences of a low glomerular number, animal models have been developed. We describe a model of postnatal food restriction in the rat in which litter size is increased to 20 pups, which leads to growth restriction. In the rat, active nephrogenesis continues until postnatal day 8, which coincides with the growth restriction in our model. Design-based stereological methods were used to estimate glomerular number and volume. Our results show an approximately 25% lower glomerular number in rats after postnatal food restriction (30,800 glomeruli/kidney) compared with control rats (39,600 glomeruli/kidney, P < 0.001). Mean glomerular volume was increased by 35% in the growth-restricted rats (P = 0.006). There was a significant negative correlation between glomerular volume and glomerular number (r = -0.76, P < 0.001). We conclude that postnatal food restriction in the rat leads to a low nephron endowment with compensatory enlargement. It is therefore a suitable model to study the effect of intrauterine growth restriction or prematurity on kidney development and the consequences of a reduced glomerular number in later life.

Animal Diseases↗

PubMed2006

Comparison of methods for counting cells in the mouse glomerulus.

BACKGROUND: Researchers have long been interested in counting the number of cells within the glomerulus. Investigators using different techniques have yielded conflicting results. The most direct method is to count the cells in serial sections from the entire glomerulus. This Exhaustive Count method is not usually practical. The disector/fractionator method counts the number of cells in a fraction of sections. The total is estimated from the product of the cell count and the reciprocal of the fraction. The Weibel-Gomez method determines the density of cells per glomerulus, then multiplies this density by glomerular volume to obtain cell number. In this study, we compared the disector/fractionator and Weibel-Gomez methods to identify a practical alternative for the time-consuming Exhaustive Count method. METHODS: Glomeruli from a normal mouse kidney were completely sectioned and images obtained. Appropriate images were used to count glomerular cell number using each method. RESULTS: The Exhaustive Count method yielded 213 +/- 22 (mean +/- SD) cells/glomerulus vs. the disector/fractionator average of 211 +/- 29 cells/glomerulus (p = 0.82). The Weibel-Gomez method average of 235 +/- 26 cells/glomerulus was statistically different from the Exhaustive Count method (p = 0.003). CONCLUSION: The Weibel-Gomez produced a 10% overestimation, whereas the disector/fractionator method was unbiased and thus a good substitute for the Exhaustive Count method.

Animals↗

PubMed2005

2,3-Dihydroxybenzoic acid attenuates kanamycin-induced volume reduction in mouse utricular type I hair cells.

The aminoglycoside kanamycin is a commonly used antibiotic, but unfortunately it is oto- and nephrotoxic in large doses. The negative effects are thought to be due to the formation of free radicals which is why strong antioxidants and iron chelators like 2,3-dihydroxybenzoic acid (DHB) are of great interest. This study estimates cellular quantitative changes in the utricular macula of mice following systemic treatment with kanamycin alone or in combination with DHB. The animals were injected with either saline, kanamycin or kanamycin + DHB for 15 days and perfusion fixed three weeks after last injection. Total volume of the utricle, as well as total number of hair and supporting cells, were estimated on light microscopic sections. Total volume and mean volume of hair cell types I and II and supporting cells were estimated on digital transmission electron micrographs. Total volume of the utricular macula, hair cell type I and supporting cells decreased significantly in animals injected with kanamycin but not in animals co-treated with DHB. Hair and supporting cell numbers remained unchanged in all three groups. In conclusion, the kanamycin-induced volume reduction of type I hair cells was attenuated by DHB.

Animals↗

PubMed2005

Stereological study of postnatal development in the mouse utricular macula.

This study describes the morphometric changes taking place in the utricular macula of mice with ages in geometric progression from 1 to 512 days after birth. By using design-based stereological methods, the total volume and surface area of the sensory epithelium as well the total number of the hair cells and supporting cells were estimated. Finally, the numerical density, volume density, and mean volume of the individual cell types were determined. The major changes were found in the number of the individual cell types during the first couple of weeks, and a mature composition of cell types was not attained until 16 days after birth. There was no change in the total number of cells and no decline in the number of hair cells within the time period studied.

Acoustic Maculae↗

PubMed2005

Glomerular number and function are influenced by spontaneous and induced low birth weight in rats.

A link exists between low birth weight and diseases in adulthood, such as hypertension, cardiovascular disease, and insulin resistance. Intrauterine growth restriction (IUGR) has been used to explain this association and has been shown to lead to a nephron endowment in humans. A reduction in glomerular number has been described in animal models with induced low birth weight as well but not in animals with spontaneous low birth weight. It therefore is debatable whether the models are suitable. The effect on glomerular number and size was studied in rats with naturally occurring IUGR and experimental IUGR, induced by bilateral uterine artery ligation. Design-based stereologic methods were used. Urinary protein excretion was determined as a measure of renal damage. Results showed a decrease of approximately 20% in glomerular number in both groups of IUGR (control 35,400, naturally occurring IUGR 30,900, and experimental IUGR 28,000 glomeruli per kidney). Mean glomerular volume was increased in both IUGR groups, which was associated with an increased proteinuria. It is concluded that IUGR leads to a nephron endowment with a compensatory glomerular enlargement. This compensation is associated with more proteinuria in the long run. Uterine artery ligation in the pregnant rat is a suitable model to study the effects of IUGR on the kidney.

Animals↗

PubMed2005

Developmental programming of aortic and renal structure in offspring of rats fed fat-rich diets in pregnancy.

Evidence from human and animal studies suggests that maternal nutrition can induce developmental programming of adult hypertension in offspring. We have previously described a model of maternal dietary imbalance in Sprague-Dawley rats whereby administration of a maternal diet rich in animal lard programmes the development of increased blood pressure, insulin resistance, dyslipidaemia, obesity and mesenteric artery endothelial dysfunction in adult offspring. To further characterize the mechanism of hypertension in this model we have examined vascular and renal structure in adult offspring of Sprague-Dawley rats fed a control diet (OC) or lard-rich diet (OHF) during pregnancy and suckling followed by a control diet post-weaning. To gain further insight, we assessed aortic reactivity and elasticity in an organ bath preparation and renal renin and Na+,K+-ATPase activity. Plasma aldosterone concentration was also measured. Stereological examination of the aorta in OHF demonstrated reduced endothelial cell volume and smooth muscle cell number compared with OC. Adult OHF animals showed increased aortic stiffness and reduced endothelium-dependent relaxation. Renal stereology showed no differences in kidney weight, glomerular number or volume in OHF compared with OC, but renin and Na+,K+-ATPase activity were significantly reduced in OHF compared with controls. Programmed alterations to aortic structure and function are consistent with previous observations that exposure to maternal high fat diets produces systemic vascular changes in the offspring. Despite normal renal stereology, altered renal Na+,K+-ATPase and renin activity offers further insight into the mechanism underlying the increased blood pressure characteristic of this model.

Animal Nutritional Physiological Phenomena↗

PubMed2005

Design-based stereological analysis of the lung parenchymal architecture and alveolar type II cells in surfactant protein A and D double deficient mice.

Alveolar epithelial type II cells synthesize and secrete surfactant. The surfactant-associated proteins A and D (SP-A and SP-D), members of the collectin protein family, participate in pulmonary immune defense, modulation of inflammation, and surfactant metabolism. Both proteins are known to have overlapping as well as distinct functions. The present study provides a design-based stereological analysis of adult mice deficient in both SP-A and SP-D (A(-)D(-)) with special emphasis on parameters characterizing alveolar architecture and surfactant-producing type II cells. Compared to wild-type, A(-)D(-) mice have fewer and larger alveoli, an increase in the number and size of type II cells, as well as more numerous and larger alveolar macrophages. More surfactant-storing lamellar bodies are seen in type II cells, leading to a threefold increase in the total volume of lamellar bodies per lung, but the mean volume of a single lamellar body remains constant. These results demonstrate that chronic deficiency of SP-A and SP-D in mice leads to parenchymal remodeling, type II cell hyperplasia and hypertrophy, and disturbed intracellular surfactant metabolism. The design-based stereological approach presented here provides a framework for the quantitative lung structure analysis in gene-manipulated mice as well as in human lung disease.

Animals↗

PubMed2005

The total length of myocytes and capillaries, and total number of myocyte nuclei in the rat heart are time-dependently increased by growth hormone.

Growth hormone (GH) can increase size and dimensions of rat hearts. The aim was to study how GH administration influences the growth of cardiac myocytes and capillaries in relation to time. Three-month-old female rats were divided into 10 groups (n=3), and injected with either GH (5mg/kg/day) or vehicle for 5, 10, 20, 40, or 80 days. From the left ventricle (LV) histological sections were made and stereological methods applied. Linear regression showed that GH time-dependently increased: LV volume (r=0.96, P<0.001), total volume of myocytes (r=0.96, P<0.001) and capillaries (r=0.64, P<0.05), total length of myocytes (r=0.90, P<0.001) and capillaries (r=0.78, P<0.001), and total number of myocyte nuclei (r=0.85, P<0.001). In conclusion, during 80 days of GH treatment the total volume and length of myocytes and capillaries, and total number of myocyte nuclei increased in a linear way. The results indicate that GH is a potent mediator of myocardial growth.

Animals↗

PubMed2005

Implant placement in bone formed beyond the skeletal envelope by means of guided tissue regeneration: an experimental study in the rat.

OBJECTIVES: The aim of the present study was to evaluate whether the placement of implants in bone formed by means of guided tissue regeneration (GTR) beyond the skeletal envelope may influence bone volume and/or structure. MATERIAL AND METHODS: Rigid, hemispherical, Teflon capsules were placed with their open part facing the lateral surface of the ramus in both sides of the mandible in 18 rats. After 1 year, the capsules were removed by a re-entry operation, and a custom-made titanium implant was placed in the augmented ramus in only one side of the jaw. Six animals were sacrificed shortly after implant surgery, another six after 3 months, and the last six after 6 months. Histological specimens of the augmented sites including the implants were prepared, and the volumes of (1) the newly formed bone (mineralized bone and marrow) (2) the soft connective tissue, and (3) the implant, in the space originally created by the capsule were estimated by a point-counting technique. Additionally the height of the augmented bone was measured. RESULTS: One year after capsule placement, the major portion of the space originally created by the capsules was filled with newly formed bone. In the test specimens, implant placement seemed to result in a denser arrangement of the augmented bone, but this event did not influence its long-term stability. Although some resorption occurred after 3 and 6 months, the vast portion of the generated bone remained stable over time in both tests and controls, and there were no differences between tests and controls at any observation periods. CONCLUSION: It is concluded that large amounts of bone can be formed beyond the skeletal envelope by means of GTR, and that this bone remains stable on a long-term basis both with and without the placement of titanium implants.

Animals↗

PubMed2004

p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies.

Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25alpha as a candidate that preferentially binds to alpha-synuclein in its aggregated state. Functionally, purified recombinant human p25alpha strongly stimulates the aggregation of alpha-synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25alpha is normally only expressed in oligodendrocytes in contrast to alpha-synuclein, which is normally only expressed in neurons. This expression pattern is changed in alpha-synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25alpha and dystopically expressed alpha-synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25alpha was detectable in the neuronal Lewy body inclusions along with alpha-synuclein. The localization in alpha-synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25alpha plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by alpha-synuclein aggregates.

Amino Acid Sequence↗

PubMed2004

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice.

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.

Animals↗

PubMed2004

Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells.

An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.

Animals↗

PubMed2004

Changes in load-bearing area after Ganz periacetabular osteotomy evaluated by multislice CT scanning and stereology.

BACKGROUND: We used a stereologic method based on 3D CT scanning to estimate the projected load-bearing surface in the hip joint. PATIENTS AND METHODS: 6 normal hip joints and 6 dysplastic hips were examined. The latter were CT-scanned before and after periacetabular osteotomy. RESULTS: We found that the average area of the projected load-bearing surface of the femoral head preoperatively was 7.4 (6.5-8.4) cm2 and postoperatively 11 (9.8-14) cm2, which was similar to the load-bearing surface in the normal control group. We performed double measurements and the coefficient of error of the mean was estimated at 1.6%. Due to overprojection, an overestimation of about 3.8% on the projected load-bearing surface occurred. Consequently, the stereologic method proved to be precise and unbiased. INTERPRETATION: Our findings indicate that this method is of value for monitoring the load-bearing area in the hip joint of patients undergoing periacetabular osteotomy.

Acetabulum↗

PubMed2004

Interactions between hyperglycemia and hypoxia: implications for diabetic retinopathy.

The primary aim of these experiments was to assess in vitro effects of hyperglycemia (30 mmol/l glucose) and hypoxia (Po(2) = 36 torr) of 2-h duration, separately and in combination, on cytosolic and mitochondrial free NADH (NADHc and NADHm, respectively) in retinas from normal rats. NADH is the major carrier of electrons and protons that fuel ATP synthesis and several metabolic pathways linked to diabetic complications. Hyperglycemia and hypoxia increase free NADHc by different mechanisms that are additive. Hyperglycemia increases transfer of electrons and protons from sorbitol to NAD(+)c, reducing it to NADHc, but does not increase NADHm. Hypoxia increases NADHm by inhibiting its oxidation. Electrons and protons accumulating in NADHm restrain transfer of electrons and protons from NADHc to NAD(+)m via the malate-aspartate electron shuttle. Hyperglycemia and hypoxia also increase glycolysis by different mechanisms that are additive, and hyperglycemia increases ATP levels in hypoxic and in aerobic retinas. The additive effects of hyperglycemia and hypoxia on accumulation of electrons and protons in a common pool of free NADHc confirm the test hypothesis and the potential of a combination of these two risk factors to accelerate the onset and progression of diabetic retinopathy (and other complications of diabetes) by augmenting metabolic pathways fueled by free NADHc.

Animals↗

PubMed2003

The number of alveoli in the human lung.

The number of alveoli is a key structural determinant of lung architecture. A design-based stereologic approach was used for the direct and unbiased estimation of alveolar number in the human lung. The principle is based on two-dimensional topology in three-dimensional space and is free of assumptions on the shape, size, or spatial orientation of alveoli. Alveolar number is estimated by counting their openings at the level of the free septal edges, where they form a two-dimensional network. Mathematically, the Euler number of this network is estimated using physical disectors at a light microscopic level. In six adult human lungs, the mean alveolar number was 480 million (range: 274-790 million; coefficient of variation: 37%). Alveolar number was closely related to total lung volume, with larger lungs having considerably more alveoli. The mean size of a single alveolus was rather constant with 4.2 x 10(6) microm3 (range: 3.3-4.8 x 10(6) microm3; coefficient of variation: 10%), irrespective of the lung size. One cubic millimeter lung parenchyma would then contain around 170 alveoli. The method proved to be very efficient and easy to apply in practice. Future applications will show this approach to be an important addition to design-based stereologic methods for the quantitative analysis of lung structure.

Culture Techniques↗