NADPH-diaphorase activity in the superficial layers of the superior colliculus of rats during aging

Neurons in the superficial layers of the superior colliculus are key elements in the visual system of rodents since they receive extensive afferent projections from retinal ganglion cells. The NADPH-diaphorase histochemical technique was used to detect differences in neuronal nitric oxide synthase (nNOS) in the superficial layers of the superior colliculus (sSC) of young adult (3 months) and aged (24 and 26 months) rats. The orientation of the dendritic processes of NADPH-diaphorase-positive neurons, cross-sectional area, and number of neurons per mm2 were analyzed. NADPH-d histochemistry revealed a high number of NADPH-d-positive cells in the stratum zonale and stratum griseum superficiale in adult and aged animals. NADPH-d-positive neurons were classified into the following morphological types: marginal, horizontal, pyriform, narrow-field vertical, wide-field vertical, and stellate. During aging, narrow field vertical and wide field vertical neurons present somatic atrophy and an increase in dendritic processes with dorsoventral orientation, whereas wide field vertical neurons show a decrease in those with lateromedial orientation. Marginal neurons undergo somatic hypertrophy at 26 months when compared with those at 3 months. The remaining types of neurons do not undergo size changes. Finally, the number of NADPH-d-positive neurons per mm2 in the various types of morphology does not significantly change with age. It is suggested to be likely that the aging process in the nitrergic neurons of the sSC does not lead to significant changes in the synthesis of NO from the constitutive NOS isoforms.     

The saucor, a new stereological tool for analysing the spatial distributions of cells, exemplified by human neocortical neurons and glial cells

The 3D spatial arrangement of particles or cells, for example glial cells, with respect to other particles or cells, for example neurons, can be characterized by the radial number density function, which expresses the number density of so-called 'secondary' particles as a function of their distance to a 'primary' particle. The present paper introduces a new stereological method, the saucor, for estimating the radial number density using thick isotropic uniform random or vertical uniform random sections. In the first estimation step, primary particles are registered in a disector. Subsequently, smaller counting windows are drawn with random orientation around every primary particle, and the positions of all secondary particles within the windows are recorded. The shape of the counting windows is designed such that a large portion of the volume close to the primary particle is examined and a smaller portion of the volume as the distance to the primary object increases. The experimenter can determine the relation between these volumina as a function of the distance by adjusting the parameters of the window graph, and thus reach a good balance between workload and obtained information. Estimation formulae based on the Horvitz-Thompson theorem are derived for both isotropic uniform random and vertical uniform random designs. The method is illustrated with an example where the radial number density of neurons and glial cells around neurons in the human neocortex is estimated using thick vertical sections for light microscopy. The results indicate that the glial cells are clustered around the neurons and the neurons have a tendency towards repulsion from each other.     

Effects of age and GDNF on noradrenergic innervation of the hippocampal formation: studies from intraocular grafts

Recent studies have suggested that factors in the target tissue influence the degree of plasticity and regeneration following aging and/or specific insults. We have investigated whether young or aged targets differ in their noradrenergic innervation from fetal locus coeruleus (LC) neurons, and also if a specific growth factor, glial cell line-derived neurotrophic factor (GDNF) can affect this innervation pattern. Tissue pieces of fetal brainstem and young (3 months) or old (18 months) iris tissue were transplanted simultaneously into the anterior chamber of the eye of adult hosts. We found that aged iris transplants became innervated to a significantly lesser degree by the cografted LC neurons than young iris transplants. Fetal hippocampal tissue was then grafted to adult hosts, and a fetal brainstem graft containing LC neurons was placed adjacent to the first graft, either at 3 or 21 months post-grafting. Thus, old/young chimeras of the noradrenergic coeruleo-hippocampal pathway were created. Aged hippocampal grafts received a much less dense innervation from co-grafted LC neurons than young hippocampal grafts. Tyrosine hydroxylase-positive-immunoreactive innervation was only found in the outskirts of aged grafts, while the young hippocampal grafts contained an even innervation pattern. The innervation density of hippocampal grafts was significantly enhanced by GDNF treatment. These findings demonstrate that target-derived factors may regulate neuronal plasticity, and that the age of the target is more important for innervation properties than the age of the neuron innervating a particular target.     

Histomorphological and quantitative immunohistochemical changes in the rat pancreas during aging

Although the endocrine pancreas is the purpose of several deep investigations, morphological data referred to the effect of aging on the gland are not homogeneous. The purpose of the current work was to analyze the changes occurring in the pancreas of aged rats, with especial reference to the islet cell populations. Six young (Y), old (O) and senescent (S) male Sprague-Dawley rats were used. The pancreas tails were processed for light microscopy and studied by means of routine stains as well as by immunohistochemical identification of insulin-, glucagon-, somatostatin-, and pancreatic polypeptide- secreting cells (Dako Envision System, DAB as chromogen). A progressive pancreatic histoarchitecture distortion was found among the aged animals. Even when the alterations were not uniformly observed, they appeared more evident and severe in the S group. The S rats showed significantly increased volume density and cell density of the B cell population, as well as larger number of islet profiles, when compared to O rats. A significant progressive increment of adipose tissue was also evident in aged animals. No abnormal changes were detected in the nonB cell populations of the different groups.
The quantitative changes found in aged animals suggest a possible compensatory reaction of the B cell population in an attempt to curb the influence of diabetogenic factors mounting with advanced age.     

Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat

To ascertain the possible implications of the nitric oxide (NO*) producing system in striatal senescence, and by using immunohistochemistry and image-processing approaches, we describe the presence of the enzyme nitric oxide synthase (NOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and nitrotyrosine-derived complexes (N-Tyr) in the striatum of adult and aged rats. The results showed neuronal NOS immunoreactive (nNOS-IR) aspiny medium-sized neurons and nervous fibres in both age groups, with no variation in the percentage of immunoreactive area but a significant decrease in the intensity and in the number of somata with age, which were not related to the observed increase with age of the striatal bundles of the white matter. In addition, NADPH-d activity was detected in neurons with morphology similar to that of the nNOS-IR cells; a decrease in the percentage of area per field and in the number of cells, but an increase in the intensity of staining for the NADPH-d histochemical marker, were detected with age. The number of neuronal NADPH-d somata was higher than for the nNOS-IR ones in both age groups. Moreover, N-Tyr-IR complexes were observed in cells (neurons and glia) and fibres, with a significant increase in the percentage of the area of immunoreaction, related to the increase of white matter, but a decrease in intensity for the aged group. On the other hand, we did not detect the inducible isoform (iNOS) either in adult or in aged rats. Taken together, these results support the contention that NADPH-d staining is not such an unambiguous marker for nNOS, and that increased protein nitration may participate in striatal aging.     

Morphometric analysis of a fibre system in the central nervous system

The authors have studied the size and composition of the rat optic nerve at three points along its course. The relative volume of myelinated nerve fibres increases that of the chiasma, whereas the relative volume of interstitium decreases. The decrease of interstitial volume is mainly related to the decrease of glial cell number. Determination of absolute volumes of myelinated fibres and interstitium shows that the volume changes are due to two factors: (a) From the eyebulb to the intermediate portion the interstitial volume decreases, whereas the absolute volume of myelinated fibres remains constant. (b) From the intermediate portion to the chiasma the volume of myelinated fibres increases significantly, whereas the further decrease of interstitial volume is small. The diameter of nerve cross-section is significantly smaller in the intermediate portion of the optic nerve than near the eyebulb and the chiasma. Here the nerve passes the canalis opticus. The density of glial cells decreases from eyebulb to the chiasma as does the density of pericytes and endothelial cells. The latter makes up only 2·3–3·9% of the density of cells in the interstitial space. The mean volume of a glial cell remains constant at about 1260 μm³ along the path of the optic nerve from eyebulb to chiasma. The number of myelinated nerve fibres is also constant along the path of the optic nerve. There are 107,000 ± 6900 fibres.     

Expression of tyrosine hydroxylase and vasopressin in magnocellular neurons of salt-loaded aged rats.

Tyrosine hydroxylase (TH) is expressed in catecholaminergic neurons. However, under certain conditions it is also ectopically expressed in magnocellular neurons of the hypothalamus. To test the hypothesis that this expression of TH is related to the cellular activation of these neurons and/or to the vasopressin (VP) expression, we studied the expression of both TH and VP in control and salt-loaded aged rats. Our results demonstrate that aged rats show a marked TH expression in VP cells which is further increased by osmotic stimulation in the absence of increase in VP synthesis in the supraoptic nucleus. The presence of TH-immunopositive dendritic swellings in the ventral part of this nucleus reveals the high state of plasticity of these neurons. Furthermore, the lack of several actors of catecholamine biosynthesis in these neurons suggests a different role for TH. This study further demonstrates an ectopic expression of TH in hypothalamic neurons of aged rats and a TH expression linked to the activation of VP neurons but unrelated to VP synthesis.     

Goblet cell number in the ileum of rats denervated during suckling and weaning.

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.     

GDNF improves cerebellar Purkinje neuron function in aged F344 rats

Aging is associated with a decline in the function of beta-adrenergic receptor responses in the cerebellum. This decline in noradrenergic receptor sensitivity may underlie some of the accompanying age-related declines in motoric learning behaviors. Glial cell line-derived neurotrophic factor (GDNF) has been reported to prevent the degeneration of noradrenergic neurons following neurotoxic lesions. Thus, it was of interest to examine if GDNF would have a beneficial effect on age-related declines in noradrenergic function. Eighteen-month-old F344 rats were injected with 500 microg GDNF in 20 microl into the cisterna magna. Three weeks following GDNF or vehicle treatment, rats were tested on a motor coordination task and then examined electrophysiologically under urethane anesthesia. GDNF did not produce an improvement in performance on an inclined balance beam or an accelerating rotorod. In young (3-month-old) F344 rats isoproterenol (ISO) will increase GABAergic inhibitions in the majority of cells examined; however, in aged rats only about 30% of neurons demonstrate this phenotype. In the aged rats treated with GDNF, ISO was able to increase GABAergic inhibitions in greater than 75% of the neurons tested, thus returning the neurons to a young phenotype. We examined the brains for expression of bcl-2, which has been shown to be increased in the aged cerebellum. GDNF was able to down-regulate this neuronal signal. Thus, intra-cisterna magna delivery of GDNF to aged rats improved beta-adrenergic receptor function and reduced stress related signaling of bcl-2 in the aged F344 rats to a level similar to that observed in young rats.     

Effects of food restriction on motoneuronal loss with advancing age in the rat

The effects of life-long food restriction on motoneuronal cell death with advancing age was studied in male Fischer rats, which had access to food only 3 days a week after weaning (FR rats). Motoneurons innervating the medial gastrocnemius muscle were labeled with retrogradely transported HRP. The number of labeled motoneurons in FR rats and rats fed ad libitum (AL rats) was similar at the age of 16 months (131.8 +/- 1.7 for FR rats vs. 133.8 +/- 4.5 for AL rats). However, at 28 months of age, AL rats had less labeled motoneurons compared to FR rats (117.0 +/- 2.4 for FR rats vs. 124.3 +/- 7.0 for FR rats). The number of type I muscle fibers in the medial gastrocnemius muscle increased significantly in AL rats during the period from 16 to 28 months of age, which might reflect motor unit reorganization following retraction of axons and/or death of innervating motoneurons. FR rats did not show statistically significant alteration. These findings were also true for the data compiled from several different experiments including those conducted for primarily different purposes in our laboratory. The results suggest that life-long food restriction retards motoneuronal cell death occurring with advancing age.     

A single pretreatment by zoledronic acid converts metastases from osteolytic to osteoblastic in the rat

Bone metastases are severe complications of cancers associated with increased morbidity, pain, risk fracture, and reduced life span for patients. Bisphosphonates emerged as a relief treatment in bone metastases. A single dose of zoledronic acid (78 μg/kg) was injected into six Copenhagen rats 4 days before receiving an intraosseous inoculation of metastatic anaplastic tumor of lymph node and lung cell (MLL) prostate cancer cells. Rat femurs were analyzed for changes by microCT and histomorphometry; trabecular volume, trabecular characteristics, osteoid parameters, osteoblastic surfaces, and osteoclast number were measured. Values were compared to a group of SHAM animals, a group of SHAM animals having received zoledronic acid and animals inoculated with MLL cells. All rats were euthanized after 1 month. MLL cells induced osteolysis in the metaphysis with extension of the tumor to soft tissues through cortical perforations. Zoledronic acid induced a marked osteosclerosis in the primary spongiosa in both SHAM and rats inoculated with MLL. Osteosclerosis was obtained in the secondary spongiosa of MLL rats. The bisphosphonate preserved cortical integrity in all animals, and no extension to soft tissues was observed in most animals. The number of osteoclasts was elevated, indicating that there was no apoptosis of osteoclasts but they became inactive. Osteosclerosis was associated with increased osteoblastic surfaces. A single zoledronic acid injection turned osteolytic metastases into osteosclerotic and preserved cortical integrity. Microsc. Res. Tech. 73:733–740, 2010. © 2009 Wiley‐Liss, Inc.     

Evidence for target-specific outgrowth from subpopulations of grafted human dopamine neurons

Clinical and experimental grafting in Parkinson's disease has shown the need for enhanced survival of dopamine neurons to obtain improved functional recovery. In addition, it has been suggested that a limited number of surviving dopamine neurons project to the dopamine-denervated host striatum. The aim of this study was to investigate if subpopulations of ventral mesencephalic dopamine neurons project to their normal targets, i.e., dorsal vs. ventral striatum. Following implantation of human ventral mesencepahlic tissue into the lateral ventricle of dopamine-depleted rats, human-derived dopamine reinnervation was achieved both in dorsal and ventral striatum. Treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a degeneration of tyrosine hydroxylase (TH)-immunoreactive nerve fibers in dorsal striatum but not in ventral areas in some animals, while MPTP was without effect in other animals. TH-immunoreactive neurons were small and appeared shrunken in animals carrying grafts affected by the MPTP treatment. In conclusion, grafted dopamine neurons projected nerve fibers into areas that they normally innervate. Thus, when searching for factors that may enhance survival of grafted dopamine neurons it is important to study which subpopulation(s) of ventral mesencephalic dopamine neurons is affected, such that a proper reinnervation may be achieved.     

Effect of age on the myosin-V immunoreactive myenteric neurons of rats ileum.

Alterations in the gastrointestinal neuromuscular function related to age have been demonstrated in human and animal models. This study analyzes the effects of the aging process on the area of the neuronal cell bodies of the myenteric plexus in the antimesenteric and intermediate regions of the ileal circumference of Wistar, 12 month-old in comparison 3 month-old animals. The ileum was removed and whole-mount preparations immunostained by the antibody anti-myosin-V were processed. The morphometric analyses were performed using a computerized image analysis system, with a subsequent distribution of neurons by size in intervals of 100 micro2. The cellular body morphometry revealed a significant increase in the size of the myosin-V- immunoreactive myenteric neurons from 12 month-old animals when compared with 3 month-old animals. However, significant differences between the regions were not observed; these observations were not age-dependent. The implications of these results in relation to the increase of the body weight, size of the small intestine, general organization of the myenteric plexus, staining method of neurons and the possible factors involved in the regulation and/or control of the volume of neronal cells due to aging, are discussed.     

Age-dependent changes of the periodontal ligament in rats

Even after the end of the natural tooth eruption, there is a continuous renewal of the periodontal collagenous fiber system, depending on functional demands. The aim of this study was to analyse the age-dependent changes and regional differences of the collagen renewal rate of the periodontal ligament in healthy rats. The study was performed by autoradiography of the molars of rats aged 1, 8, and 18 months, where collagen was labelled by intravenously applied 3H-proline. After an 8-hour incorporation period, the animals were killed. For comparative examinations, molar roots were subdivided into cervical, middle, and apical thirds. Structural and quantitative analyses were performed by light microscopy and autoradiography, using an image-analysing computer-assisted operating unit that determined the 3H-proline-labelled collagen by photometry based on extinction measurement. With increasing age of the animals, the number of silver grains (3H-proline-blackened collagen) was reduced and the quantitative evaluation indicated a reduction of 3H-proline in the periodontal ligament. The lowest level of 3H-proline activities was observed in the middle, and the highest level in the apical root third, independent of age. All preparations revealed condensations of silver grains, which were located in the region of the periodontal ligament adjacent to the alveolar bone, but did not reveal any preferred position with regard to the dental topography. With progressive age, the uptake of 3H-proline in the periodontal ligament was reduced by about 20 to 30%, a result that corresponds to a decrease in collagenous fiber production. Collagen was mainly formed in the apical and cervical root third, starting from the alveolar bone side, presumably in response to functional strain.     

Comparison of transsynaptic viral labeling of central nervous system structures from the uterine horn in virgin, pregnant, and lactating rats

Using the transneuronal viral tracing method, the central nervous system (CNS) connections of the uterine horn were studied in virgin, pregnant, and in lactating rats. The frequency of viral labeling in the brain and the distribution of virus-infected neurons from the uterine horn were compared among groups. There was a marked difference in the frequency of viral labeling in the brain stem. In virgin rats more than half of the brain stems (5 out of 9) were labeled. In contrast, in pregnant animals viral-labeled neurons were detected in only a few cases (3 out of 16) and almost each brain stem of the lactating group was labeled (12 out of 13). A similar, less marked difference was observed in the hypothalamus. The pattern of distribution of infected neurons was similar in each group. In the brain stem, the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, gigantocellular and paragigantocellular nucleus, ventrolateral medulla, A5 cell group, and caudal raphe nuclei were the most frequently labeled structures. In the diencephalon, viral-infected neurons were detected primarily in the hypothalamic paraventricular nucleus. The telencephalon was devoid of infected cells. Data suggest that the CNS control of the uterine horn varies depending on reproductive status. The low frequency of brain labeling in pregnant rats may be related to the almost complete lack of sympathetic fibers in the uterus prior to parturition and the very high frequency of labeling in lactating animals to the postpartum hyperinnervation of the uterus.     

Procedure of RNA extraction which does not extract DNA and proteins from nervous tissue cells.

Several schemes of acidic extraction of RNA taken from biochemical or cytochemical literature were compared by means of ultraviolet and visible cytospectrophotometry. Basing on the data obtained an extraction scheme has been recommended which consists in the treatment of nervous tissue sections with 16 per cent perchloric acid at 4 degrees C for 48 h. This extraction has been shown to extract completely cellular RNA without extracting DNA and proteins from the nervous tissue cells. Using this scheme, ultraviolet cytospectrophotometry of neurons and glial cells was performed twice, before and after the selective RNA extraction, which enabled a calculation of RNA concentration by the difference of the absorption values. In mice, after 3 h-swimming, and in cats, after 2-week daily injections of adrenaline, a number of quantitative and qualitative RNA changes were found out in nerve and glial cell of mouse spinal cord and cat sympathetic ganglion with the aid of the recommended scheme of RNA extraction.     

Distribution of nestin protein: immunohistochemical study in enteric plexus of rat duodenum

The intermediate nestin filament is expressed in neural stem cells, neuroectodermal tumors and various adult tissues under situations that reproduce developmental phases, e.g., physiological renewal of certain cell types, tissue regeneration, and healing or revascularization. In the human gastrointestinal tract, nestin has been reported in glial cells and interstitial cells of Cajal. We examined by immunohistochemistry the appearance and distribution of nestin protein in enteric ganglia of rat duodenum. Through the myenteric and submucosal plexuses, a high number of nestin-positive cells were visualized in this specie. The nestin-positive cells were smaller and more numerous than enteric neurons. They were present both within and around ganglia. The results of this study suggest that the rat enteric glial cells (EGCs) are rich in nestin, a protein usually associated with dividing or migrating cells and the dynamic reorganization of nestin filaments during the cell cycle. EGCs could function as enteric stem cells.