The effects of neonatal forebrain cholinergic lesion on adult hippocampal neurogenesis.

Previous work in our laboratory indicated that cholinergic denervation by intraventricular infusion of 192-IgG-saporin on postnatal day 7 (N192S) reduced the number of cells in the dentate gyrus expressing doublecortin, a marker for immature neuroblasts. In addition, there was a suggestion that N192S impaired the neurogenic response to environmental enrichment (EE). The purpose of the present study was to further characterize the impact of N192S on the proliferation, differentiation and survival of newborn cells in the dentate gyrus. After 42 days in EE or standard housing, all rats received injections of 5-bromo-2-deoxyuridine (BrdU) to label dividing cells. They were sacrificed either one day (to assess cell proliferation) or 28 days later (to assess survival and differentiation of BrdU-labelled cells). EE failed to increase neurogenesis, thereby preventing determination of the effects of N192S on EE-induced neurogenesis. However, N192S by itself reduced the number of BrdU(+) cells 1 day after BrdU exposure, but did not alter the number of cells expressing the cell cycle marker Ki-67. The number of BrdU(+) cells 28 days after BrdU exposure was not affected by N192S. Confocal analysis of BrdU(+) cells double-immunofluorescently stained to detect NeuN or S100B indicated that N192S did not alter the proportion of new cells that adopted a neuronal or glial identity. The most plausible explanation for these results is that N192S accelerates the death of newborn cells, but does not change their overall survival rate or phenotypic differentiation.

Melatonin promotes neurogenesis in dentate gyrus in the pinealectomized rat.

It was previously shown that pinealectomy causes delayed loss of pyramidal neurons in rat hippocampal layers CA1/3 and that this is reversed by melatonin supplementation. Here, we used immunohistologic detection of doublecortin, a protein expressed in newborn neurons, to determine if melatonin supplementation promotes neurogenesis after pinealectomy. It was found that melatonin supplementation significantly increased the number of doublecortin immunoreactive neurons in the dentate gyrus over the postsurgical intervals of 2, 4, 6, 8, 10 and 17 months. The increase was most evident at 6 months postsurgery and thereafter, and was apparent despite a severe decline in doublecortin-labeled cells over the 17 month postsurgical interval in all groups of rats. Doublecortin immunoreactive cells were not observed in the pyramidal layer itself. These results indicate that melatonin promotes neurogenesis in the dentate gyrus of pinealectomized rats. However, it is equivocal that these newborn neurons migrate to the pyramidal layer and account for the reappearance of neurons at this location in these rats. This study provides further evidence for a role of melatonin in promoting neurogenesis, adding another role to its already remarkably pleiotropic profile. The scope and significance of this newly discovered role remains to be determined.

Developmental forebrain cholinergic lesion and environmental enrichment: behaviour, CA1 cytoarchitecture and neurogenesis.

Intraventricular injections of 192 IgG saporin in 7-day-old rat severely reduced hippocampal cholinergic innervation as reflected by both decreased acetylcholinesterase staining and immunoreactivity for the p75 neurotrophin receptor. It was determined if this altered the effects of environmental enrichment on spatial learning, hippocampal CA1 cell cytoarchitecture as reflected by the Golgi stain, and neurogenesis in the dentate gyrus as indicated by doublecortin immunoreactivity. At weaning, lesioned and control rats were either group housed in large, environmentally enriched cages or housed two per standard cage for 42 days. When subsequently assessed with a working-memory spatial navigation task, both lesioned and control rats showed enhanced learning as a result of enrichment. Quantitative analysis of Golgi stained sections indicated that enrichment did not affect CA1 dendritic branching, total dendritic length or dendritic spine density. However, the lesion reduced the number of apical branches, spine density on intermediate to distal apical dendrites, and the length of basal branches. It also reduced the number of doublecortin immunoreactive neurons in the dentate gyrus and appeared to prevent their increase due to environmental enrichment. It is concluded that developmental cholinergic lesioning does not attenuate neurobehavioral plasticity, at least as reflected by the behavioral consequences of enrichment. It does, however, attenuate neurogenesis in the dentate gyrus, like adult-inflicted cholinergic lesions. As previously found for cortical neurons, it also reduces CA1 pyramidal cell dendritic complexity and spine density in adulthood. The results have implications for the loss of synapses that occurs in both developmental and aging-related brain disorders involving cholinergic dysfunction.

Chronic and acute melatonin effects in gerbil global forebrain ischemia: long-term neural and behavioral outcome.

Melatonin attenuates the short-term consequences of brain ischemia in several animal models. However, there is scant information regarding its efficacy for improving the long-term outcome. To further address that issue, we subjected gerbils to 5-min bilateral carotid occlusion. Some gerbils received acute peri-surgical administration of melatonin while others received continuous melatonin in their water. The gerbils' brains were histologically assessed at 20 wk postsurgery. Chronic but not acute melatonin attenuated ischemia-induced hyperactivity at 3 days postsurgery. Twenty weeks postsurgery, the ischemic gerbils showed varying degrees of bilateral loss of hippocampal CA1 pyramidal cells and elevation of glial fibrillary acidic protein immunoreactivity there. Both the cell loss and the immunoreactivity were markedly asymmetrical for some gerbils. Neither acute nor chronic melatonin altered this pattern of CA1 cell loss and glial immunoreactivity increase. Ischemia increased the number of CA1 cells that were immunoreactive for doublecortin (DCX), a marker for newborn neurons. This increase in CA1 DCX expression was not affected by either melatonin treatment. However, both acute and chronic melatonin reduced the number of DCX immunoreactive neurons in the dentate gyrus. Thus, neither acute nor chronic melatonin altered the long-term neural outcome of forebrain ischemia, although chronic administration seemed to attenuate the short-term behavioral effect. It is suggested that persistently high brain levels of melatonin may be essential for long-term neuroprotection against ischemia. The possibility that melatonin may modulate hippocampal neurogenesis merits further exploration both in normal animals and in models of brain insult.

Comparison of a reference region model with direct measurement of an AIF in the analysis of DCE-MRI data.

Models have been developed for analyzing dynamic contrast-enhanced (DCE)-MRI data that do not require measurements of the arterial input function (AIF). In this study, experimental results obtained from a reference region (RR) analysis are compared with results of an AIF analysis in the same set of five animals (four imaged twice, yielding nine data sets), returning estimates of the volume transfer constant (Ktrans) and the extravascular extracellular volume fraction (ve). Student's t-test values for comparisons of Ktrans and ve between the two models were 0.14 (P=0.88) and 0.85 (P>0.4), respectively (where the high P-values indicate no significant difference between values derived from the two models). Linear regression analysis indicated there was a correlation between Ktrans extracted by the two methods: r2=0.80, P=0.001 (where the low P-value indicates a significant linear correlation). For ve there was no such correlation (r2=0.02). The mean (absolute) percent difference between the models was 22.0% for Ktrans and 28.1% for ve. However, the RR parameter values were much less precise than the AIF method. The mean SDs for Ktrans and ve for the RR analysis were 0.024 min-1 and 0.06, respectively, vs. 0.002 min-1 and 0.03 for AIF analysis.

A comparison of T2*-weighted magnitude and phase imaging for measuring the arterial input function in the rat aorta following intravenous injection of gadolinium contrast agent.

The arterial input function (AIF) is important for quantitative MR imaging perfusion experiments employing Gd contrast agents. This study compared the accuracy of T(2)*-weighted magnitude and phase imaging for noninvasive measurement of the AIF in the rat aorta. Twenty-eight in vivo experiments were performed involving simultaneous arterial blood sampling and MR imaging following Gd injection. In vitro experiments were also performed to confirm the in vivo results. At 1.89 T and TE=3 ms, the relationship between changes in 1/T(2)* in blood (estimated from MR signal magnitude) and Gd concentration ([Gd]) was measured to be approximately 19 s(-1) mM(-1), while that between phase and [Gd] was approximately 0.19 rad mM(-1). Both of these values are consistent with previously published results. The in vivo phase data had approximately half as much scatter with respect to [Gd] than the in vivo magnitude data (r(2)=.34 vs. r(2)=.17, respectively). This is likely due to the fact that the estimated change in 1/T(2)* is more sensitive than the phase to a variety of factors such as partial volume effects and T(1) weighting. Therefore, this study indicates that phase imaging may be a preferred method for measuring the AIF in the rat aorta compared to T(2)*-weighted magnitude imaging.

Chronic mild stress exacerbates the effects of permanent bilateral common carotid artery occlusion on CA1 neurons.

The effect of chronic mild stress (CMStress) was examined in an animal model of chronic cerebral hypoperfusion. Eight-month-old male Sprague-Dawley rats underwent permanent bilateral occlusion of the carotid arteries (2VO) or sham surgery. At 7 days postsurgery, animals from these groups were randomly assigned to undergo CMStress consisting of relatively mild stressor exposure 6 days a week for 6 weeks or a no-stress regimen. They were perfused 24 h thereafter and stereology was used to estimate the total number of hippocampal CA1 and CA3 pyramidal cells. Glial fibrillary acid protein (GFAP) immunoreactivity in the hippocampus was also measured. Degenerating neurons were quantified with the Fluoro-Jade B staining technique. CMStress significantly potentiated CA1 cell loss in 2VO rats (17% loss), compared to a 7% loss of CA1 cells in nonstressed 2VO rats. CMStress had no effect on CA3 cell number. CMStress also caused a significant reduction in GFAP-immunoreactive astrocyte density in CA1, CA3, and the hilus of both sham and 2VO rats. Fluoro-Jade staining was absent, indicating that cell loss probably occurred in the early stage of combined 2VO and CMStress. It was concluded that CMStress exacerbates the consequences of chronic cerebral hypoperfusion on CA1 probably by reducing astrocytes, thereby increasing extracellular glutamate and/or diminishing free radical defense systems. These findings have particular relevance to understanding the contribution of chronic stress to Alzheimer's disease, which, in its premorbid stage, is characterized by cerebral hypoperfusion, and, in its clinical stage, is characterized by CA1 cell loss.

Neonatal 192 IgG-saporin lesion of forebrain cholinergic neurons: focus on the life span?

The cholinergic immunotoxin 192 IgG-saporin can be used to effect selective, substantial and permanent lesions of basal forebrain neurons in the neonatal rat. Human neurodevelopmental disorders such as Rett and Down syndromes are characterized by early cholinergic dysfunction and cognitive impairment. Hence, the study of the neonatal 192 IgG-saporin lesioned rat should illuminate the role of cholinergic dysfunction in these human disorders. To date, we and others have failed to observe notable effects of this neonatal lesion on learning and memory, even when combined with a severe lesion of noradrenergic forebrain innervation. As well, attention seems not to be affected. However, complex problem solving (intelligence?) is compromised by the cholinergic lesion. There also appears to be reduced cortical dendritic branching indicative of synapse loss but further research is needed to characterize this. Even if the synapse loss due to neonatal cholinergic lesion is modest and thus insufficient to cause a significant neurodevelopmental dysfunction, its consequences may be devastating during old age.

Retinal and optic nerve degeneration after chronic carotid ligation: time course and role of light exposure.

BACKGROUND AND PURPOSE: Carotid artery disease can cause chronic retinal ischemia, resulting in transient or permanent blindness, pupillary reflex dysfunction, and retinal degeneration. This experiment investigated the effects of chronic retinal ischemia in an animal model involving permanent carotid occlusion. The time course of retinal pathology and the role of light in this pathology were examined. METHODS: Sprague-Dawley rats underwent permanent bilateral occlusion of the common carotid arteries or sham surgery. Half of the animals were postsurgically housed in darkness, and half were housed in a 12-hour light/dark cycle. Animals were killed at 3, 15, and 90 days after surgery. Stereological techniques were used to count the cells of the retinal ganglion cell layer. Thy-1 immunoreactivity was assessed to specifically quantify loss of retinal ganglion cells. The thicknesses of the remaining retinal sublayers were measured. Optic nerve degeneration was quantified with the Gallyas silver staining technique. RESULTS: Permanent bilateral occlusion of the common carotid arteries resulted in loss of the pupillary reflex to light in 58% of rats. Eyes that lost the reflex showed (1) optic nerve degeneration at 3, 15, and 90 days after surgery; (2) a reduction of retinal ganglion cell layer neurons and Thy-1 immunoreactivity by 15 and 90 days; and (3) a severe loss of photoreceptors by 90 days when postsurgically housed in the light condition only. CONCLUSIONS: Ischemic damage to the optic nerve caused loss of pupillary reflex and death of retinal ganglion cells in a subset of rats. Subsequently, light toxicity induced death of the photoreceptors.