John Hengemihle

Hippocampal neuron and synaptophysin-positive bouton number in aging C57BL/6 mice.

A loss of hippocampal neurons and synapses had been considered a hallmark of normal aging and, furthermore, to be a substrate of age-related learning and memory deficits. Recent stereological studies in humans have shown that only a relatively minor neuron loss occurs with aging and that this loss is restricted to specific brain regions, including hippocampal subregions. Here, we investigate these age-related changes in C57BL/6J mice, one of the most commonly used laboratory mouse strains. Twenty-five mice (groups at 2, 14, and 28-31 months of age) were assessed for Morris water-maze performance, and modern stereological techniques were used to estimate total neuron and synaptophysin-positive bouton number in hippocampal subregions at the light microscopic level. Results revealed that performance in the water maze was largely maintained with aging. No age-related decline was observed in number of dentate gyrus granule cells or CA1 pyramidal cells. In addition, no age-related change in number of synaptophysin-positive boutons was observed in the molecular layer of the dentate gyrus or CA1 region of hippocampus. We observed a significant correlation between dentate gyrus synaptophysin-positive bouton number and water-maze performance. These results demonstrate that C57BL/6J mice do not exhibit major age-related deficits in spatial learning or hippocampal structure, providing a baseline for further study of mouse brain aging.

Stereological analysis of astrocyte and microglia in aging mouse hippocampus

Recent evidence suggests neuroglia-mediated inflammatory mechanisms may stimulate neurodegenerative processes in mammalian brain during aging. To test the hypothesis that the number of microglia and astrocytes increase in the hippocampus during normal aging, unbiased stereological techniques were used to estimate total cell number in hippocampal subregions (CA1, dentate gyrus and hilus) of male C57BL/6J mice of different ages: 4-5 months, 13-14 months and 27-28 months. Immunocytochemical visualization for microglia and astrocytes were via Mac-1 and GFAP antibody, respectively. Estimates of total microglia and astrocyte number were assessed using the optical fractionator. No statistically significant age differences were found in the numbers of microglia or astrocytes in the hippocampal regions sampled. These findings suggest that age-related increases in the total numbers of hippocampal microglia and astrocytes is not causal for observed age-related increases in cytokine response.

Behavioral assessment of aging in male Fischer 344 and brown Norway rat strains and their F1 hybrid.

Male Fischer-344 (F344) and Brown Norway (BN) rats 7-, 13-, and 24-month-old and their F344 x BN hybrid (F1) 7-, 13-, 24- and 31-month-old were tested in a behavioral battery (15-min and 24-h locomotor activity, inclined screen, rod suspension, rotorod, shock-motivated learning in a straight runway and 14-unit T maze). Necropsy was performed 3 days later and the results rated for pathology (i.e., severity of lesions observed). Age-related performance declines were observed in all behavioral tests except 15-min locomotor activity. Strain effects were observed in 15-min (BN more active than F344 and F1) and 24-h locomotor activity test (F344 more active than BN and F1 strains); rotorod performance (F344 fell more than BN and F1); and in all measures [errors (E), runtime (RT)], shock frequency (SF), and duration (SD)] in the 14-unit T maze (F344 worse than BN, BN worse than F1). T maze performance of 31-month-old F1 rats was deficient in RT, SD, and SF but E performance was equivalent to that of 7-month-old F1 rats. In a second experiment, only 7- and 31-month-old F1 rats were tested in the 14-unit T maze and the results obtained in Experiment 1 were replicated. Gross necropsy revealed age and strain effects in the number of lesions observed and the mean ratings of pathology. The 24-month-old F344 rats exhibited the greatest number of lesions and had the highest ratings (generally observed as chronic nephrosis and enlarged spleens characteristic of mononuclear cell leukemia). BN rats exhibited a high incidence of hydronephrosis at all age levels. While experiencing less obvious pathology, F1 rats experienced a significant number of lesions in the 31-month-old group. Pathology ratings correlated with behavioral performance but only for a few tests (e.g., SD and RT in 14 unit T maze in 24-month-old F344). Thus, behavioral performance declined with age and the battery of tests differentiated between the strains tested (in general, F344 worse than BN; BN worse than F1). The correlation of pathology ratings at gross necropsy with behavior did not appear to be systematic, suggesting that morbidity was not responsible for the age-related performance declines. However, more extensive evaluation of the relationship of age-related changes in health status to behavior with larger samples of rats is suggested.

EFFECTS OF ESTROGEN AND RALOXIFENE ON NEUROGLIA NUMBER AND MORPHOLOGY IN THE HIPPOCAMPUS OF AGED FEMALE MICE

Hormone replacement therapy with the gonadal steroid estrogen or synthetic agents such as raloxifene, a selective estrogen receptor modulator, may affect cellular function in brains of postmenopausal women. In vitro studies suggest that 17beta estradiol and raloxifene can alter the microglial and astrocyte expression of immuno-neuronal modulators, such as cytokines, complement factors, chemokines, and other molecules involved in neuroinflammation and neurodegeneration. To directly test whether exogenous 17beta estradiol and raloxifene affect the number of glial cells in brain, C57BL/6NIA female mice aged 20-24 months received bilateral ovariectomy followed by s.c. placement of a 60-day release pellet containing 17beta estradiol (1.7 mg), raloxifene (10 mg), or placebo (cholesterol). After 60 days, numbers of microglia and astrocytes were quantified in dentate gyrus and CA1 regions of the hippocampal formation using immunocytochemistry and design-based stereology. The results show that long-term 17beta estradiol treatment in aged female mice significantly lowered the numbers of astrocytes and microglial cells in dentate gyrus and CA1 regions compared with placebo. After long-term treatment with raloxifene, a similar reduction was observed in numbers of astrocytes and microglial cells in the hippocampal formation. These findings indicate that estrogen and selective estrogen receptor modulators can influence glial-mediated inflammatory pathways and possibly protect against age- and disease-related neuropathology.

Stereological estimation of total microglia number in mouse hippocampus

Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimers disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.

Chronic nimodipine treatment in aged rats: Analysis of motor and cognitive effects and muscarinic-induced striatal dopamine release

Nimodipine is a calcium channel blocker reported to have beneficial effects on treatment of ischemic damage as well as the potential for retarding aspects of brain and behavioral aging when provided chronically to rats. We treated aged male F-344 rats (24 months) with nimodipine in SC pellets in the following doses: 0 (controls), 20 mg (low-dose), or 40 mg (high-dose) replenished after 6 weeks. After 3 months of treatment, surviving rats and a group of young controls (6 months) were tested in a behavioral battery involving exploratory activity in an open field and in a runwheel cage as well as motor abilities required for remaining on an inclined screen, suspended from a wire, and balanced on a rotorod. Rats were also pretrained for one-way active avoidance in a straight runway before being trained in a 14-unit T maze. During 20 trials rats were required to negotiate each of 5 maze segments within 10 s to avoid foot shock (0.8 mA). Nimodipine treatment produced no significant effects on body weight, food intake, or survival of aged rats. Analysis of behavioral results indicated significant age-related decline in performance of all tasks except in open-field behavior. Nimodipine treatment had no significant effects on behavioral performance of aged rats except in maze learning. Rats on the high-dose regimen performed significantly better than aged controls in the maze. The results indicate that chronic nimodipine treatment of aged rats had no toxic effects and might be beneficial for preventing age-related decline in learning performance.

Chronic treatment with human recombinant erythropoietin increases hematocrit and improves water maze performance in mice

Erythropoietin is a glycoprotein produced endogenously in the kidney, which stimulates red blood cell production. We evaluated the effects of chronic treatment with recombinant human erythropoietin (epoetin alfa: EPO) on the performance of 6-month-old male C57BL/6J mice in a spatial learning task, the Morris water maze. Mice were treated with either EPO (1.5 U injected SC every other day) or vehicle (PBS also injected SC every other day). Results indicated that the treatment had no effect on maze performance after 8 weeks, but after 19 weeks the EPO-treated mice showed better performance compared to controls as measured by mean distance (centimeters) to reach the goal platform. The improved performance in EPO-treated mice at 19 weeks was accompanied by an increased hematocrit. After 32 wk of EPO-treatment, the hematocrit returned to baseline levels even though the size and density of the red blood cells were increased.

Cognitive enhancement. New strategies for stimulating cholinergic, glutamatergic, and nitric oxide systems.

The development of treatments for AD is being pursued along many diverse lines. While the ACh hypothesis has generated abundant development efforts, little clinical progress has been achieved to date. Recent efforts aimed at developing more potent, more specific, and safer ChE inhibitors appear to offer greater potential for therapeutic success than achieved to date. Treatments aimed at the NMDA Glu system lag much further behind in their development. Progress in this area must be tempered by the potential for glutamate excitotoxicity mediated through this neurotransmitter system. Development of indirect agonists operating at the glycine and polyamine modulatory sites on the NMDA receptor might offer the safest alternative to applying more direct agonists. While a great degree of interest had been generated by the reports of NO involvement in signal transduction through the NMDA system, this area of research has been complicated by conflicting reports regarding NO involvement in learning and LTP. Moreover, the interaction of drugs acting on NOS with the vascular effects mediated by eNOS has also complicated development of drugs that act specifically on the neural actions of NO. This area will continue to receive extensive research attention; but similar to the development of Glu agonists, attention must be given to the potential neurotoxic effects of overstimulating this system. Perhaps targeting other presynaptic mechanisms that effect glutamate release might be a safer strategy to pursue. Considerable progress has been made over the last two decades in identifying the genetic and neural mechanisms involved in AD. Progress in developing treatments will remain highly correlated with this effort, and with basic research geared to comprehending how memories are formed and why neurons degenerate and regenerate.

Chronic treatment of aged mice with L-deprenyl produces marked striatal MAO-B inhibition but no beneficial effects on survival, motor performance, or nigral lipofuscin accumulation

Male C57BL/6J mice were provided I-deprenyl (at 0, 0.5 mg/kg or 1.0 mg/kg per day) in their drinking water beginning at 18 months of age. A battery of motor tests, including open-field, tightrope, rotorod, inclined screen, runwheel, and rotodrum tests, was administered before treatment and then 6 months later at 24 months of age. A subsample of mice was retested again at 27 months of age. An untreated group of 9-month-old mice served as young controls. Deprenyl treatment reduced striatal MAO-B activity by up to 60% after 6 months on treatment but had no significant effects on striatal catecholamine levels. No significant effects of deprenyl treatment were observed on body weight, fluid intake, or survival of the mice. Chronic deprenyl treatment also did not affect motor performance in any test, except rotodrum performance at 27 months of age, which was significantly better in the 1.0 mg/kg group treated group compared to controls. No age or deprenyl effects were observed with respect to cell counts in the substantia nigra. However, nigral cells containing lipofuscin increased with age, but this neurohistochemical parameter was also unaffected by deprenyl treatment.

Similarities in the age-related hippocampal deposition of periodic acid-Schiff-positive granules in the senescence-accelerated mouse (SAM P8) and C57BL/6 mouse strains

With advancing age clusters of abnormal granules positive for periodic acid-Schiff appear in the hippocampus of C57BL/6 (B6) mice and the senescence-accelerated mouse (SAM) P8. The granules can also be visualized with a polyclonal antibody to a 110,000 mol. wt laminin-binding protein and stain specifically with a monoclonal antibody to heparan sulfate proteoglycan. The present study used light and electron-microscopic analysis to compare the staining and morphological properties of these granules in SAM P8 hippocampus with those in B6 hippocampus at different ages. The results of the light-microscopic analysis revealed that granules in SAM P8 and B6 had similar morphology, staining characteristics and distribution patterns, and appeared to have a close association with astrocytic process. The onset of granules in SAM P8 mice (at two to three months of age) was earlier than that observed in B6 mice (at four to six months of age), but the maximum incidence was similar in both strains. Electron-microscopic analysis revealed that the granules in SAM P8 and B6 mice also had a very similar ultrastructure. Granules in both strains were surrounded by a discontinuous membrane and contained mostly crystalline-like, degenerated material. The successive ultrastructural changes from the exterior to interior of the granules suggest that the degenerative process was initiated outside the granules and that degenerative structures migrate inward. Astrocytes and heparan sulfate proteoglycan are closely associated with beta-amyloid deposits in Alzheimers disease. The presence of astrocyte-associated heparan sulfate proteoglycan-positive material in aged SAM P8 and B6 mice might model age-related alterations in glia function possibly involved in human cerebral amyloidogenesis.