HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:12-20 (2008)
© 2008 The Gerontological Society of America

Aging Reduces Hypoxia-Induced Microvascular Growth in the Rodent Hippocampus

Jeremy P. Ingraham, M. Elizabeth Forbes, David R. Riddle and William E. Sonntag

1 Program in Neuroscience and Departments of 2 Neurobiology and Anatomy and 3 Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina.
4 Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Science Center, Oklahoma City.

Address correspondence to William Sonntag, PhD, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Science Center, 975 NE 10th Street, BRC-1303, Oklahoma City, OK 73104. E-mail: william-sonntag{at}ouhsc.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The effect of aging on microvascular density and plasticity in the rodent hippocampus, a brain region critically important for learning and memory, was investigated in F344xBN rats. Capillary density and angiogenesis were measured in three regions of the hippocampus in young and old rats and in old rats administered growth hormone, a treatment that improves cognitive function in older animals. Animals were housed under control conditions or in hypoxic conditions (11% ambient oxygen levels) to stimulate vascular growth. Our results indicate that aging is not associated with a reduction in hippocampal capillary density. However, aged animals demonstrate a significant impairment in hypoxia-induced capillary angiogenesis compared to young animals. Growth hormone treatment to aged animals for 6 weeks did not alter hippocampal capillary density and did not ameliorate the age-related deficit in angiogenesis. We conclude that aging significantly reduces hippocampal microvascular plasticity, which is not improved with growth hormone therapy.

Key Words: Rat—Hippocampus—Growth hormone—Capillary density—Angiogenesis

AGING is associated with deficits in several aspects of cognitive function. In particular, data from human, nonhuman primate, and rodent studies demonstrate specific impairments in learning and memory with advancing age (1,2). Although recent investigations have advanced our understanding of age-related alterations in brain function, the etiology of these impairments appears to be complex and remains incompletely understood. Many previous studies of these age-related deficits have focused on alterations in various aspects of neuronal anatomy and physiology, including dendrite structure (3) and proliferative capacity (4). However, proper functioning of these principle cells is critically dependent on the cerebrovasculature to supply oxygen and energy substrates needed to fuel their activities. Age-related alterations in the structure or function of the cerebral vasculature could hinder the delivery of these substances to the brain and therefore contribute to decreased neural function.

The density of microvasculature, including precapillary arterioles and capillaries, in the brain is a necessary component of several fundamental aspects of cerebrovascular function. Microvascular density is an important determinant of the capacity for blood flow through a region of brain tissue, and there is a strong correlation between capillary density and local regional blood flow (5). The density of microvessels in the brain also reflects the surface area that is available for the exchange of oxygen, nutrients, and waste products between brain tissue and blood and determines the diffusion distance these substrates must traverse in the course of this exchange. Furthermore, several laboratories have reported the presence of specific growth factors that are produced in the vasculature, such as nerve growth factor (NGF), insulin-like growth factor 1 (IGF-I), and brain-derived neurotrophic factor (BDNF), suggesting that vasculature exerts a trophic influence on surrounding tissues (6–8). Therefore, maintenance of vascular density within the brain is crucial for proper blood flow, access of tissue components to blood-borne metabolic supplies, and trophic support. Although a number of previous investigations have compared cerebral capillary density in young and aged brains (9), the results of those studies include reports of increased, decreased, and unchanged capillary density with aging. Thus, these studies have provided no compelling consensus on how aging affects this parameter, and further investigation is needed to address this important issue.

In addition to capillary density, plasticity of the microvasculature is an important component of cerebrovascular function. Microvascular plasticity is necessary for matching sustained alterations in metabolic demand with blood flow and to restore blood flow after pathological insults, such as stroke, where alternate delivery pathways must be developed to ensure blood reaches a specific region of the brain. In addition, recent evidence suggests that neuronal plasticity in the adult brain may be intimately associated with the microvasculature such that neurogenesis occurs at a "neuroangiogenic interface" where vascular and neural cell growth are regulated by similar mechanisms (10). Several studies suggest that vascular plasticity is reduced with aging in some tissues, such as skin and hind-limb vasculature (11–14), but few of these investigations have focused on plasticity of cerebral microvessels, and no studies have assessed age-related alterations in microvascular plasticity within the hippocampus, a brain region critical for learning and memory function.

In the present study, we assessed capillary density within three subregions of the hippocampus (CA1, dentate gyrus [DG], and CA3) in adult and aged rats under control conditions and following 4 weeks of chronic hypoxia, a physiological challenge that has been shown to elicit significant capillary angiogenesis in the adult rodent brain (15,16). Additionally, some aged rats were treated with twice-daily injections of growth hormone, which we have previously demonstrated results in increased hippocampal synaptic plasticity, increased hippocampal glucose utilization, and improved performance on hippocampally-dependent tasks of spatial learning (17,18).


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twenty-five young (4-month-old) and aged (30-month-old) F344xBN rats were obtained from the National Institute on Aging colony at Harlan Sprague Dawley (Indianapolis, IN). The aged rats were randomly divided into two groups with one group (Old+GH) receiving twice-daily subcutaneous injections of recombinant porcine growth hormone (300-µg injection; Alpharma, Victoria, Australia), whereas the others received vehicle only (Old). The young animals also received injections of vehicle on the same schedule. Injections began 2 weeks before the animals were transferred from their home cages to the specially designed chambers (described in the Hypoxia section) in which they were housed for the remainder of the study. Injections continued throughout the duration of the experiment for a total of 6 weeks. Animals were housed on a 12-hour light/dark cycle with food and water provided ad libitum. Animal care was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and this study was approved by the Institutional Animal Care and Use Committee.

Hypoxia
Exposure to normobaric hypoxia was achieved through the use of custom-designed animal chambers. The chambers consisted of a standard polycarbonate cage (19 in x 10.5 in x 8.5 in) with a removable Plexiglas top made air-tight with weather-stripping. An air–gas access port was provided on one end of the cage, and vents were provided on the opposite end. Air flow through the chambers was provided by air pumps. Nitrogen was mixed with room air to a final ambient oxygen level of 11% inside the chambers. An oxygen sensor (Maxtec, Salt Lake City, UT) was introduced through a fitted port into the chambers one to two times per day to assess the oxygen level inside the chamber, and adjustments to nitrogen gas flow were made as necessary. Hypoxia was induced gradually by decreasing the ambient oxygen level in the chambers by 1.5% per day for the first 7 days and was maintained at 11% from day 7 through day 30. Chambers were cleaned every other day to reduce ammonia levels. During cleaning and injections, animals were exposed to room air for very brief periods (<5 minutes). Normoxic animals were kept in similar chambers in which the ambient oxygen level was maintained at 21% throughout the experiment.

Measurement of Capillary Density
At the end of the study, animals were anesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg), and blood was collected for determination of hematocrit and serum IGF-I levels. The animals were then perfused through the left ventricle at a rate of 30 mL/min with 37°C phosphate-buffered saline (PBS) for 2 minutes followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer for 10 minutes. Brains were immediately removed and postfixed in buffered 4% paraformaldehyde overnight. They were cryoprotected in a series of graded sucrose solutions, blocked at the frontal pole and cerebellum, and frozen in OCT compound for cryostat sectioning. Cryostat sections of 40 µm thickness were cut through the entire extent of the hippocampus and stored free-floating at –20°C in cryoprotectant solution (25% ethylene glycol, 25% glycerol, 50% 0.05 M phosphate buffer) for subsequent immunostaining and analysis.

From each brain, three cryostat sections from the dorsal hippocampus were chosen to be immunostained and used for vessel density analysis. Selection of these three sections was standardized to common anatomical markers so that vessel analysis was performed in similar anatomical space in all animals. The first section was 1.2 mm caudal to the rostral pole of the hippocampus; the second and third sections were 800 µm and 1600 µm caudal to the first, respectively. Immunostaining of these sections for visualization of vessel morphology was accomplished using a primary antibody to Rat Endothelial Cell Antigen (RECA) and the avidin–biotin horseradish peroxidase system with diaminobenzidine (DAB) for final visualization. Briefly, sections were washed in PBS for 15 minutes, after which endogenous peroxidase activity was quenched with 0.1% H2O2 in PBS for 30 minutes. Sections were washed again and blocked in PBS containing 5% horse serum and 0.25% Triton X for 1 hour. After incubation in blocking solution containing primary antibody (mouse anti-RECA at 0.1 µg/mL; Serotec, Raleigh, NC) overnight at 4°C, sections were washed and incubated for 1.5 hours in secondary antibody solution containing 1:400 horse anti-mouse biotinylated antibody (rat adsorbed; Vector Laboratories, Burlingame, CA) with 3% horse serum in PBS. Following another wash, the sections were incubated in avidin–biotin-peroxidase complex (ABC Elite Kit; Vector Laboratories) for 1 hour and washed again. Antibody labeling was visualized using a 3,3'-DAB substrate kit (Vector Laboratories). After the visualization reaction was complete, the sections were washed again and stored in cryoprotectant at –20°C.

Capillary density was assessed by measuring capillary length per unit volume in CA1, DG, and CA3 of each animal. Measurement was performed using the Neurolucida system for quantitative morphology (MicroBrightField, Colchester, VT) and an Olympus BX50 microscope fitted with Nomarski differential interference contrast (DIC) optics. This computerized system allowed concurrent visualization, tracing, and measurement of capillaries through the entire 40-µm thickness of the tissue section. Sections were mounted free-floating in cryoprotectant solution onto microscope slides and cover-slipped. In each section, a sampling box of approximately 500 µm x 300 µm was outlined within each region of interest (Figure 1). The square area of this box was then multiplied by the section thickness to calculate the volume of the sampling box. Within each of these sampling boxes, all capillaries (defined as stained vessels ≤10 µm in diameter) were viewed with a 40x objective (numerical aperture = 0.75) and traced in their entirety using the Neurolucida tracing function. The total length of all capillaries within a sampling box was then divided by the volume of the sampling box to arrive at the density value for that sampling box. Density measurements from the three hippocampal sections were averaged to estimate the capillary length per volume of each subregion of the hippocampus.


Figure 01
View larger version (140K):
[in this window]
[in a new window]

  		Figure 1. Example of sampling method used for measurement of capillary density in three subregions of the hippocampus. For each animal, this sampling method was applied to three cryostat sections from within the dorsal hippocampus. The density of capillaries in a given subregion was calculated by averaging the measured densities from the three sections. DG = dentate gyrus

 
In addition to measuring capillary density, the Neurolucida system was also used to assess the total volume of each region within the dorsal hippocampus. For this measurement, seven sections, 320 µm apart, were selected from within the same dorsal hippocampal space analyzed for capillary density. In each section, the entire extent of each subregion was outlined using the tracing function of the software to determine its area. The area of each region was then multiplied by the section thickness to calculate the volume of the subregions in that section. The volume measurements from all seven sections were then used to calculate the total volume of each subregion within the dorsal hippocampus.

Measurement of Serum IGF-I
To verify biological activity of the subcutaneously injected growth hormone, blood collected at the time of death was used to determine circulating levels of IGF-I as previously described (19). Briefly, IGF-I (Bachem, Torrance, CA) was radiolabeled using the lactoperoxidase, glucose oxidase method and purified on a Sep-Pak silica cartridge (Waters, Milford, MA). Serum IGF-I was extracted in acid-ethanol and measured by radioimmunoassay. Materials for analysis of IGF-I were generously provided by Dr. A. Parlow from the National Hormone and Peptide Program.

Data Analysis
Initial analysis of the data consisted of two-way analysis of variance to determine if there was an effect of age, hormone treatment, or hypoxia on capillary density in each subregion of the hippocampus. If a main effect was detected, the Student–Newman–Keuls post hoc analysis was used to determine differences between groups. A difference between groups was considered statistically significant if the p value of the comparison was <.05.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
General
As reported in other studies using hypoxia in rodent models (15,16), the hypoxic animals in this study exhibited two characteristic physiological responses: modest weight loss and polycythemia. Following 4 weeks of normobaric hypoxia, the Old and Old+GH animals weighed 528 ± 6.8 g and 532 ± 19.7 g, respectively, compared to the normoxic Old and Old+GH animals (593 ± 18.4 g and 583 ± 20.4 g, respectively). This represents a 10% loss of body weight for both of these hypoxic groups. In the young animals, hypoxia did not result in significant weight loss (normoxic Young = 328 ± 10.7 g; hypoxic Young = 335 ± 19.8 g). Regardless of age, all hypoxic animals demonstrated a similar, significant increase in hematocrit compared to their normoxic counterparts (Figure 2A).


Figure 02
View larger version (33K):
[in this window]
[in a new window]

  		Figure 2. Hematocrit and circulating insulin-like growth factor-1 (IGF-I) values. A, There was no effect of aging or growth hormone treatment on hematocrit under control conditions (solid bars). In all age groups, hypoxia resulted in a significant increase in hematocrit (*p <.001 vs normoxic animals within age group). The Old, hypoxic animals exhibited a significantly higher hematocrit compared to hypoxic Young and Old+GH animals (#p <.02). B, There was no effect of aging or hypoxia on serum IGF-I levels (light gray vs black bars). Under control conditions, administration of growth hormone to aged animals significantly increased serum IGF-I (*p <.03 vs young and aged saline-treated animals). There was a similar trend under hypoxic conditions, but this trend was not statistically significant. Data in A and B represent 3–5 animals/group. Error bars = standard error of the mean

 
Under control conditions, administration of growth hormone to aged animals increased serum IGF-I 19.0% above that of young, saline-treated animals (p =.028) and 19.4% above that of aged, saline-treated animals (p =.025) (Figure 2B, solid bars). There was a similar response in the hypoxic animals, but this trend did not reach statistical significance (Figure 2B, striped bars). These results demonstrate that the subcutaneously delivered growth hormone was biologically active in the aged animals receiving this therapy. No differences in serum IGF-I were detected between the Young and Old saline-treated groups, regardless of oxygen status (Figure 2B, light gray vs black bars)

During the course of the study, one hypoxic Old animal suffered a cortical infarct and one hypoxic Old+GH animal exhibited severe hydrocephalus. These animals were excluded from data analysis.

Capillary Density
Because our analysis of capillary density is sensitive to alterations in hippocampal volume as well as differences in capillary length, initial analyses considered the volume of each subregion of the hippocampus. Analysis of these data revealed no difference in the volume of any hippocampal subregion among Young, Old, and Old+GH animals under control conditions (data not shown, p =.105–.805), and hypoxia did not alter hippocampal volumes in any age group (data not shown, p =.063–.852).

Examples of hippocampal sections from the six experimental groups compared in this study are shown in Figure 3. The photomicrographs show portions of the CA1 and DG regions for comparison, and analysis of capillary density is summarized in Figure 4. The data indicate that under control conditions there was no effect of aging on capillary density in any of the subregions analyzed (Figure 4A–C, solid bars). The aged animals to which growth hormone was administered exhibited marginally higher capillary density throughout the hippocampus compared to the saline-treated aged animals; however, this trend was not statistically significant for any of the three subregions.


Figure 03
View larger version (86K):
[in this window]
[in a new window]

  		Figure 3. Representative photomicrographs of hippocampal vasculature. Images include the CA1 and dentate gyrus (DG) subregions (CA3 regions are not shown). Vessels were immunostained using a primary antibody to Rat Endothelial Cell Antigen (RECA). Analysis revealed no effect of aging or growth hormone administration under normoxic conditions. Following hypoxia, young animals exhibited significantly higher capillary density in all three hippocampal subregions compared to both groups of aged animals

 

Figure 04
View larger version (37K):
[in this window]
[in a new window]

  		Figure 4. Results of capillary density analysis in CA1 (A), dentate gyrus (DG) (B), and CA3 (C) subregions of hippocampus. Under control conditions, no effect of aging or growth hormone treatment was detected in any hippocampal subregion (solid bars). Following 4 weeks of hypoxia, only young animals experienced significant capillary angiogenesis in every subregion (*p <.001, normoxic vs hypoxic). Old animals exhibited significant vessel growth in DG and CA3 (*p <.001 and p <.02, respectively, normoxic vs hypoxic) whereas the Old+GH group showed increased capillary density only in the DG (*p <.04, normoxic vs hypoxic). In all subregions, young animals demonstrated higher posthypoxic capillary density than both groups of aged animals (#p <.02 vs Young, hypoxic). There were no differences in posthypoxic capillary density between the Old and Old+GH groups in any subregion. Data in A–C represent 3–5 animals/group. Error bars = standard error of the mean. D, Percent increase in capillary density between the normoxic and hypoxic animals of each age group. Young animals show robust vessel growth in response to hypoxia compared to both groups of aged animals

 
Following 4 weeks of chronic hypoxia, young animals exhibited significant capillary angiogenesis in all subregions of the hippocampus and showed significantly higher capillary density in these subregions compared to both groups of aged animals (Figure 4A–C, hatched bars). In contrast, Old animals showed significant angiogenesis only in DG and CA3, whereas the Old+GH group only showed significant capillary growth in the DG. There were no significant differences in posthypoxic capillary density between saline-treated and growth hormone–treated aged animals in any hippocampal subregion. When the results from each experimental age group (Young, Old, Old+GH) were expressed as the percent difference in capillary density between the normoxic and hypoxic animals (Figure 4D), the differences in the angiogenic responses of these groups were evident. Young animals were able to increase capillary density in all subregions of the hippocampus by 35%–40%. In contrast, aged animals showed regional variability in capillary growth, with a maximum increase of 20% in the DG, 15% in CA3, and only 6%–7% in CA1. The aged animals to which growth hormone was administered also showed regional variation in angiogenesis with hypoxia and exhibited overall less capillary growth than either the Young or Old groups.


   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study focused on two critical aspects of cerebrovascular status in the aging brain, the density of capillaries and the capacity for capillary growth. These variables were measured in the rodent hippocampus, a brain structure intimately involved in learning and memory function. Our results demonstrate that, under basal conditions, aging is not associated with decreased capillary density in the hippocampus. However, when challenged with 4 weeks of chronic hypoxia, aged animals exhibit an impaired capacity for capillary growth. The data also demonstrate that administration of growth hormone, a treatment that has previously been shown to improve hippocampal function in aged animals, does not alter capillary density and does not increase hypoxia-induced capillary angiogenesis in aged animals.

Capillary Density
The length of capillary per unit volume of brain tissue represents at least three fundamental parameters of cerebrovascular function. First, capillary density determines the inherent capacity for blood flow through a given volume of brain tissue. Other factors being equal, a higher density of vessels will result in greater perfusion volume. Second, the density of microvasculature reflects the amount of surface area that is available for the exchange of oxygen and nutrients between blood and brain parenchyma. The brain has limited storage capacity for these essential items and relies on the vascular system for a constant and consistent supply. The capillary wall is the interface across which this exchange takes place; therefore, the amount of capillary present in a given volume of tissue determines the extent of this vital interface. Lastly, capillary length per volume, along with capillary diameter, dictates the distance that exists between microvessels in a given volume of tissue. This intercapillary distance reflects how far oxygen and nutrients must diffuse from the vasculature to reach a given cell in the tissue and thus impacts the rate at which this exchange can take place. In addition to the fundamental relevance of microvascular density to cerebrovascular function, recent evidence suggests that vasculature exerts a trophic effect on surrounding tissue through the synthesis and secretion of several neural growth factors including NGF, IGF-I, and BDNF (6–8). Any alteration in the density of the vasculature could therefore impact trophic support as well. Therefore, it is conceivable that age-related alterations in microvascular extent could contribute significantly to impaired cognitive function.

A number of previous studies have compared the density of capillaries in young and aged brains. These investigations include measures of capillary density in a wide range of brain regions in both humans and animal models [see (9) for review]. Unfortunately, these studies have reported disparate conclusions, even within the same brain region of the same experimental animals, and have provided no clear consensus on the effect of aging on cerebral capillary density. For example, in several studies of the rat occipital cortex, aging was associated with an increase (20), no change (21), or a decrease (22) in capillary density. Although there are numerous possibilities to explain the disparity of these reports, it is clear that additional investigations are needed to address the effect of aging on cerebrovascular density. To this end, the current study used a rigorous, computer-aided, three-dimensional technique to analyze the density of capillaries in specific regions of the rodent hippocampus, a brain area intimately involved in learning and memory function and in which we have previously demonstrated significant age-related dysfunction (17,18). Using this approach we were able to simultaneously visualize and trace all capillary structures that were present within defined areas of a 40-µm-thick brain section. This allowed the direct sampling of capillary density in each hippocampal subregion. Only the amount of sampling (i.e., the number of sections used and the size and number of sampling boxes used) presented any limitation to directly measuring the true capillary length per volume of this brain structure. Using this methodology, we show that aging is, in fact, not associated with altered capillary density in the rodent hippocampus.

The conservation of capillary density found in the current study suggests that, in aged animals, the inherent capacity for blood flow and nutrient exchange in this brain region is not limited by capillary density. However, there are other structural alterations in microvessels that occur with aging that could compromise both blood flow and exchange capacity in the brain [reviewed in (23) and (24)]. For example, there are reports of increased basement membrane thickness in the capillaries of aged brains (25,26), which could inhibit the transport of diffusable and transported substances across the blood–brain barrier. There also appears to be an increase in the tortuosity or twisting of microvessels in the aged brain (27), which could result in decreased perfusion through these vessels. The extent to which these alterations in microvascular ultrastructure impact neuronal physiology remain to be elucidated, and further investigation is needed to assess their impact on cognitive function.

Microvascular Plasticity
Previous investigations have demonstrated that aging is associated with an impaired capacity for wound healing (28,29) as well as reduced angiogenic capacity in a number of different vascular beds. For example, vascular invasion of subcutaneously implanted polyvinyl alcohol sponges is reduced in aged mice (14) and rats (13), and aged rabbits exhibit impaired angiogenesis in response to femoral artery resection (11). In the brain, increasing age is associated with reduced cortical capillary plasticity following stroke (30) and in response to environmental enrichment (31). Our study extends these previous findings and is the first to report an age-related deficit in angiogenesis in the rodent hippocampus following exposure to chronic hypoxia. The hypoxia model used in this study is especially advantageous for evaluating cerebral angiogenesis because it provides a potent, highly controllable, and noninvasive stimulus for vascular growth that is also a physiologically relevant challenge to aged animals, as reduced oxygen delivery to the brain is a key component of several pathological conditions including stroke and chronic heart failure.

We propose that a reduction in hippocampal vascular plasticity with aging, as our data illustrate, could contribute to a decline in cognitive function through several possible mechanisms. Impaired microvascular plasticity may reduce metabolic support for the physiological mechanisms that underlie the processes of learning and memory formation. These mechanisms, such as long-term potentiation (LTP) and long-term depression (LTD), which are electrophysiological correlates of learning and memory, undoubtedly alter metabolic demand within the hippocampus. To adequately match metabolic supply with this new level of demand, the vasculature within this region must alter delivery capacity. Impaired vascular plasticity could limit this adaptation, resulting in impaired learning and memory. Although direct evidence for this hypothesis is lacking in the hippocampus, a similar finding was demonstrated in the aged rat cortex following exposure to an enriched environment (31). Additionally, recent studies indicate that the continual replacement of granule layer neurons in the DG is important for hippocampal function (32) and that a reduction in granule cell replacement in the adult hippocampus may decrease performance on some learning and memory tasks (33). A study by Palmer and colleagues (10) revealed that the dividing cells in the subgranular zone that generate new granule layer neurons are found in clusters associated with blood vessels and that approximately 37% of dividing cells in this region express endothelial markers (10). These findings were the first to suggest that neuronal and vascular plasticity may be intimately interconnected. More recent literature has shown that neural and vascular physiologies are indeed tightly coupled and that these systems share many common regulatory mechanisms [see (34) for review]. Therefore, there is emerging evidence for a close association between a decline in hippocampal vascular plasticity and decreased neuronal plasticity that contributes to age-related memory impairments.

Growth Hormone Treatment
Previous reports from our laboratory have demonstrated that, in aged animals, systemic treatment with growth hormone, which increases circulating levels of IGF-I, and administration of IGF-I directly to the brain both result in positive changes in hippocampal physiology, including improved performance on hippocampally dependent tasks of spatial learning (35), increased synaptic plasticity (17), and increased glucose utilization (18). In addition, growth hormone and IGF-I have been shown to have an integral role in the growth, maintenance, and repair of the vasculature. Blood vessels have receptors for both growth hormone and IGF-I (7,36), and these hormones have been shown to promote endothelial cell proliferation and migration, tube formation, and angiogenesis in a number of tissues (37,38). These data suggested that growth hormone administration to aged animals may also impact microvascular density and plasticity within the hippocampus. The current study shows, however, that 6 weeks of growth hormone administration does not affect capillary density in the aged hippocampus and does not ameliorate the age-related deficit in hypoxia-induced angiogenesis. It is possible that growth hormone or IGF-I did not contribute significantly to hypoxia-specific microvascular growth and therefore provided no therapeutic benefit to the aged animals in this study. It is also possible that the duration of hormone treatment in the current study was insufficient to facilitate improvements in microvascular status, and further investigation is needed to elucidate whether prolonged treatment with growth hormone may provide some improvement in angiogenic capacity in the aging brain.

Conclusion
In this study we assessed the effect of aging and growth hormone treatment to aged animals on capillary density and angiogenesis in the rodent hippocampus. We report that aging does not alter capillary density but significantly reduces hypoxia-stimulated angiogenesis in this brain region. Additionally, administration of growth hormone to aged animals did not alter capillary density or angiogenic capacity. Further investigation is underway in our laboratory to elucidate the mechanisms underlying the age-related decline in microvascular growth in this brain region.


   Acknowledgments
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This study was supported by National Institutes of Health grant PO1 AG11370 (to W.E.S.).

We thank Rhonda Ingram, Colleen Bennett, Tracy Moore, and Justin Galasso for their technical assistance as well as Dr. Delrae Eckman, Dr. Judy Brunso-Bechtold, and Dr. Carol Milligan for their assistance with project planning and data analysis. In addition, we thank Dr. Al Parlow for providing materials for radioimmunoassay of rat IGF-I and Dr. Haiying Chen for assistance with statistical analysis.


   Footnotes
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received May 16, 2007

Accepted September 19, 2007


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gallagher M, Pelleymounter MA. Spatial learning deficits in old rats: a model for memory decline in the aged. Neurobiol Aging. 1988;9:549-556.[Medline]
  2. Nilsson LG. Memory function in normal aging. Acta Neurol Scand Suppl. 2003;179:7-13.[Medline]
  3. Grill JD, Riddle DR. Age-related and laminar-specific dendritic changes in the medial frontal cortex of the rat. Brain Res. 2002;937:8-21.[Medline]
  4. Lichtenwalner RJ, Forbes ME, Bennett SA, Lynch CD, Sonntag WE, Riddle DR. Intracerebroventricular infusion of insulin-like growth factor-I ameliorates the age-related decline in hippocampal neurogenesis. Neuroscience. 2001;107:603-613.[Medline]
  5. Gjedde A, Diemer NH. Double-tracer study of the fine regional blood-brain glucose transfer in the rat by computer-assisted autoradiography. J Cereb Blood Flow Metab. 1985;5:282-289.[Medline]
  6. Creedon D, Tuttle JB. Nerve growth factor synthesis in vascular smooth muscle. Hypertension. 1991;18:730-741.[Abstract/Free Full Text]
  7. Delafontaine P, Bernstein KE, Alexander RW. Insulin-like growth factor I gene expression in vascular cells. Hypertension. 1991;17:693-699.[Abstract/Free Full Text]
  8. Leventhal C, Rafii S, Rafii D, Shahar A, Goldman SA. Endothelial trophic support of neuronal production and recruitment from the adult mammalian subependyma. Mol Cell Neurosci. 1999;13:450-464.[Medline]
  9. Riddle DR, Sonntag WE, Lichtenwalner RJ. Microvascular plasticity in aging. Ageing Res Rev. 2003;2:149-168.[Medline]
  10. Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol. 2000;425:479-494.[Medline]
  11. Rivard A, Fabre JE, Silver M, et al. Age-dependent impairment of angiogenesis. Circulation. 1999;99:111-120.[Abstract/Free Full Text]
  12. Swift ME, Kleinman HK, DiPietro LA. Impaired wound repair and delayed angiogenesis in aged mice. Lab Invest. 1999;79:1479-1487.[Medline]
  13. Wang H, Keiser JA, Olszewski B, et al. Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF. Int J Mol Med. 2004;13:581-587.[Medline]
  14. Sadoun E, Reed MJ. Impaired angiogenesis in aging is associated with alterations in vessel density, matrix composition, inflammatory response, and growth factor expression. J Histochem Cytochem. 2003;51:1119-1130.[Abstract/Free Full Text]
  15. LaManna JC. Rat brain adaptation to chronic hypobaric hypoxia. Adv Exp Med Biol. 1992;317:107-114.[Medline]
  16. Harik N, Harik SI, Kuo NT, Sakai K, Przybylski RJ, LaManna JC. Time-course and reversibility of the hypoxia-induced alterations in cerebral vascularity and cerebral capillary glucose transporter density. Brain Res. 1996;737:335-338.[Medline]
  17. Ramsey MM, Weiner JL, Moore TP, Carter CS, Sonntag WE. Growth hormone treatment attenuates age-related changes in hippocampal short-term plasticity and spatial learning. Neuroscience. 2004;129:119-127.[Medline]
  18. Lynch CD, Lyons D, Khan A, Bennett SA, Sonntag WE. Insulin-like growth factor-1 selectively increases glucose utilization in brains of aged animals. Endocrinology. 2001;142:506-509.[Abstract/Free Full Text]
  19. Sonntag WE, Lenham JE, Ingram RL. Effects of aging and dietary restriction on tissue protein synthesis: relationship to plasma insulin-like growth factor-1. J Gerontol. 1992;47:B159-B163.[Abstract]
  20. Bar T. Morphometric evaluation of capillaries in different laminae of rat cerebral cortex by automatic image analysis: changes during development and aging. Adv Neurol. 1978;20:1-9.[Medline]
  21. Knox CA, Oliveira A. Brain aging in normotensive and hypertensive strains of rats. III. A quantitative study of cerebrovasculature. Acta Neuropathol (Berl). 1980;52:17-25.[Medline]
  22. Amenta F, Ferrante F, Mancini M, Sabbatini M, Vega JA, Zaccheo D. Effect of long-term treatment with the dihydropyridine-type calcium channel blocker darodipine (PY 108-068) on the cerebral capillary network in aged rats. Mech Ageing Dev. 1995;78:27-37.[Medline]
  23. Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer's disease. Prog Neurobiol. 2001;64:575-611.[Medline]
  24. Kalaria RN. Cerebral vessels in ageing and Alzheimer's disease. Pharmacol Ther. 1996;72:193-214.[Medline]
  25. Burns EM, Kruckeberg TW, Gaetano PK. Changes with age in cerebral capillary morphology. Neurobiol Aging. 1981;2:283-291.[Medline]
  26. Keuker JI, Luiten PG, Fuchs E. Capillary changes in hippocampal CA1 and CA3 areas of the aging rhesus monkey. Acta Neuropathol (Berl). 2000;100:665-672.[Medline]
  27. Moody DM, Brown WR, Challa VR, Ghazi-Birry HS, Reboussin DM. Cerebral microvascular alterations in aging, leukoaraiosis, and Alzheimer's disease. Ann N Y Acad Sci. 1997;826:103-116.[Medline]
  28. Mustoe TA, O'Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg. 2006;117:(7 Suppl): 35S-41S.[Medline]
  29. Mogford JE, Sisco M, Bonomo SR, Robinson AM, Mustoe TA. Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing. J Surg Res. 2004;118:190-196.[Medline]
  30. Szpak GM, Lechowicz W, Lewandowska E, Bertrand E, Wierzba-Bobrowicz T, Dymecki J. Border zone neovascularization in cerebral ischemic infarct. Folia Neuropathol. 1999;37:264-268.[Medline]
  31. Black JE, Polinsky M, Greenough WT. Progressive failure of cerebral angiogenesis supporting neural plasticity in aging rats. Neurobiol Aging. 1989;10:353-358.[Medline]
  32. Lemaire V, Koehl M, Le Moal M, Abrous DN. Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proc Natl Acad Sci U S A. 2000;97:11032-11037.[Abstract/Free Full Text]
  33. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E. Neurogenesis in the adult is involved in the formation of trace memories. Nature. 2001;410:372-376.[Medline]
  34. Park JA, Choi KS, Kim SY, Kim KW. Coordinated interaction of the vascular and nervous systems: from molecule- to cell-based approaches. Biochem Biophys Res Commun. 2003;311:247-253.[Medline]
  35. Markowska AL, Mooney M, Sonntag WE. Insulin-like growth factor-1 ameliorates age-related behavioral deficits. Neuroscience. 1998;87:559-569.[Medline]
  36. Hansson HA, Jennische E, Skottner A. Regenerating endothelial cells express insulin-like growth factor-I immunoreactivity after arterial injury. Cell Tissue Res. 1987;250:499-505.[Medline]
  37. Gould J, Aramburo C, Capdevielle M, Scanes CG. Angiogenic activity of anterior pituitary tissue and growth hormone on the chick embryo chorio-allantoic membrane: a novel action of GH. Life Sci. 1995;56:587-594.[Medline]
  38. Sato Y, Okamura K, Morimoto A, et al. Indispensable role of tissue-type plasminogen activator in growth factor-dependent tube formation of human microvascular endothelial cells in vitro. Exp Cell Res. 1993;204:223-229.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS