Age-associated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy.

Abstract

The effects of normal aging on the primate brain are incompletely understood. Although both human and nonhuman primates demonstrate clear functional declines in selective attention, "executive" functions, and some components of declarative memory with aging, most studies have failed to demonstrate extensive neuronal atrophy or loss as a substrate for these degenerative changes in primates. In particular, extensive age-related neuronal loss in memory-related brain regions such as the hippocampus and entorhinal cortex has not been found. However, it is possible that neuronal loss or atrophy might occur in subcortical nuclei that modulate the activity of neocortical regions, thereby accounting for altered cognitive function with aging. In the present study, we describe, to our knowledge for the first time, a significant and extensive decline in the number and size of immunolabeled neurons in subcortical cholinergic basal forebrain regions of aged rhesus monkeys, the best animal model of human aging, by using stereological methods. Notably, the loss of subcortical cholinergic neuronal markers in aged monkeys was nearly completely reversed by human nerve growth factor gene delivery. These findings (i) identify reversible cellular atrophy as a potential mechanism contributing to age-related cognitive decline in primates, (ii) suggest, when considered with other studies, that subcortical brain regions exhibit greater vulnerability to the effects of aging than cortical regions, and (iii) indicate that neurotrophin gene transfer may be an effective means of preventing neuronal atrophy or degeneration in age-related neurodegenerative disorders.

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Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 10893–10898, September 1999
Neurobiology
Age-associated neuronal atrophy occurs in the primate brain and
is reversible by growth factor gene therapy
(aging?Alzheimer’s disease?nerve growth factor?stereology?cholinergic systems)
D. E. SMITH*, J. ROBERTS†, F. H. GAGE‡, AND M. H. TUSZYNSKI*§¶
*Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0626;†California Regional Primate Research Center, University of
California, Davis, CA 95616-8542;‡Salk Institute for Biological Studies, La Jolla, CA 92037; and§Veterans Affairs Medical Center, San Diego, CA 92161
Communicated by Larry R. Squire, University of California, San Diego, CA, July 2, 1999 (received for review February 11, 1999)
ABSTRACT
brain are incompletely understood. Although both human and
nonhuman primates demonstrate clear functional declines in
selective attention, ‘‘executive’’ functions, and some components
of declarative memory with aging, most studies have failed to
demonstrate extensive neuronal atrophy or loss as a substrate for
these degenerative changes in primates. In particular, extensive
age-related neuronal loss in memory-related brain regions such
as the hippocampus and entorhinal cortex has not been found.
However,itispossiblethatneuronallossoratrophymightoccur
in subcortical nuclei that modulate the activity of neocortical
regions, thereby accounting for altered cognitive function with
aging. In the present study, we describe, to our knowledge for the
first time, a significant and extensive decline in the number and
size of immunolabeled neurons in subcortical cholinergic basal
forebrainregionsofagedrhesusmonkeys,thebestanimalmodel
ofhumanaging,byusingstereologicalmethods.Notably,theloss
of subcortical cholinergic neuronal markers in aged monkeys
was nearly completely reversed by human nerve growth factor
gene delivery. These findings (i) identify reversible cellular atro-
phy as a potential mechanism contributing to age-related cog-
nitive decline in primates, (ii) suggest, when considered with
other studies, that subcortical brain regions exhibit greater
vulnerabilitytotheeffectsofagingthancorticalregions,and(iii)
indicate that neurotrophin gene transfer may be an effective
means of preventing neuronal atrophy or degeneration in age-
related neurodegenerative disorders.
The effects of normal aging on the primate
Declines in cognitive function accompany normal aging in hu-
mans and nonhuman primates (1, 2). Impaired function in
selective attention, ‘‘executive’’ functions, and some components
of declarative memory constitute a regular feature of aging in
both humans and in the best animal model of human aging, the
rhesus monkey (1, 3). However, the structural basis, if any, for
age-related declines in cognition is unclear. To date, studies of
neuronal changes with aging in human and nonhuman primates
haverevealedmodestifanyreductioninneuronalnumberorsize
in several cortical brain regions, including the hippocampus (4),
entorhinal cortex (5), and other neocortical regions (6–9). The
failure to identify an age-related change in neuronal number has
led to the suggestion that synaptic, dendritic, metabolic, or
neurophysiological rather than somal structural changes underlie
age-related declines in cognitive function (1, 2, 10–15). However,
an alternative hypothesis is that structural degenerative changes
may occur in subcortical neuronal populations that project to and
regulate the function of hippocampal and cortical circuitry. To
date, this possibility has not been carefully examined in primates.
Neurons located in the subcortical basal forebrain region
provide virtually all cholinergic innervation to the hippocampus
and neocortex (16). These basal forebrain subcortical cholinergic
neurons, classified as Ch1-Ch4 neurons (16), play an important
role in regulating neuronal activity in cortical circuits, and per-
turbation of this system has been related to deficits in attention
and specific aspects of memory (17, 18). The intermediate
division of the Ch4 region (Ch4i) projects to several cortical
regions and is the primary source of cholinergic innervation to
classic memory-mediating structures of the cerebral cortex, in-
cluding the entorhinal cortex (16). To investigate whether sub-
cortical brain regions that influence cognition exhibit vulnerabil-
ity to the aged state, neuronal number and size were quantified
in Ch4i in the basal forebrain by using unbiased stereological
methods. Comparisons were made between aged and nonaged
rhesus monkeys. In addition, a subset of aged monkeys received
grafts of nerve growth factor (NGF)-secreting autologous fibro-
blasts to the Ch4 region to determine whether spontaneous
age-related neurodegenerative events in the cholinergic basal
forebrain, if present, were sensitive to and reversible by growth
factor therapy. Previously it has been reported that degenerating
basalforebraincholinergicneuronsaresensitivetoNGF(19–25).
Findings of the present study demonstrate, to our knowledge for
the first time, that aging in the primate brain is accompanied by
significant and quantitatively extensive neuronal atrophy in sub-
cortical cholinergic neuronal populations. Further, the sponta-
neous age-related atrophy of these neurons is sensitive to and
reversible by neurotrophin gene therapy.
METHODS
Four groups of rhesus monkeys were studied: four adult nonaged
nonoperatedsubjects(meanage9.4?1.1yr;threemalesandone
female), three aged nonoperated subjects (mean age 25.1 ? 2.5
yr; all males), four aged monkeys that received intraparenchymal
grafts of autologous fibroblasts genetically modified to produce
and secrete human NGF (mean age 22.6 ? 0.8 yr; one male and
three females), and four aged monkeys that received intraparen-
chymal control grafts of autologous fibroblasts genetically mod-
ified to produce the reporter gene ?-galactosidase (?-gal) (mean
age 23.3 ? 0.8 yr; three males and one female). All subjects were
born at the California Regional Primate Center and spent the
majority of their lives in 5-acre field enclosures containing social
groups of 50–60 animals. Before the present study, animals were
maintained under similar social conditions and were not involved
in behavioral studies, surgical procedures, or pharmacological
experiments. All animal care procedures adhered to American
Association for the Accreditation of Laboratory Animal Care
and institutional guidelines.
For NGF delivery to aged subjects, autologous fibroblasts
genetically modified to secrete human NGF were prepared as
previously described (26, 27). Briefly, autologous fibroblasts
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: Ch4i, the intermediate division of the Ch4 region;
NGF, nerve growth factor; ?-gal, ?-galactosidase; ChAT, choline
acetyltransferase; AChE, acetylcholinesterase.
¶To whom reprint requests should be addressed. E-mail: mtuszyns@
ucsd.edu.
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obtained from skin biopsies were genetically modified in vitro to
produce and secrete the active ?-portion of human NGF. Trans-
duction procedures used replication-incompetent retroviral vec-
tors derived from Moloney murine leukemia virus. Transduced
cells were selected by growth in G418. Production of biologically
active NGF was verified by neurite outgrowth from PC12 cells
(28). Production of full length NGF mRNA was determined by
Northern blot. NGF protein produced from cells was assayed by
NGF ELISA sensitive to 5 pg?ml (29). In vitro before grafting,
autologous fibroblasts from NGF-grafted monkeys secreted a
mean of 12 ? 2 ng human NGF?106cells?day. Optimal NGF-
producingbulkcloneswereamplifiedtonumberssufficientfor in
vivo grafting. Aged control-grafted monkeys received grafts of
identical autologous fibroblasts, with the exception that the NGF
gene was replaced by the reporter gene Escherichia coli ?-gal,
whichlacksneurotrophicactivity.Thus,cellsinNGF-graftedand
control-grafted subjects differed by a single gene. Cells were
harvested in vitro, and a total of 5.0 ? 106cells were grafted into
each subject adjacent to the region of the intermediate division
ofthecholinergicbasalforebrain(seesurgerydescriptionbelow).
Surgery. Monkeys underwent preoperative MRI scans to
visualize basal forebrain targets. After stereotaxic coordinates
were generated from MRI scans, each monkey received intra-
parenchymal grafts of autologous NGF-secreting or control
fibroblasts. Monkeys were preanesthetized with 25 mg?kg ket-
amine i.m. and were anesthetized with isofluorane. Cells were
grafted into each of five sites equally spaced over the rostral-
caudal extent of the Ch4 region bilaterally (10 grafts total per
animal). Grafts were targeted to a position slightly dorsal to but
within 500 ?m of the Ch4 nucleus. Ten-microliter cell suspen-
sions, 1.0 ? 105cells??l, were grafted into each site with a
25-gauge Hamilton syringe at a rate of 1 ?l?min, for a total of 10
milliongraftedcellsperanimal.Postoperatively,allsubjectswere
observed closely for signs of discomfort or toxicity and received
theanalgesicbuprenorphine.Graftedmonkeyssurvivedfor3mo
and were then sacrificed by transcardial perfusion as described
below.
The duration of in vivo gene expression was examined in two
additional rhesus monkeys. One monkey (age 10 yr) received a
suspension graft of 25 ?l of autologous fibroblasts transduced to
express human NGF into the parietal cortex. The stereotaxic
coordinatesoftheinjectionsitewerenoted,andthisregionofthe
brain was freshly dissected 8 mo later and immediately frozen to
?80°C until subsequent processing for NGF content by ELISA
(29). A second monkey received a control graft of fibroblasts to
the parietal cortex that were not genetically modified. This graft
too was freshly dissected 8 mo later and immediately frozen for
two-site ELISA.
Histology and Stereology. Animals were sedated with 25
mg?kg i.m. ketamine and were then deeply anesthetized with
Nembutal (30 mg?kg i.p.). All reflex responses to cutaneous
stimulation were verifiably absent before perfusion procedures
were begun. Subjects were perfused transcardially for 1 hr with a
4% solution of paraformaldehyde in 0.1 M phosphate buffer at
4°C followed by 5% sucrose solution. Brains were stereotaxically
blocked in the coronal plane.
Serialcoronalsectionsthroughthebrainwerecutonafreezing
microtomesetat40?m.Everysixthsectionwasprocessedforp75
immunoreactivity (monoclonal hybridoma cell line generated by
M. Bothwell, University of Washington, dilution 1:100) as pre-
viously described (21). p75 has been reported to exhibit 95%
coassociation with cholinergic neurons of the basal forebrain and
labels no other neuronal types in this brain region (30). An
additional 1-in-6 sections was Nissl stained for quantification of
totalneuronalnumberandtoevaluategraftpositionandsurvival.
Stereological procedures were used to quantify the number of
cholinergic neurons in p75-immunolabeled sections and the total
number of neurons in Nissl-stained sections. In both p75-
immunolabeled and Nissl-stained sections, neurons were quan-
tified in Ch4i. The following anatomical boundaries were used to
demarcateCh4i:(i)therostralboundaryofCh4iismarkedbythe
appearanceoftheansapeduncularisasitbeginstopenetrateCh4,
beginning dorsomedially and traversing diagonally through Ch4i
(dorsomedial to ventrolateral); (ii) the caudal boundary of Ch4i
is marked by the completion of the passage of the ansa pedun-
cularisthroughCh4atitsventrolateralextentandthesubsequent
groupingofcholinergicneuronsintoasinglecluster,theposterior
division (Ch4p); (iii) the ventral boundary of Ch4i at its rostral
pole is formed by the Ch3 cell group. The cells of this group are
smaller than those of Ch4, fusiform in shape with the long axis
oriented along the ventral surface of the brain, and appear
hypochromiconp75labeling.Theventralboundaryatthecaudal
aspect of Ch4i is formed by the ventral surface of the brain; and
(iv) the dorsal boundary of Ch4i is formed by the globus pallidus.
There were no significant differences among the four animal
groups in the mean number of tissue sections containing Ch4i
(8.6 ? 0.5) used for stereological quantification (ANOVA P ?
0.86).
Stereological counts were performed on every sixth section
through the entire extent of Ch4i. The stereology equation of
West (31) was used to calculate total neuronal numbers:
n ? Q
? 1
ssf↵
? 1
asf↵
? 1
tsf↵
where n, calculated total number of neurons; Q ? sum of
counted points (i.e., neurons); ssf, section-sampling fraction, in
this case 1?6; asf, area-sampling fraction or the fraction of the
total area that is contained within counting frames, in this case
5%; and tsf, tissue sampling fraction, or the fraction of the
thickness of each section within which neurons are counted, in
this case 75%.
FIG. 1.
region in nonaged monkey exhibits normal distribution of cholinergic
neurons. Ch4id: Ch4 nucleus, intermediate-dorsal component; Ch4iv:
Ch4 nucleus, intermediate-ventral component. M, medial. Bar ? 450 ?m
in a–d. (Inset) Normal p75-immunolabeled neurons and neurite density
at 20-fold higher magnification. (b) Ch4i region in aged nongrafted
monkey, showing fewer neurons that appear more pale than those of
nonaged subjects. Inset indicates lower neuronal and neurite density than
observed in nonaged monkey. (c) In contrast, p75-labeled neurons in
monkeyswithcontrolgraftsexhibitnoevidentmorphologicalresponseto
the adjacent graft. Neurons resemble those of nongrafted aged subjects
in extent of loss of neuronal labeling and neurite density. (d) Ch4i region
in aged NGF-grafted monkey shows restoration of neuronal and neuritic
density. NGF-secreting fibroblast graft (G) is dark because of dense
penetration by p75-labeled cholinergic axons. Many neuronal soma
orient in the direction of the NGF-secreting cell source and extend
axons into the graft. Inset demonstrates these changes at 20-fold higher
magnification.
p75 immunolabeling in aged and nonaged monkeys. (a) Ch4i
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The optical fractionator method (31) was used to conduct
stereology. Identical stereology parameters were used in all
quantification procedures by using sampling frames pseudoran-
domly generated from the NEUROZOOM stereology program (32)
or the STEREOINVESTIGATOR program (MicroBrightField,
Colchester, VT) for p75-immunolabeled and Nissl-stained neu-
rons, respectively: fraction (percent of area), 5%; counting frame
size x, 66.46 ?m; y, 53.73 ?m; section thickness, 40 ?m.
These parameters were chosen to minimize the coefficient of
error of the estimate while maximizing the efficiency of sampling
by using the nomogram of Gunderson (33).
Thestereologycomputerprogramscontrolledmovementfrom
onecountingframetothenextbymovingaLudlmotorizedstage
mounted on an Olympus (New Hyde Park, NY) Vanox AHBT-3
microscope. Ch4i neurons were counted by using a ?60 high
numerical aperture (1.40) oil objective (total magnification
?600).Immunolabeledcellswereincludedinthecountif(i)they
were p75 positive; (ii) the cell nucleus was within the counting
frame (or touching the inclusion boundary) but did not touch the
exclusion boundary; and (iii) the nucleus was best in focus within
the inclusion volume (i.e., the top 12.5% and bottom 12.5% were
excluded and the nucleus was best in focus within the volume
between the two exclusion volumes). Cells in Nissl-stained sec-
tions were counted if (i) they were hyperchromic and either
polygonal or fusiform in morphology, as previously described for
neurons in the Ch4i region (16); (ii) the cell nucleus was within
the counting frame (or touching the inclusion boundary) but did
not touch the exclusion boundary; and (iii) the nucleus was best
in focus within the inclusion volume. Thus, unbiased estimates of
neuronal numbers per subject were generated and compared
among groups. (Immunolabeling was noted to be uniform
through the z axis of sections from young and aged brains,
allowing accurate comparisons between subjects.)
p75?Choline Acetyltransferase (ChAT) Double Labeling and
Quantification. To determine whether changes in p75 immuno-
labeling were paralleled by changes in a more general cholinergic
marker, ChAT, double labeling for p75?ChAT was performed.
The ratio of p75?ChAT neurons in each subject was then
calculated, and comparisons were made between groups. Two
coronal sections per animal, chosen to correspond to points
exactly 1?3 of the distance through the rostral-caudal extent of
Ch4i and 2?3 of the distance through the rostral-to-caudal extent
of Ch4i, were processed for p75?ChAT double immunolabeling.
Doublelight-levelimmunocytochemistrywasperformedbyusing
the p75 monoclonal antibody (described above) and a polyclonal
ChAT antibody (Chemicon; 1:1,000 dilution). Primary antibody
incubation procedures were performed sequentially on consec-
utive days. After incubation in appropriate secondary antibodies,
p75 immunoreactive cells were visualized with Vector Red (Vec-
tor Laboratories) and ChAT immunoreactive cells were labeled
with Vector SG (Vector Laboratories). Vector Red produces a
red reaction product and Vector SG, a blue-gray reaction prod-
uct. Thus, double-labeled cells were readily distinguished by a
deep purple color reaction product (see Results). Stereological
sampling of single- and double-labeled cells with NEUROZOOM
software was performed by using the criteria that cells were (i)
p75 positive, ChAT positive, or both; (ii) the cell nucleus was
within the counting frame (or touching the inclusion boundary)
butdidnottouchtheexclusionboundary;and(iii)thenucleuswas
best in focus within the inclusion volume. Cells were counted as
singlelabeledforp75ifonlytheredreactionproductwaspresent,
singlelabeledforChATifonlytheblue-grayreactionproductwas
visiblewithinthesoma,anddoublelabeledifthecellswerepurple
(see Results). Numbers of p75-, ChAT-, and double-labeled cells
intheCh4iregionofeachsubjectwereexpressedasaratiooftotal
neuron numbers sampled.
All grafts were also labeled for acetylcholinesterase (AChE)
histochemistry as previously described (34, 35) to detect the
presence and persistence of cholinergic axonal penetration into
NGF-secreting cell grafts.
Quantification of Neuronal Area. Stereological methods were
used to determine differences in p75-immunolabeled neuronal
area between groups. Cells were included in the area quantifi-
cationiftheymetthemorphologicalcriteriadescribedabove.The
cycloid method was used to measure the cross-sectional area (36,
37). Briefly, a cycloid grid was overlaid on each sampled section
in the NEUROZOOM program. The computer mouse was used to
‘‘click’’onintersectionsbetweensampledprofiles(Ch4ineurons)
and cycloids. The NEUROZOOM program then calculated the
cross-sectional area for that neuron. Cycloid spacing was set at 5
?m.Quantificationwasperformedbyusinga?60highnumerical
aperture (1.40) oil objective. One hundred total neurons per
animal were randomly sampled by using the NEUROZOOM soft-
warefromeveryp75-labeledsectionthroughthefullextentofthe
Ch4i nucleus. The mean cross-sectional area for Ch4i neurons
was calculated for each animal, and means were averaged to
derive values for each group.
FIG. 2.
decline in the number (ANOVA P ? 0.05) and size (P ? 0.05) of p75-immunolabeled neurons compared with nonaged monkeys. Aged monkeys that
have received grafts of control fibroblasts that express the reporter gene ?-gal show losses in cellular markers identical to those observed in nongrafted
subjects. In contrast, aged recipients of NGF-secreting cell grafts show significant amelioration of losses in immunolabeled cell numbers and size. Post
hoc testing reveals significant losses only in the aged nongrafted and ?-gal-grafted groups (?).
Neuronalquantification.Quantificationofneuronalnumber(a)andsize(b)innonagedandagedmonkeys.Agedmonkeysexhibitasignificant
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Statistics.MultiplegroupcomparisonsweremadebyANOVA
with post hoc analysis by using Fisher’s least square difference.
Biological significance was established at the 95% confidence
level. Data are presented as mean ? SEM.
RESULTS
Normal Cell Numbers and Size. The number of cholinergic
neurons in the normal intermediate division of the Ch4 region in
four nonaged rhesus monkeys was quantified by using stereologi-
cal methods and was found to contain a mean of 45,700 ? 6,200
p75-immunolabeled neurons (Figs. 1 and 2). The majority of
these neurons also colocalized for ChAT: 93.9 ? 0.7% of p75-
immunolabeled neurons were labeled for ChAT (Fig. 3), consis-
tent with previous reports on colocalization of these two markers
(30). Mean somal area of Ch4i neurons immunolabeled for p75
was 501 ? 9 ?m2in nonaged subjects.
Nissl counts of all neurons in the Ch4i region were also
performedbyusingstereologicalmethods.Cells(60,300?3,400)
with neuronal morphology were present within the same ana-
tomicalboundariesinwhichp75-labeledneuronswerequantified
(Table 1). Thus, at least 74.7 ? 6.6% of neurons in Ch4i are
cholinergic.
Normal Aging Results in a Decline in Cell Number and Size.
Agingresultedinasignificantdeclineinboththenumberandsize
of neurons immunolabeled for p75 (Figs. 1, 2). The number of
p75-labeled neurons was significantly reduced in three nonoper-
ated aged monkeys by 43% compared with nonaged monkeys, to
26,000 ? 2,100 neurons (ANOVA P ? 0.05). The size of
remaining p75-labeled neurons also significantly declined by
10.4% in nonoperated aged monkeys, to 449 ? 9 ?m2(ANOVA
P ? 0.05). Thus, aging resulted in a significant reduction in the
number and size of basal forebrain cholinergic neurons labeled
for a specific marker of basal forebrain cholinergic neurons, the
p75 low-affinity neurotrophin receptor. The shrinkage in size of
remaining neurons suggested that cholinergic neurons were un-
dergoing a general state of atrophy rather than isolated down-
regulation of p75 expression. This general state of cholinergic
atrophy was confirmed by ChAT?p75 double immunolabeling,
which revealed that declines in p75 expression were paralleled by
declines in ChAT expression: in aged primates, 92.6 ? 1.2% of
p75-labeled neurons also labeled for ChAT. However, the num-
ber of Nissl-stained neurons did not significantly decline in aged
unoperated subjects compared with nonaged monkeys (57,200 ?
3,900 neurons, P ? 0.62; Table 1), indicating that reductions in
immunolabeledcellnumbersreflectedatrophyratherthandeath.
Reversibility of Age-Related Cholinergic Neuronal Atrophy by
NGF Gene Transfer. All NGF-grafted and ?-gal-grafted animals
showedsurvivingcellulartransplants(Fig.1candd).Age-related
declines in p75-labeled neuronal number and size were reversed
in the four recipients of NGF-secreting cell grafts (Figs. 1, 2).
Meanp75-immunolabeledneuronalnumberinagedrecipientsof
NGF-secreting cells was restored to within 7.7% of values in
nonaged subjects (42,200 ? 2,600 neurons), an amount that was
not significantly different from nonaged subjects on post hoc
analysis. Similarly, neuronal size in recipients of NGF-secreting
cells recovered to within 3.5% of measurements in nonaged
subjects (484 ? 9 ?m2), an amount that also did not differ from
nonaged subjects on post hoc analysis. The four aged recipients
of control ?-gal-secreting cell grafts exhibited significant declines
of 43.9% in neuronal number (25,700 ? 4,400) and 13% in
neuronal size (435 ? 19 ?m2) compared with nonaged monkeys,
amounts that were indistinguishable from findings in the unop-
erated aged subjects. These findings in the aged grafted-control
groupindicatethatthegraftingprocedureitselfneitherworsened
nor improved age-associated changes in neuronal parameters.
p75?ChAT double labeling revealed that 89.5 ? 0.5% of
neurons immunolabeled for p75 were also labeled for ChAT in
the NGF graft recipient group, compared with 92.6 ? 0.3% of
neurons in the control graft group. This finding supports the
observationthatNGFrestoresoverallcholinergicfunctionrather
thanmerelyup-regulatingp75expression:ifp75expressionalone
had been augmented by NGF grafts, an increase in the ratio of
p75toChATwouldoccur,ratherthantheslightdecreasethatwas
actually observed. Once again, Nissl counts in aged subjects
suggested that neurons in cholinergic brain regions were atrophic
but not dead: the number of Nissl-stained neurons in aged
NGF-treated animals (53,100 ? 2,400) and aged ?-gal-grafted
animals (55,000 ? 5,600) did not differ significantly from num-
bers in nonaged subjects (ANOVA ? 0.62). Collectively, these
data indicate that the stage of aging examined in this study is
associated with neuronal atrophy, and that cellularly delivered
neurotrophic factors ameliorate this atrophy.
Cholinergic Sprouting Toward the Intraparenchymal NGF
Source. Cholinergic neurons in aged monkeys exhibited somal
orientation toward NGF-secreting grafts and extended p75-
labeled axons into all of these grafts (Fig. 1c). This neurotropic
FIG. 3.
the cholinergic basal forebrain is single labeled for p75. (b) A different
neuroninthesamehistologicalsliceissinglelabeledforChAT.(c)Athird
neuron in the same histological slice is double labeled for p75 and ChAT.
Bar ? 20 ?m in a–c.
Technique of double labeling for p75?ChAT. (a) A neuron in
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response was not evident in subjects that received control grafts
(Fig. 1d). Thus, a parenchymal NGF source elicits cholinergic
process outgrowth in aged subjects in addition to ameliorating
somal atrophy. Grafts of genetically modified cells were identi-
fiable in living monkeys on postoperative MRI scans (Fig. 4).
Duration of in Vivo Transgene Expression. Eight months after
in vivo grafting, a freshly dissected NGF graft and surrounding
host tissue contained 25 ng human NGF?gm tissue, exceeding by
25-fold the highest physiological levels of NGF production in the
cortex. Fresh dissection of a control graft revealed no detectable
humanNGF.Inaddition,humanNGF-secretinggraftsinallfour
of the aged NGF-grafted subjects were densely penetrated by
cholinergic axons on AChE labeling, suggesting that NGF pro-
duction persisted through the 3-mo experimental period in these
subjects (data not shown). Control grafts were rarely penetrated
by AChE-labeled axons.
DISCUSSION
Findings of the present study indicate that aging in primates is
associated with extensive atrophy of a population of subcortical
neurons that modulate cortical activity. The normal number of
immunolabeled cholinergic cells in the intermediate division of
theprimatebasalforebraincholinergiccomplexis45,700?6,200
neurons, and the normal size of these neurons is 501 ? 9 ?m2.
Loss of immunolabeling for p75 occurs in 43% of cholinergic
neurons with aging, with parallel reductions in the transmitter
ChAT. Remaining cholinergic neurons exhibit a 10% shrinkage
in area. These changes in subcortical neurons contrast with
reportsofeitherlimitedornochangeincellnumberandsizewith
aging in cortical regions in primates, including the hippocampus,
entorhinal cortex, presubiculum, and frontal cortex (e.g., refs. 1,
4, 6–9). However, these age-related changes in subcortical cho-
linergicneuronsrepresentatrophyratherthandeath,becausethey
are reversible by delivery of the NGF gene at the moderate stage
of aging represented by this study.
Interestingly, a recently published stereological study also
found significant age-related declines in a different subcortical
neuronal system in aged rhesus monkeys: 40% of nigral neurons
exhibit spontaneous degeneration with aging (38). Taken to-
gether with findings of the present study, the possibility emerges
that subcortical systems may be selectively vulnerable to aging.
Indeed, preliminary data from the same set of animals examined
in the present study indicate a lack of cell loss with aging in
entorhinal cortical layers II–III. The possibility that subcortical
neural systems are selectively vulnerable to aging compared with
corticalsystemscanbefurtherexploredbyexaminingage-related
changesinotherneuronalpopulationssuchasthelocuscoeruleus
and nucleus raphae. These studies are in progress.
Previousstudieshavereportedage-relatedreductionsinmark-
ersofbasalforebraincholinergicsystemsinrodents,althoughthe
extent of these reductions has varied considerably between stud-
ies.Someofthesedifferencesarelikelyattributabletoprescreen-
ing of the study population for cognitive performance, the types
ofmarkersutilizedtostudycellnumber(immunolabelingvs.Nissl
stains), methods of neuronal quantification (stereological vs.
nonstereological), and the species of rodent. For example, Ba ¨ck-
man et al. (39) reported cell shrinkage with aging in rodents, but
no reduction in number of p75-immunolabeled cholinergic neu-
rons. Other studies in aged rodents have reported losses of
immunolabeled basal forebrain cholinergic neurons, but only if
rats are divided into ‘‘aged-impaired’’ and ‘‘aged-unimpaired’’
groups on the basis of spatial memory performance. Under such
circumstances, aged-impaired subjects exhibit reductions in num-
bers of AChE-stained, ChAT-immunolabeled, or p75-immuno-
labeled neurons (20, 40, 41), and these reductions vary in extent
from 16% (40) to 40% (20). On the other hand, two recent
stereological studies of the rodent basal forebrain (42, 43) have
reported cell atrophy with aging that more closely approximates
findings of the present primate study. Smith and Booze (42)
reported a 30% reduction in the number of ChAT-immunola-
beled and Nissl-stained neurons in the basal forebrain of rodents
that were not prescreened for spatial memory performance,
whereas Martinez-Serrano et al. reported a 40% reduction in the
number of p75-labeled cholinergic neurons in aged rodents that
were preselected for impaired spatial memory function (44). The
extent of cholinergic neuronal atrophy in aged primates observed
inthepresentexperimentismostconsistentwiththemoresevere
reports of atrophy in rodents, because (i) primate reductions in
FIG. 4.
cholinergic nuclei are visible on MRI as circular regions of attenuated signal (arrows). (b) The same slice after histological processing reveals healthy
graft (G) tissue that is robustly penetrated by p75-labeled cholinergic axons. Bar ? 450 ?m.
Cell grafts can be visualized and monitored in the living primate. (a) Grafts of NGF-expressing cells in bilateral basal forebrain
Table 1.
Ch4i region
Stereological quantification of Nissl-stained neurons in
Group
No. Nissl-stained
neurons (SEM)
Nonaged
Aged intact
Aged ?-gal grafted
Aged NGF grafted
60,300 ? 3,400
57,200 ? 3,900
55,000 ? 5,600
53,100 ? 2,400
ANOVA P ? 0.62
Neurobiology: Smith et al.Proc. Natl. Acad. Sci. USA 96 (1999) 10897