1. Holly M. Brothers & Gary L. Wenk
The role of neuroinflammation in the determination of regional and neuronal vulnerability in Alzheimer’s disease
Holly M. Brothers & Gary L. Wenk
Department of Psychology
The Ohio State University
Nothing to Disclose

2. Studies in our laboratory demonstrate that regional and cellular vulnerability in the brain are influenced by an interaction between: neuroinflammation, duration of inflammation, and gender. 1 1

3. What initiates neuroinflammation? What are its consequences?
Microglial activation is an early event in the pathogenesis of AD that appears in brain regions that later show the greatest degree of atrophy.
Mutant Proteins
Synaptic Loss/ Neuronal Death
Neuroinflammaiton is:
Common to all neurodegenerative diseases
Sufficient to cause dementia (dementia pugilistica)
Epidemiology suggests that reducing neuroinflammaiton (NSAIDs) early in this process is protective
Not sporatic, is initiated and perpetuated by mutant proteins associated with AD as well as cell death
Most importantly, the distribution of activated immune cells is correlated with those regions that degenerate the most.
A number of risk factors for AD have been identified, including age, ApoE4 genotype, Down’s syndrome, and history of head injury.
A number of pathophysiologic abnormalities have also been identified in AD. The histopathologic hallmarks of AD include the deposition of -amyloid in senile plaques and blood vessel walls as well as the presence of neurofibrillary tangles in the brain.
In addition, the levels of several neurotransmitters are greatly reduced. Depletion of ACh represents the most important event. The dramatic and global reduction of ACh and other important neurotransmitters is almost certainly responsible for the broad and profound clinical manifestations of AD (Samuels and Davis, 1997).
Biochemical evidence suggests that inflammation and/or the deleterious effects of free radicals may also play a role in the pathogenesis of AD (Rogers, 1995; Rogers et al., 1992; Smith et al., 1991). Because oestrogen is important in the maintenance of cholinergic neurones, it is also believed that postmenopausal oestrogen deficiency may play a role in the disease (Gibbs and Aggarwal, 1998).
The leading approaches to AD therapy today are based on cholinergic mechanisms. Future therapies to alter disease course will target neuro-fibrillary tangle formation and -amyloid metabolism.
Dementia Symptoms
Inflammation
Cagnin et al., 2001

4. Chronic neuroinflammation model
Infusion of lipopolysaccharide (LPS, 0.15µl/hr, 6µg/day) into the IVth ventricle or basal forebrain
cannula tract
LPS
In order to study the effects of chronic neuroinflammaiton, we use a model created by Beatrice Hauss-Wzyneriac…
Hippocampus is cradled between the third ventricle on the inside and the lateral ventricles. SN, unlike hippocampus, is not in direct contact with a ventricle.
Lipopolysaccharide (LPS)- an endotoxin from the cell wall of gram-negative bacteria.
Osmotic minipump- slow, consistent drug release.

5. Regional microglia activation
LPS 21 days → activated microglia in the hippocampus and temporal lobe.
2 Days
aCSF + Sal, 4 weeks
21 Days
LPS + Sal, 4 weeks

6. Is there a pathological consequence?
Fewer cresyl violet stained cells counted by stereology in entorhinal cortex layers II and III of rats infused with LPS for 37 days 1 2 3 4 5 6 7 8 9 *
Total Number of Neurons (x104)
aCSF
LPS
Cresyl violet staining of neurons,
Does the paper say ‘large’ or define size anywhere?
Hauss-Wegrzyniak et al., 2002

7. Is there a pathological consequence?
Volumetric MRI analysis revealed that 42 days of LPS infusion caused a significant shrinkage of the temporal lobes and hippocampus and increase in the volume of the lateral ventricles.
aCSF
LPS
T2-weighed MRI images
Hippo and EC outlined in white (outlined in orange hidden behind).
Hauss-Wegrzyniak et al., 2000

8. Is there a functional consequence?
Impaired LTP induction in vivo in dentate gyrus of rats infused with LPS for 37 days
Hauss-Wegrzyniak et al., 2002

9. What underlies a loss of LTP?
Glutamate is necessary for LTP
Glutamate dysfunction disrupts LTP
Glutamate → NMDAR activation → Ca++ entry → Arc induction
Mg+
Glutamatergic neuron
Ca++
Arc
5 min
30 min
Under normal circumstances, when learning occurs, only about 2% of the neurons in the hippocampus are activated – this is called “sparse encoding.”
Arc
in DG
Rosi et al., 2005

10. Chronic neuroinflammation activates microglia (blue) and over-activates Arc (red)
a-CSF
Arc + hippocampal neurons:
aCSF: ~2%
LPS: ~4%
LPS
Double immunostaining for Arc protein (red) in granule cells and OX-6 protein (activated microglia, blue) in granule cells and hilus in the DG. (green marks nuclei and yellow results the nuclei of Arc positive cells)
Evidence of excessive Ca++ entry and supports idea of excessive glutamate
Rosi et al., 2005

11. What other regions are vulnerable to chronic neuroinflammation?
LPS infused into the nucleus basalis magnocellularis (14, 37, 74 or 112 days) produced a time-dependent, but not dose-dependent, decline in the number of Choline acetyltransferase (ChAT)-immunoreactive cells.
In frontal Cortex???
Willard et al., 1999

12. SN dopaminergic neurons are also vulnerable to chronic inflammation
LPS + Sal
aCSF + Sal a
7x basal levels of resting microglia
demonstrates a link between microglia activation and vulnerable regions, depending on disease state

13. Does Gender influence the response to neuroinflammation?
Female rats infused 42 days with LPS have ~1/2 the number of activated microglia as males.
Estrogen replacement did not protect. ‡
1600 ‡
1400
1200
1000
+ #
Figure 5. Semiquantitative determination of the number of OX-6-positive
microglia in the hippocampus, amygdala, lateral hypothalamus, zona
incerta, central thalamic nucleus, cingulate gyrus/retrosplenial cortex, piriform/
entorhinal cortex, and parafascicular cortex was made by using four
comparable regions from each rat from each group. The boundaries of the
regions were defined with reference to an atlas of the rat brain (Pellegrino
et al., 1979) and are shown in detail in Figure 1. Chronic lipopolysaccharide
(LPS) activated microglia, which was not reduced by estrogen (E2; a
mixture of 25% 17-estradiol and 75% cholesterol) replacement therapy.
Intact females receiving LPS infusion had significantly fewer activated
microglia. Oil was 100% cholesterol as vehicle. CSF cerebrospinal fluid
infusion. Asterisks indicate a significant difference compared with all
CSF-infused groups ( p .001); plus sign indicates a significant difference
compared with the LPS intact group ( p .001); pound sign indicates a
significant difference compared with LPS-infused ovariectomized rats
( p .02).
Number of Activated Microglia
800
600
400
200
aCSF LPS
aCSF LPS
aCSF LPS
Intact
OVX
OVX + Estrogen
Marriott et al., 2002

14. Neuroinflammation did not impair the performance of intact females
Microglia activation correlates with impaired water maze performance (r = 0.481, p<0.03)
Neuroinflammation did not impair the performance of intact females
1200
1000
800
Path Length (cm)
600
Figure 2. Performance of young female rats in the Morris water maze
task. In the absence of ovaries, chronic neuroinflammation impaired performance
(** p .002) and chronic estrogen therapy impaired performance
(* p .001), as compared with intact rats or CSF oil-treated rats.
The combined presence of these two conditions produced a significantly
greater impairment ( p .001) than either condition alone (i.e., as
compared with the CSF E2 and LPS oil groups). Oil was 100%
cholesterol as vehicle. CSF cerebrospinal fluid infusion; LPS lipopolysaccharide
infusion; E2 estrogen (a mixture of 25% 17-estradiol
and 75% cholesterol) treatment.
aCSF OVX
400
LPS
aCSF
200
Mean +
SEM 1 2 3 4
Day of Testing
Marriott et al., 2002

15. * In the absence of ovaries:
Chronic neuroinflammation impaired performance.
Chronic estrogen impaired performance.
Together, ovariectomy and LPS were synergistic.
1200
1000 +
LPS OVX + E2
LPS OVX
aCSF OVX + E2
aCSF OVX
LPS
aCSF
800
Path Length (cm) *
600
Microglia activation correlates with impaired performance (r = 0.481, p<0.03)
Neuroinflammation did not impair the performance of intact females
In the absence of ovaries:
Chronic neuroinflammation impaired performance.
Chronic estrogen impaired performance.
Together, ovariectomy and LPS were synergistic.
Figure 2. Performance of young female rats in the Morris water maze
task. In the absence of ovaries, chronic neuroinflammation impaired performance
(** p .002) and chronic estrogen therapy impaired performance
(* p .001), as compared with intact rats or CSF oil-treated rats.
The combined presence of these two conditions produced a significantly
greater impairment ( p .001) than either condition alone (i.e., as
compared with the CSF E2 and LPS oil groups). Oil was 100%
cholesterol as vehicle. CSF cerebrospinal fluid infusion; LPS lipopolysaccharide
infusion; E2 estrogen (a mixture of 25% 17-estradiol
and 75% cholesterol) treatment.
400
200
Mean +
SEM 1 2 3 4
Day of Testing
Marriott et al., 2002

16. Regional and cellular vulnerability is influenced by neuroinflammation, time and gender.
Distribution of activated microglia changes over time and concentrates within regions prone to neurodegeneration.
The loss of forebrain acetylcholine neurons and is time-dependent.
Chronic neuroinflammation is likely responsible for the selective vulnerability of specific neural systems and brain regions associated with age-related neurodegenerative changes.
Female rats show no neuroinflammation-induced cognitive impairment unless ovarian function is lost.
The pattern of activated microglia evolves over time, from being uniformly distributed during the first few days to being concentrated within temporal lobe and basal forebrain regions after four weeks of LPS infusion.
The loss of forebrain acetylcholine neurons is time-dependent.
In contrast to males, female rats demonstrate a unique pattern of microglia activation and show no cognitive impairment in the presence of chronic neuroinflammation. The loss of ovarian function impairs performance in females with neuroinflammation.
Chronic neuroinflammation is responsible for the selective vulnerability of specific neural systems and brain regions associated with age-related neurodegenerative changes.

17. Dr. Beatrice Hauss-Wegrzyniak
Dr. Gary L. Wenk
Dr. Beatrice Hauss-Wegrzyniak
Dr. Susanna Rosi
ICAD 2009
Dr. Lisa Marriott
Dr. Yannick Marchalant
Holly M. Brothers
Supported by NIA
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