The authors have declared that no competing interests exist.
Conceived and designed the experiments: AJK DJ. Performed the experiments: DJ VZ CIFJ PJD MPCM A. Hafkemeijer ALJ CLMN. Analyzed the data: DJ VZ CIFJ ALJ A. Hafkemeijer CLMN AV JJA. Contributed reagents/materials/analysis tools: AJK A. Heerschap PJD AV JJA. Wrote the paper: DJ AJK.
Proton magnetic resonance spectroscopy (1H MRS) is a valuable tool in Alzheimer’s disease research, investigating the functional integrity of the brain
The cause of Alzheimer’s disease (AD) is still largely unknown despite many years of extensive research. Since AD is characterized by the presence of neurofibrillary tangles and amyloid-β (Aβ) containing aggregates, it has been suggested that the Aβ peptide is a major contributor to the neurodegenerative processes in AD
Besides Aβ, several other potential causal mechanisms have been proposed since the discovery of AD. Large epidemiological studies have revealed that many risk factors for AD are vascular-related, causing chronic cerebral hypoperfusion and cerebrovascular pathology
Rather than one sole mechanism, it is much more likely that AD is a multifactorial disease, caused by a combination of these factors, compromising the functional integrity of the brain. One method of determining the functional integrity of the brain, or specific brain regions, is to examine their metabolism by using proton magnetic resonance spectroscopy (1H MRS). 1H MRS allows the non-invasive
Similar to AD patients, the most consistent finding in transgenic animal models is a reduction of NAA, although this was found to occur at different ages in different transgenic species apparently depending on the interplay of mouse strain, transgene and disease progression
The present longitudinal study set out to characterize the neurochemical profile of the hippocampus, measured by 1H MRS, in the brains of AβPPswe-PS1dE9 and wild-type mice at 8 and 12 months of age. Furthermore, we wanted to determine whether alterations in hippocampal metabolite levels coincided with behavioral changes, cognitive decline and neuropathological features, to gain a better understanding of the underlying neurodegenerative processes. Moreover, the extracellular amyloid-β plaque load, TBS-T soluble Aβ levels and high-molecular weight Aβ aggregate levels were determined in the brains of the 12-month-old AβPP-PS1 mice used in the present study
The experiments were performed according to Dutch federal regulations for animal protection and were approved by Veterinary Authority of the Radboud University Nijmegen Medical Centre (Permit Number: RU-DEC2008-126). All efforts were made to minimize suffering of the animals.
The AβPPswe-PS1dE9 founders were obtained from Johns Hopkins University, Baltimore, MD, USA (D. Borchelt and J. Jankowsky, Dept. of Pathology) and a colony was established at the Radboud University Nijmegen Medical Centre, the Netherlands. In short, mice were created by co-injection of chimeric mouse/human AβPPswe (mouse AβPP695 harboring a human Aβ domain and mutations K595N and M596L linked to Swedish familial AD pedigrees) and human PS1dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter elements. The two transfected genes co-integrate and co-segregate as a single locus
Male transgenic AβPP-PS1 mice and their wild-type littermates underwent behavioral testing and MRI measurements at 8 months of age, and again at 12 months of age. In total 25 mice were used: at 8 months of age, 15 wild-type and 10 AβPP-PS1 mice, and at 12 months of age, 9 wild-type and 7 AβPP-PS1 mice. Due to some technical problems during the experiments, not all mice could be used for the statistical analyses for each measure. For example, some mice were excluded from further analyses of the 1H MRS data, since the spectra obtained did not meet the inclusion criteria. The body weights of the mice were determined one day before the start of the behavioral tests, and again on the day of the MRI measurements.
Behavioral testing was performed in the following order: First open field, followed by Morris water maze (MWM), and finally the reversal MWM (rMWM). All testing sessions were performed during the light phase (between 9 a.m. and 5 p.m.) and were recorded for computer-assisted analysis using Noldus Ethovision 3.1 software (Noldus Information Technology B.V., Wageningen, the Netherlands). All behavioral testing was performed in the same room, homogenously illuminated by normal fluorescent room light at 60 lux.
To analyze explorative and anxiety-related behavior, mice were placed individually in the center of a square open field (50×50×50 cm) with white Plexiglas walls, and were observed for 30 minutes. The duration (seconds) of walking, wall leaning, rearing, sitting and grooming were scored and later analyzed in three blocks of 10 minutes. These open field parameters were defined as described previously
To investigate spatial learning abilities, mice were tested in the Morris water maze (MWM). In short, mice were placed in a pool (104 cm diameter) filled with water (21–22°C; made opaque by the addition of milk powder) at different starting positions and trained to find a submerged platform by using distant visual cues in the room. These spatial cues were present on the four walls of the test room at a distance of 0.5 meter. The 8 cm diameter round platform was submerged 1 cm below the water surface and placed in the middle of the northeast (NE) quadrant at a distance of 26 cm from the wall. During all trials the researcher was present and always located at the same location in the room (close to the SW quadrant).
Acquisition (spatial learning): Mice were trained to find the location of the submerged escape platform in 4 acquisition trials (maximal swimming time 120 s; 30 s on the platform; inter-trial interval 60 min) per day during 4 consecutive days. The latency time (s) to find the hidden platform was scored. Starting positions during the 4 trails/day were: S, N, E, W. After the 2 min swim the mice were placed back in their home cage, and a paper towel was available inside the cage for additional drying.
Probe (spatial memory): All mice performed a single probe trial 60 min after the last trial on day 4, in which the platform was removed from the swimming pool. Mice were allowed to swim for 60 s and the time spent swimming and searching in the NE quadrant (where the platform had been located), total swimming distance, mean velocity and total number of platform crossings (at the former platform location) were recorded.
Four days after the standard MWM probe trial, a simplified reversal MWM
MR measurements were performed on a 7T/300 mm horizontal-bore MR spectrometer interfaced to a ClinScan console (Bruker Biospin, Ettlingen, Germany). An integrated circular polarized transmit 1H volume coil (200 mm/154 mm outer/inner diameter) combined with a circular polarized receive 1H surface coil was used for signal reception. During the experiments, mice were anesthetized with 2% isoflurane (Abott, Cham, Switzerland) in a mixture of N2O and oxygen (1∶2) through a nose cone. Mice were placed in a stereotactic holder to prevent unwanted movement during the scanning. Body temperature was maintained at a physiological level with heated airflow and was monitored with a rectal optical temperature probe. Respiration of the animal was monitored using a pneumatic cushion respiratory monitoring system (Small Animal Instruments Inc, NY, USA). Multislice turbo spin echo images in the coronal, transversal and longitudinal orientation were acquired to visualize the anatomy and the morphology of the mouse brain structures. Imaging parameters were: FOV = 25×25 mm, matrix size = 256×256, slice thickness = 0.5 mm, TE = 46 ms, and TR = 3500 ms.
Metabolite concentrations in the hippocampus were determined using proton magnetic resonance spectroscopy (1H MRS) with a single voxel technique. The spectroscopic volume of interest (VOI) of 1.0×1.0×1.6 mm was positioned in the hippocampus (
Quantification of the metabolite concentration was performed using a the Linear Combination (LC) model software package (LCModel™, S. Provencher, Oakville, Canada). The quantification algorithm of LCModel™ applies linear combinations of model spectra to calculate the best fit of the experimental spectrum. The model spectra (dataset of prior knowledge) are calibrated to match the magnetic field strength, sequence type and sequence parameters used for data acquisition. The final analysis is performed in the frequency domain with raw data (free induction decay (FID)) as the input. The unsuppressed water spectrum was used to estimate the absolute concentration of the metabolites, of which simulated model spectra, generated by NMRSIM™ (Bruker Biospin, Ettlingen, Germany) were taken into the analysis.
The criteria to select reliable metabolite concentrations were based on the Cramér-Rao lower bounds (CRLB), which are estimates of the S.D. of the fit for each metabolite
Seven metabolites fulfilled the criteria: choline+glycerophosphocholine+phosphocholine (tCho), creatine+phosphocreatine (tCre), glutamate (Glu), glutamine+glutamate (Glx),
Directly following the MR measurements at 12 months of age, mice were sacrificed by cervical dislocation, and subsequently brains were removed from the skull after decapitation. Brains were weighed and cut mid sagittal for immunohistochemistry and biochemistry. One hemisphere was snap frozen in liquid nitrogen and then stored at −80°C, before further biochemical processing. The other hemisphere was immersion fixated in 4% paraformaldehyde for 24 hours, and subsequently stored in 0.1 M phosphate buffered saline (PBS, pH = 7.3) with 1% sodium azide at 4°C. Before cutting, the brain tissue was cryoprotected by immersion in 30% sucrose in 0.1 M phosphate buffer (PB, pH = 7.3). Six series of 40 µm coronal sections were cut through the brain using a sliding microtome (Microm HM 440 E, Walldorf, Germany). For every staining, one complete series with 240 µm distance between the sections was used. Immunohistochemistry was performed using standard free-floating labeling procedures
Presynaptic boutons were visualized with anti-synaptophysin antibody (1∶20,000; monoclonal rabbit anti-synaptophysin clone EP1098Y, Abcam Inc., Cambridge, UK) using one subseries of brain sections per animal. Synaptophysin is localized in small synaptic vesicles of the presynaptic terminal and functions in the regulation of exocytosis
Immature neurons were visualized with anti-doublecortin antibody (1∶3000; polyclonal goat anti-doublecortin (C18): sc-8066, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using one subseries of brain sections per animal. Doublecortin is a microtubule-associated protein that is exclusively found in somata and processes of migrating and differentiating neurons
Quantification of presynaptic boutons and doublecortin-positive immature neurons was performed using a Zeiss Axioskop microscope, equipped with hardware and software from Microbrightfield (Williston, VT, USA). Appropriate sections were digitized and photomicrographed using a computer-assisted analysis system, Stereo Investigator (Microbrightfield). Brain regions were based on the mouse brain atlas of Franklin and Paxinos
To determine the amount of synaptophysin-immunoreactive presynaptic boutons (SIPBs) in the hippocampus and cortical regions, appropriate sections were digitized and photomicrographed using an 100× oil immersion objective. SIPBs were analyzed in the hippocampal regions stratum radiatum (SR) of the cornu ammonis (CA)1 area, stratum lucidum (SL) of the CA3 area, inner molecular layer (IML) and outer molecular layer (OML) of the dentate gyrus (DG), and in the cortical regions prelimbic area (PLA) and anterior cingulate gyrus (ACg). These regions were chosen because of their large amyloid load in AD patients and transgenic mouse models for AD and their importance in learning and memory
A: In the hippocampus SIPBs were analyzed in the inner (yellow) and outer (red) molecular layer of the dentate gyrus, stratum radiatum (SR) of the CA1 area (blue), and stratum lucidum (SL) of the CA3 area (green). Scale bar = 200 µm. B: SIPBs were quantified with an 100× objective using image analysis from digitized photomicrographs of the synaptophysin-immunoreactivity. Scale bar = 10 µm.
For the assessment of immature neurons in the hippocampus as a measure for neurogenesis (
A: Image taken using an 10× objective. Scale bar = 100 µm. B: Image taken with an 40× objective. Scale bar = 50 µm.
The extracellular Aβ load, soluble Aβ levels and insoluble high-molecular weight Aβ aggregate levels were determined in the brains of the 12-month-old AβPP-PS1 mice, as has been described elsewhere
In short, Aβ deposits were visualized using WO-2 antibody (1∶20,000, mouse anti-human Aβ4–10, a kind gift of K. Beyreuther, University of Heidelberg, Germany) using one subseries of brain sections per animal. Donkey anti-mouse biotin (1∶1500, Jackson ImmunoResearch was used as secondary antibody. Extracellular Aβ plaque load was quantified in the hippocampus, prelimbic area (PLA) and anterior cingulate gyrus (ACg) with a computer-assisted analysis system (Stereo Investigator, Microbrightfield) using Cavalieri’s probe (
A: The Aβ plaque load was quantified in the prelimbic area at level +1.98 up to +1.78 anterior to bregma, B: in the anterior cingulate gyrus at level +1.10 up to +0.86 anterior to bregma, and C: in the dentate gyrus (DG), CA1 and CA3 areas of the hippocampus at level −2.18 up to −2.46 posterior to bregma, using one appropriate section per animal. Images were taken using a 2.5× objective. Scale bar = 500 µm.
For biochemical Aβ analyses, frozen hemibrains were homogenized in Tris buffered saline with 1% Triton X-100 (TBS-T) plus protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) and centrifuged at 16,000
Data are expressed as mean±SEM and were analyzed with SPSS for windows 16.0 software (SPSS Inc. Chicago, IL, USA). The repeated measures ANOVA was used for the open field parameters (with the repeated measure: time) and the acquisition phase of the MWM and rMWM (with the repeated measure: trial block), followed by a Bonferroni post hoc to analyze possible interactions between time/trial block and genotype. If interactions between time/trial block and genotype (between-group-factors) were present, the data were split for the concerning factor and thereafter analyzed again with the repeated measures ANOVA. Multivariate ANOVA’s were conducted with between group factor: genotype, to analyze possible differences between wild-type and AβPP-PS1 mice in the probe trials of the MWM and rMWM, the body weight, brain weight, metabolite concentrations, and the amount of SIPBs and immature neurons. Aging effects in the AβPP-PS1 mice are represented as relative values compared to the wild-type mice (set as 100%) of the corresponding age-group, and were analyzed with between group factor: age. Correlation analyses with the Aβ measures were performed using bivariate Pearson’s correlation method. For clarity reasons, F-values are not displayed. Statistical significance was set at
All mice were weighed one day before starting the behavioral test battery and again on the day of the MR measurements. Since body weights within the groups did not change significantly between those two time points, the mean weight was used for further statistical analyses. Both at 8 months and 12 months of age, AβPP-PS1 mice had a tendency towards a higher body weight than wild-type mice, but it did not reach statistical significance. At 8 months of age, overall mean body weight of the AβPP-PS1 mice was 29.79±0.65 g compared to 28.24±0.42 g of the wild-type mice (
Both relative and absolute brain weights were not affected by genotype (
During the 30 min observation at 8 months of age, both in wild-type and AβPP-PS1 mice the time spent walking (
Different open field parameters were measured within a 30 min period, and analyzed in three 10 min trial blocks. A and B: AβPP-PS1 mice (n = 5) did not differ from wild-type mice (n = 8) at 8 months of age (A), but spent slightly less time walking than wild-type mice at 12 months of age, # trend
8-month-old AβPP-PS1 mice were more active in the open field than wild-type mice:
AβPP-PS1 mice traveled a longer distance (
Like at 8 months of age, the time spent walking (
Compared to wild-type mice, 12-month-old AβPP-PS1 mice spent more time wall leaning (
Aging effects in the AβPP-PS1 mice were analyzed as relative values compared to the wild-type mice (set as 100%) of the corresponding age. With age, the time that AβPP-PS1 mice spent walking decreased, from 105.7%±3.8 at 8 months of age to 79.5%±7.1 at 12 months of age (
Altogether, these data show increased activity but decreased active exploration (rearing) in 8-month-old AβPP-PS1 mice compared to wild-type mice. This increased activity and decreased rearing disappear at 12 months of age, due to habituation to the open field and/or the older age of the animals. Instead, 12-month-old AβPP-PS1 mice show increased anxiety-related behavior compared to wild-type mice, as indicated by the increased time spent in the corners and decreased time spent in the center of the open field, and increased time spent wall leaning, which is a type of exploration but with an anxiety-related behavioral component
Both AβPP-PS1and wild-type mice showed a decrease in escape latency during training at 8 months of age (
Spatial learning was measured in a 4-day acquisition phase, by determining the latency to find a hidden platform in the NE quadrant. Spatial memory was tested in the probe phase in which the percentage of time spent in the target NE quadrant was measured and the total number of platform crossings (where formerly the platform had been located). A: Both 8-month-old wild-type (n = 14) and AβPP-PS1 mice (n = 10) showed a decrease in latency during training. Overall latencies tended to be higher in AβPP-PS1 mice, although it did not reach statistical significance, # trend
During the probe trial at 8 months of age, no differences were found between wild-type and AβPP-PS1 mice in the time spent in the platform quadrant (NE) (
Both wild-type and AβPP-PS1 animals learned to find the platform during acquisition at 12 months of age (
AβPP-PS1 mice spent less time in the NE target quadrant compared to their wild-type littermates at 12 months of age (
Aging effects in the AβPP-PS1 mice were analyzed as relative values compared to the wild-type mice (set as 100%) of the corresponding age. Escape latencies during training in the MWM decreased with age (
Both wild-type and AβPP-PS1 animals learned to find the new SW platform location during acquisition at 8 months of age (
Spatial learning with an extra episodic memory component was measured in a 2-day acquisition phase, by determining the latency to find a hidden platform in the SW quadrant. Spatial memory was tested in the probe phase in which the percentage of time spent in the target SW quadrant was measured and the total number of platform crossings (where formerly the platform had been located). A: Both 8-month-old wild-type (n = 14) and AβPP-PS1 mice (n = 10) showed a decrease in latency during training. Overall latencies did not differ between the genotypes. B: During the probe trial, the 8-month-old wild-type and AβPP-PS1 mice traveled a similar distance. C: No differences were observed between the 8-month-old mice in the percentage of time spent in the target SW quadrant, although only wild-type mice deviated from 25% chance performance level. D: No differences were observed between the 8-month-old mice in the frequency of platform crossings E: Only 12-month-old wild-type mice (n = 9) showed a decrease in latency during training. 12-month-old AβPP-PS1 mice (n = 7) did not improve their performance during acquisition. However, overall latencies did not differ between the genotypes. F: During the probe trial, the 12-month-old AβPP-PS1 mice traveled a slightly longer distance than wild-type animals, although it did not reach statistical significance, # trend
No differences were found between wild-type and AβPP-PS1 in the time spent in the SW quadrant during the probe trial at 8 months of age (
At 12 months of age, the wild-type animals showed a significant decrease in escape latency over time in the acquisition phase (
During the probe trial at 12 months of age, AβPP-PS1 mice tended to spent less time in the target SW quadrant than wild-type mice (
Aging effects in the AβPP-PS1 mice were analyzed as relative values compared to the wild-type mice (set as 100%) of the corresponding age. Escape latencies during training in the rMWM did not change with age and repetition of the rMWM test (
During the probe trial, the swim distance increased with age (
No differences were observed in the neurochemical profile of the hippocampus of 8-month-old wild-type and AβPP-PS1 mice (
A: Representative 1H MR spectra acquired from the hippocampus of a 12-month-old wild-type mouse. B: Representative 1H MR spectra acquired from the hippocampus of a 12-month-old AβPPswe-PS1dE9 transgenic mouse. Notice the decreased NAA peak in AβPP-PS1compared to wild-type. C: At 8 months of age, no differences between wild-type (n = 13) and AβPP-PS1 mice (n = 8) were observed in the hippocampal neurochemical profile. D: At 12 months of age, AβPP-PS1 mice (n = 4) had significantly lower concentrations of NAA than wild-type mice (n = 7), *
At 12 months of age, AβPP-PS1 mice had significantly lower concentrations of NAA (
Aging effects in the AβPP-PS1 mice were analyzed as relative values compared to the wild-type mice (set as 100%) of the corresponding age. With age, the concentration of NAA decreased from 94.7%±6.0 at 8 months of age to 65.1%±8.4 at 12 months of age (
At 12 months of age, there were no significant differences in the amount of synaptophysin-immunoreactive presynaptic boutons (SIPBs) between AβPP-PS1 and wild-type mice (
The amount of SIPBs were quantified in the hippocampal inner (IML) and outer molecular layer (OML) of the dentate gyrus, the stratum radiatum (SR) of the CA1 area, and the stratum lucidum (SL) of the CA3 area, and in the cortical prelimbic area (PLA) and anterior cingulate gyrus (ACg). No differences in the amount of SIPs between 12-month-old wild-type (n = 9) and AβPP-PS1 mice (n = 6) were observed in any region analyzed (
Correlation analyses with the extracellular amyloid-β deposition, TBS-T soluble Aβ levels and high-molecular weight Aβ aggregate levels found in the brains of 12-month-old AβPP-PS1 mice
No significant interactions were found between any of the Aβ measures and the open field data (
A significant negative correlation was found between the Aβ plaque load in the hippocampus and the tCho levels measured with 1H MRS (
A significant negative correlation was found between the TBS-T soluble Aβ42 levels and the amount of SIPBs in the PLA region (
Finally, we found a significant negative correlation between the amount of immature neurons and the level of high-molecular weight Aβ40 (
The present longitudinal study set out to characterize the neurochemical profile of the hippocampus, measured by 1H MRS, in the brains of AβPPSswe-PS1dE9 mice at 8 and 12 months of age as compared to age-matched wild-type littermates. Furthermore, we wanted to determine whether alterations in hippocampal metabolite levels coincided with behavioral changes, cognitive decline and neuropathological features, to gain a better understanding of the underlying neurodegenerative processes. Moreover, we determined the extracellular amyloid-β load, TBS-T soluble Aβ levels and high-molecular weight Aβ aggregate levels of the 12-month-old AβPP-PS1 mice in our laboratory
In agreement with previous results from our lab
In line with previous results from our lab, AβPP-PS1 mice also showed impaired performance in the MWM and rMWM both at 8 and 12 months of age as compared to wild-type littermates
Standard measures of performance in the MWM as used in the current study, such as escape latency during acquisition training, time spent in platform quadrant and the number of platform crossings during a probe trial, may depend on other factors than visuo-spatial learning ability and memory capacity alone. Longer escape latencies during spatial navigation may be caused by slower swim speed, although we do not expect this to be a confounding factor, since wild-type and AβPP-PS1 mice did not display any significant differences in swim speed during the probe trials at any age. Furthermore, several groups have reported no differences in swim speed during MWM training between C57BL6/J wild-type and AβPPswe-PS1dE9 mice at any age tested
This is in line with a study by O’Leary and Brown, in which the search strategies used by 16-month-old AβPP-PS1and wild-type mice during visuo-spatial navigation in the Barnes Maze were analyzed
Synaptic loss is a pathological hallmark of AD, and it is the best correlate of cognitive impairment
Recent studies suggest that synapse loss might not be an early event in the progression of AD, as a decrease in synapses is only seen in later stages of the disease, where especially tau pathology is more widespread
In transgenic animals models, significant reduction of NAA levels and elevation of
It has been demonstrated that AβPPswe-PS1dE9 mice may exhibit various neurobiological abnormalities in the hippocampus before 8 months of age. Such abnormalities include inflammatory processes involving clusters of activated microglia and astrocytes, and TNF-α expression
To summarize, in this paper we characterized the neurochemical profile of the hippocampus, measured by 1H MRS at 7 Tesla, in the brains of 8- and 12-month-old AβPPswe-PS1dE9 mice as compared to age-matched wild-type animals. Our results show that at 8 months of age no alterations in hippocampal metabolite levels could be detected, while behavioral changes and cognitive decline were present in the AβPP-PS1 mouse model. At 12 months of age, a decrease in hippocampal NAA levels, reflecting reduced neuronal integrity, correlated with more severe behavioral and cognitive impairment in AβPP-PS1 mice as compared to wild-type animals. Furthermore, correlation analyses suggest a possible role of Aβ in inflammatory processes, synaptic dysfunction and impaired neurogenesis.
1H MRS could potentially provide unique information about the underlying degenerative processes, because metabolite levels are sensitive to different
We would like to thank Henk Arnts and Janneke Mulders for their excellent care giving of our mice. The authors would also like to acknowledge Ilse Arnoldussen, Xiaotian Fang, Anne Rijpma and Maximilian Wiesmann for their laboratory work.