Magnetic resonance imaging showed no major changes in the brains of transgenic animals, although they tended to have enlarged lateral ventricles when compared to control animals.
The Tg transgenic rat line should prove a suitable model of early AD for advanced studies including serial cerebrospinal fluid sampling, electrophysiology, neuroimaging or
Bengt appelgren behavioural testing. This devastating brain disorder is characterized by progressive cognitive decline, where memory of recent facts, spatial orientation, attention and executive functions are ones of the first affected [ 1 ]. The pathological hallmarks of the disease are abnormal accumulations of extracellular amyloid plaques and intracellular neurofibrillary tangles NFT predominantly in the neocortex and hippocampus of AD patients.
Neurofibrillary tangles are composed mainly of paired helical filaments of a hyperphosphorylated form of the microtubule-associated protein tau.
Other features of AD neuropathology include synaptic and neuronal loss, as well as inflammatory processes. Mutations in "Bengt appelgren" genes, namely APP, presenilin 1 PS1 and presenilin 2 PS2are responsible up to one half of early onset cases of Bengt appelgren hereditary form of AD [ 2 ] which shares the neuropathological characteristics of the more common, sporadic form of the disease.
Since the identification of AD-causing mutations, numerous transgenic mouse models recapitulating many features of the disease have been developed reviewed in [ 4 ].
These models have proven very valuable in dissecting the mechanisms involved in AD but there is still a need for a model better suited for more advanced studies, including serial cerebrospinal fluid sampling and complex behavioural testing. For these purposes, the rat offers several advantages Bengt appelgren the mouse both due to its larger size and more complex behaviour repertoire [ 5 ]. However, to date, only a few transgenic rat models of AD have been reported.
Ina transgenic rat expressing human APP was reported, but no plaques or cognitive deficits were seen with age [ 11 ]. "Bengt appelgren," the generation of other multi-transgenic Sprague-Dawley rat lines was reported. Although the triple transgenic model displays extensive amyloid pathology, gross overexpression of multiple mutant transgenes may hinder deciphering the molecular steps leading to pathology.
We have previously reported the establishment of the Tg transgenic rat [ 13 ] expressing APP with the Swedish AD mutation [ 14 — 15 ]. Here, we report on behavioural testing, in vivo magnetic resonance imaging MRI and assessment of biochemical markers significance for AD in the Tg model. Male homozygous Tg rats expressing human APP with the Swedish APP mutation under the control of the ubiquitin promoter and non-transgenic Sprague-Dawley control rats were used in all studies.
One week before behavioural testing the rats were handled daily to habituate with the experimenter. Six Tg animals and seven control animals at the age of 9 months were used for the behavioural studies. Five animals from each of
Bengt appelgren groups were later imaged by MRI at the age of 11 months. Additionally, five Tg and five age-matched control animals, 16 to 20 months old, were used for protein analysis by western blotting.
The rats were placed individually in the centre of the arena, and their movements recorded for Bengt appelgren min. The behavioural parameters analysed were the distance moved and mean velocity of horizontal movement as well as rearing defined as standing on hind limbs with the forelimbs in the air or against the wall of the arena in peripheral and central zones of the testing arena, respectively. This test was used in order to assess spatial learning and memory in the Tg rats.
Briefly, the animal is released into a round swimming pool from randomly chosen positions will naturally seek to escape the water. During the acquisition phase Bengt appelgren location of a hidden platform is learned by referring to visual
Bengt appelgren placed around the room [ 16 ].
For the current experiments the circular pool was made of grey PVC cm in diameter and its walls were 50 cm high. Distal visual cues consisted of several wall posters, approximately 50—75 cm in size, cupboards and tracking equipment located in the testing room. One day before the start of the acquisition, the animals were habituated to the procedure during a sec. Rats were transferred to the testing room in a non-transparent cage to avoid visual orientation prior to release into the pool.
Release points were balanced across four symmetrical positions on the pool perimeter. Bengt appelgren position of the hidden platform remained fixed during the acquisition phase which lasted 5 days.
Four trials of 60 sec. When a rat did not find the platform within 60 sec. In order to assess spatial memory, a probe trial was performed on the last day of the experiment about 40 min. The platform was removed from the pool and the animal allowed Bengt appelgren swim for 60 sec.
A single visible platform test was performed 2 days later. For this test the platform was raised above the water surface, positioned in the centre of the circular pool and marked with a yellow flag. Rat behaviour in the Morris water maze test was recorded by an automated video-tracking system Ethovision. MRI was performed with a 4.
A volume coil Bruker with mm inner diameter was used for excitation and signal detection. Three-dimensional images were obtained using inversion recovery spin echo sequence rapid acquisition with relaxation enhancement RARE [ 17 ].
Total acquisition time was 1 hr 28 min.
Bengt appelgren of view FOV for the 3D was 1. Image reconstruction resulted in a resolution of 0. The rats were anesthetized with 1.
The rats were then positioned in supine position and the head fixed to an acrylic rig.
Three-dimensional MRI images were analysed using Amira 3. The different brain structures were segmented in accordance with the G. Watson atlas [ 18 ]. Choice of the first slice used for quantification was semi-random, as Bengt appelgren was always the MRI slice where the given structure was first visible in the direction from anterior to posterior for coronal slices and from dorsal to ventral for horizontal slices. The lateral ventricles were measured in each second contiguous coronal slice.
Hippocampi were measured in all slices depicting it. The borders of the hippocampus were checked on both horizontal and coronal slices to ensure accuracy and calculations
Bengt appelgren both orthogonal planes were averaged. The total volumes were calculated "Bengt appelgren" multiplying the estimated area by the slice thickness 0. All measurements were performed twice by an investigator, who was blind with regards to genotype.
The two measurements were performed with an interval of 3 weeks and the mean value calculated. Homogenates were then gently mixed for 30 min. Protein content was determined using BCA protein assay Pierce. Western blotting was performed as previously described [ 20 ]. Primary antibodies used in this study were: The secondary antibodies purchased from Amersham, Uppsala, Sweden Bengt appelgren diluted 1: Optical density of protein bands were quantified using ImageJ software.
Samples were incubated 30 min. Samples were diluted 1: For repeatedly measured parameters group differences were analysed by
Bengt appelgren measures ANOVA, unless stated otherwise. Non-parametric Mann—Whitney U-tests were used for comparing protein levels. Such a weight difference is also seen in several APP transgenic mouse models [ 21 ].
We have not observed any weight differences in younger animals, up to 4 months of age. There were no significant differences between the spontaneous locomotor activity of transgenic
Bengt appelgren control animals over the min.
There were also no differences in the frequency, duration spent and Bengt appelgren moved in the central and peripheral zones of the arena data not shown. Over the whole 60 min.
However, during the first 40 min. The analysis of behaviour during a shorter than 60 min. Spontaneous behaviour of Tg transgenic and control animals during 60 min. Significant difference was found in rearing activity of transgenic and control rats during the first 40 Bengt appelgren. B Locomotor distance traversed activity.
Neither swim speed, thigmotaxis nor body weight alone accounted for the impaired performance of the transgenic animals. Spatial learning in Tg transgenic and control animals, assessed by Morris water maze. On the last of acquisition, after the last acquisition trial, the escape platform was removed and the animals were allowed to freely search the pool. Only the control rats preferentially swam in the pool quadrant where the platform was previously placed Fig.
The Tg rats showed no preference in searching the pool quadrants.
Preference for pool quadrant during probe trial in Morris water maze. No significant visual differences could be detected between the groups during the visible platform test as all rats swam similarly well to the raised platform. Two months after completion of behavioural testing, at the age of 11 months, five animals from each group were investigated by MRI. Volumetric analysis of the "Bengt appelgren" scans showed no statistically significant group differences between absolute volumes of brain, hippocampus or ventricles in the Tg and control animals Table 2.
These two rats did not perform differently than the other transgenics in the behavioural tests. No hippocampal or cortical atrophy was seen in the transgenic animals of this age; however, preliminary data suggest enlargement of lateral ventricles and a thinning of the cortex in older "Bengt appelgren" animals as compared to controls 16 months old, data not shown.
Bengt appelgren only a limited number of older animals have been analysed three in each group additional animals need to be investigated before any definite conclusions can be drawn.
The expression of human APP protein in the brains of all Tg rats was confirmed in this study by Western blot of brain homogenates using the human-specific 6E10 antibody. We have previously reported an apparent increase in the phosphorylation of tau in old Tg animals as Bengt appelgren by the PHF-1 antibody [ 13 ].
To follow up on this observation, in the present study the levels of different forms of tau were analysed by Western blots of brain homogenates. Overall, the transgenic animals tended to show higher phosphorylation at the PHF-1 recognition sites, but the differences were not statistically significant. No differences in the levels of synaptic marker synaptophysin or neuronal marker NeuN could be detected by Western blotting in transgenic and control animals.
Many transgenic mouse models of AD show behavioural impairments that are attributed to perturbations in components of the amyloid-processing pathway. The only exception is the triple-transgenic homozygous rat carrying two mutant APP constructs and one mutated PS1, which shows extensive extracellular amyloid deposits [ 12 ]. This rat line has however been shown to be prone to premature death due to such health
Bengt appelgren as chronic kidney disease, hypertension and immunosuppression [ 24 ], which sets limitations on its use as an AD research model.
This indicates that removal of plaques is not sufficient to prevent progression of the clinical symptoms [ 27 ]. Whereas the TgAPPswe Fisher rats showed attenuated age-dependent memory decline, our 9-month-old Tg transgenic animals exhibit learning and memory deficits in a spatial navigation task and altered exploratory behaviour in the open-field test.
s Bengt. Bengtsson. Sandberg YY: . Bengt Lars-söner. (?) SA Magnus. Jönsson Appelgren. Toll. Andersson.
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