MATERIALS AND METHODS - Ischemic Vulnerability in the Brain of an Alzheimer’s Mouse Model

3x TG-AD mice (n=8 ischemia/5 non-ischemia/1 positive control) and wild-type (WT) (C57BL/6J) mice (n=7 ischemia/5 non-ischemia) were used in this study.

Thirteen 14-month-old female mice carrying the APPSwe, tauP301L, and PS1M148V transgenes (3x TG-AD) were used. Twelve WT littermates served as controls. Mice had ad libitum access to food and water, were kept in cages maintained at humidity level of 55±10% and a constant temperature of 22±2°C, and maintained under 12-h/12-h light/dark cycles (lights on from 7:00 to 19:00). All animal protocols were approved by the Institutional Animal Management and Use Committee of Ajou University School of Medicine.

B. Surgery

Female 3xTG and C57BL/6J mice were anesthetized with 2% isoflurane and maintained at 1.5% isoflurane via a face mask. Rectal temperature was measured during surgery and maintained at 37°C on a heating pad. Short-term cerebral ischemia was induced by transient bilateral common carotid artery occlusion (tBCCAO). Briefly, the BCCAO was isolated and occluded with a microvascular clip.

Fifteen minutes following ischemia induction, the clips were removed from both arteries to allow blood recirculation.

C. Behavioral tests

1. Morris water maze test

The Morris water maze (MWM) was used to assess changes in cognition and behavior among groups. Three days after tBCCAO, the mice underwent the MWM test for a period of 5 days. The MWM pool was 2 m in diameter and filled with white water at a temperature of 25 ± 1°C and a depth of 25 cm. During capture training, a translucent acrylic platform (diameter 10 cm) was placed in the center of the northeast quadrant at a depth of 1.5 cm below the water surface. The pool had spatial clues and was located in the center of the experiment room, which was equipped with a video camera attached to the ceiling that was used to record the swimming path.

The mice were tested 3 times a day for 5 consecutive days. In each of the 3 tests, the mouse was gently released into the water, facing the tank wall, at four different starting positions that were equally spaced around the pool wall.

Mice were allowed 60 seconds to find the hidden platform.

Upon reaching the platform, the mouse was left for 10 seconds. If the mouse was unable to find the platform within 60 seconds, the training ended and the maximum score of 60 seconds was assigned. In such cases, the mouse was

guided by hand to the hidden platform, where it was allowed to remain for 15 seconds. The latency to escape to the hidden platform was recorded as a metric of spatial learning performance. In order to assess spatial memory, a probe test was performed 24 hours after the last training test. In this test, we removed the platform from the pool, and mice swam freely for 60 seconds before they were removed from the water.

2. Y maze test

The Y maze test was performed as described previously.

Spatial cognition was investigated using spontaneous alternation in a Y-type maze device, a horizontal maze consisting of three arms (40 cm in length, 3 cm in width, 12 cm in height), with the arms at an angle of 120° to each other. Animals were initially placed in the center of the maze, and the number of arm entries made in a period of 8 minutes were manually recorded for each animal.

Spontaneous alternation was defined as the consecutive entry into all 3 arms. The maze was thoroughly washed with water to remove any residual odor after the completion of a test before the next mouse was placed in the maze.

The percentage of alternations was defined as % = (number of alternations)/ (all entries in entry) × 100. The total number of arm entries was also used as an indicator of walking activity.

D. Tissue processing

After surgery, mice were anesthetized and transcardially perfused with 30 ml of 1X phosphate-buffered saline (PBS) followed by 30 ml of 4% paraformaldehyde (PFA).

Perfused brains were extracted, cut along the midline, post-fixed in 4% PFA for 24 hours, and dehydrated with 30% sucrose. The brain was followed by OCT processing, and 20-μm coronal sections were cut using a Leica cryostat.

The brain tissue was stored in a stock solution at 4°C.

E. Cresyl violet staining

Neuronal damage to the hippocampus was visualized by cresyl violet staining. Brain sections were mounted on coated slidesthat were allowed to dry at room temperature overnight. Brain sections were stained with 0.1% cresyl violet solution for 5 minutes. The stained brain slices were washed in distilled water. The brain slices were then dehydrated with ethanol, immersed in xylene for 5 minutes, and mounted on slides in a Vecta mounting solution.

F. Immunohistochemistry

Brain sections were mounted on coated slides and washed with 1X phosphate buffered saline for 5 minutes.

Endogenous peroxidase activity was inhibited by adding 0.3 % hydrogen peroxide for 5 minutes followed by 0.25%

Triton x-100 for 10 minutes. After washing the brain sections, they were blocked with 10% normal horse serum for 1 hour and allowed to react with primary antibodies (anti-Iba-1, 1:1000 dilution; Wako Chemicals GmbH, Neuss, Germany; anti-GFAP, 1:500 dilution; DAKO, Glostrup, Denmark; anti-active cleaved caspase-3, 1:20 dilution, Millipore, Darmstadt, Germany; anti-AT8, 1:100 dilution Thermo Fisher Scientific, Malvern, PA, USA; anti-APP-Y188, 1:750 dilution, Abcam, Princeton, NJ, USA) overnight at 4℃. Subsequently, brain sections were incubated with biotinylated secondary antibody (1:200 dilution, Vector Laboratories, Inc., Burlingame, CA, USA) for 2 hours and reacted with an avidin–biotin-complex reagent (ABC, Vector Laboratories, Inc., Burlingame, CA, USA) for 1 hour. Nickel-enhanced 3,3′-diaminobenzidine (DAB, Vector Laboratories, Inc., Burlingame, CA, USA) was used for visualization of immunoreactivity.

G. Immunofluorescence staining

Brain sections were mounted on coated slides and washed with 1X phosphate buffered saline for 5 minutes.

Endogenous peroxidase activity was inhibited by adding 0.3 % hydrogen peroxide for 5 minutes followed by 0.25%

Triton x-100 for 10 minutes. After washing the brain sections, they were blocked with 10% normal horse serum for 1 hour and incubated with Anti-APP-Y188 (1:750

dilution, Abcam, Princeton, NJ, USA) in combination with anti-NeuN (1:500 dilution, Millipore, Darmstadt, Germany) antibodies overnight for 4°C. Sections were then incubated with Alexa Fluor 488 (1:200 dilution, Invitrogen, Eugene, OR, USA) or Alexa 555 (1:500 dilution, Invitrogen) secondary antibody. Slides were embedded with a Vecta shield mounting solution (Vector Laboratories, Inc.) containing 4′,6-diamidino-2-phenylindole (DAPI).

H. Thioflavin S

Brain sections were mounted on coated slides and washed with 1X phosphate buffered saline. Brain sections were stained with 1% thioflavin-S (T1892, Sigma, St.

Louis, MO) solution for 5 minutes. Brain sections were then differentiated in 70% ethanol for 5 minutes and rinsed twice in dH2O. Slides were embedded with a Vecta shield mounting solution (Vector Laboratories, Inc.) containing 4′,6-diamidino-2-phenylindole (DAPI).

I. Statistical analyses

The data are presented as means ± standard error of the mean (S.E.M.) and statistically analyzed using a Student’s t-test in GraphPad’s Prism program. Differences were considered to be significant for P values <0.05.

III. RESULTS A. Behavioral testing after tBCCAO

The Morris water maze (MWM) test was used to assess the effect of tBCCAO on learning memory in the AD mice.

The TG mice showed no improvement in latency in the MWM test over time after exposure to ischemia, whereas the WT mice showed decreased latency as the trial proceeded, irrespective of exposure to ischemia (Fig. 1A).

In addition, in the ischemia group, TG mice differed significantly from WT mice in terms of the time spent in the platform zone after removal of the platform (P = 0.0123;

Fig. 1B). These results suggest that ischemia had a greater effect on learning memory in TG than in WT mice. To measure the willingness to explore the new environment, the behavior spontaneous alternation behaviors were measured using Y maze (Fig. 1C). However, there was no statistical difference in Y maze alteration tests (Fig. 1D).

Figure 1. Effect of tBCCAO on memory as measured by the Morris water maze test and spontaneous alternation test in the Y maze. A. The escape latency of all trials, performed over 5 days, was significantly different between the ischemia TG group vs. ischemia WT (**P = 0.0039, *P = 0.0215 vs. Ischemia WT). B. The platform zone-crossing time was recorded during a trial with the platform removed.

Ischemia TG mice spent a significantly shorter amount of time in the platform zone than did Ischemia WT mice (*P =

0.0123 vs. Ischemia WT). C. Y Maze. D. There were no significant changes between WT groups and TG groups.

B. Neuronal cell death

Staining of cresyl violet and caspase-3 was performed to confirm neuronal cell death due to ischemia. Neuronal cell death shown by cresyl violet staining was greater in the ischemic TG group compared to ischemic WT group (P

= 0.0135) (Fig. 2A-B). Apoptosis stained by the caspase-3 did not show a significant difference between ischemic TG and ischemic WT groups (Fig. 2C). In addition, apoptosis was greater in the ischemia TG group than in the ischemia WT group, although the results of the caspase-3-positive cell count confirmed only an increasing tendency in the ischemia TG group (Fig. 2D). This finding reveals that a greater extent of necrosis and apoptosis was induced in ischemia TG mice and that these mice are more vulnerable to ischemia.

Figure 2. Effect of tBCCAO on neuronal cell death.

A. Cresyl violet staining of ischemic WT and TG mouse brains. In non-ischemic status, neuronal cells in the hippocampus looks smaller in TG group compared to WT group. Magnification, 100×. B. Cell count of cresyl violet-positive neuronal cells in hippocampal CA1 between ischemia WT and TG groups with cerebral ischemia. TG mice showed significantly more neuronal cell death than WT mice. Data are presented as the mean ± standard error of the mean. *P = 0.0135 vs. ischemia WT. C. Cleaved caspase-3 immunoreactivity was increased in the

hippocampal CA3 region in the ischemia TG group. D. Cell count of the 3,3′-diaminobenzidine (DAB)-positive cleaved caspase-3-positive cells in the hippocampal CA3 region between the WT and TG groups with cerebral ischemia. There were no significant differences in the number of these cells across ischemia groups.

C. Changes in Alzheimer’s pathology 1. Amyloid plaques

The thioflavin S-positive signal was evaluated in the cortex, hippocampus, and amygdala region. Amyloid plaques were not obviously observed in 14-month-old TG mice with or without cerebral ischemia (Fig. 3A). However, the thioflavin S-positive signal reflected the extracellular accumulation of amyloid aggregates in the amygdala of 18-month-old mice. (Fig. 3B).

Figure 3. Amyloid plaques’ thioflavin S expression in TG. A.

Amyloid plaques were not seen in 14-month-old TG mice.

B. Amyloid plaques were observed in the amygdala of 18-month-old TG mice. Magnification, 100×.

2. Amyloid precursor protein

The APP (Y188) signal was evaluated in the cortex, amygdala, and hippocampal CA1 regions. Positive signals were obtained in both TG mice but not observed in both WT mice. In the ischemia group in particular, atrophy was observed in the ischemic lesion (Fig. 4A-C). In the ischemia TG group, the intensity of Y188 staining tended to be reduced, but the number of Y188-positive cells in hippocampal CA1 was not significantly different between the control and ischemia groups (Fig. 4D). Double-staining for Y188 and NeuN confirmed that the Y188 signal was derived from neuronal amyloid (Fig. 4E).

Figure 4. Amyloid precursor Y188 expression. A-C. The amyloid precursor Y188 was expressed in TG mouse brains.

A. Cortex B. Amygdala C. Hippocampal CA1. D. There were no significant differences in the number of Y188-positive cells between control and ischemia TG groups’ hippocampal CA1. E. Merged images of Y188 and NeuN immunostaining confirms that Y188 staining reflects neuronal amyloid.

Magnification, 400×.

3. Tau protein

Staining with AT8 allowed the observation of tau protein.

Tau-positive signals were evaluated in the cortex and hippocampus. Positive cells increased in the ischemia WT group compared with the control WT group. The ischemia TG group also showed an increase in positive cells compared with the control group (Fig. 5A-B). The density of control/ischemia TG was measured, and the density of ischemia TG was found to be significantly increased (P = 0.0271) (Fig. 5C). These results indicate that ischemia caused hyperphosphorylation of tau leading to activation of glia cells, resulting in the staining of microglia and astrocytes to confirm glial changes.

Figure 5. Tau protein AT8 expression. A. Tau AT8 expression was increased in the hippocampal CA1 region of both TG mouse brains. B. Although tau AT8 expression was increased in the cortex in ischemic WT and TG mouse brains, this increased expression appears to be markedly greater in ischemia TG mice. C. Quantitative analysis of AT8 density in hippocampal CA1 confirmed that ischemia TG was significantly increased compared to control TG (P

= 0.0271). Magnification, 400×.

D. Glial changes

Immunohistochemistry experiments were conducted to investigate differences in neuropathological changes. Iba-1 expression in WT control and TG hippocampi were not significantly different between WT and TG group with and without cerebral ischemia, respectively. Nevertheless, the density of Iba-1 positive cells in mice with ischemia was significantly greater than in those without. WT mice exposed to ischemia demonstrated activation of microglia in the hippocampus while microglia were activated in the cortex as well as in the hippocampus of TG mice exposed to ischemia. Iba-1 expression density was quantified to compare Iba-1 expression in each group. The staining density for Iba-1 was significantly increased in both WT and TG mice after ischemia, as compared to that in control WT and TG mice. However, the amount did not differ between TG and WT mice exposed to ischemia (Fig. 6A-B). In the hippocampi of control WT and TG mice, normal astrocyte morphology was observed. In contrast, in WT mice exposed to ischemia, we observed astrogliosis in a region showing signs of cell death. We also observed a cavitation phenomenon in astrocyte-rich regions also showing signs of cell death in TG mice. The GFAP intensity results showed a significant difference between WT and TG groups for brain ischemia (P = 0.0231). Thus, mice exposed to ischemia were more vulnerable to

neuropathologic changes than were control mice, and TG mice were more vulnerable than WT mice (Fig. 6C-D).

Figure 6. Immunohistochemistry of hippocampal lesions. A.

Iba-1 expression was increased in the hippocampal CA1 region in the ischemic brains of both TG and WT groups after tBCCAO, suggesting increased microglial activation. B.

Iba-1 density was increased in the ischemia TG group compared to the ischemia WT group. However, there were no significant differences in the number of these cells across ischemia groups. C. GFAP expression of the hippocampal CA1 region in ischemic brain. D. Quantitative analysis of GFAP intensity confirmed that ischemia TG was significantly increased compared to ischemia WT. Data are

presented as the mean ± standard error of the mean. *P = 0.0231 vs. ischemia WT. Magnification, 400×.

IV. DISCUSSION

In this study, we investigated whether AD is particularly vulnerable to brain ischemia by using 3xTG-AD mice triple-transgenic for the APPSwe, tauP301L, and PS1M148V mutations (Sterniczuk, Antle et al. 2010). A significant cognitive decline was observed in the aged mice of the ischemic TG group when we tested their cognitive abilities using the MWM test after inducing cerebral ischemia. Furthermore, observation of the hippocampal CA1 region indicated that cell death was significantly increased in the TG mice as compared to their WT counterparts, indicating that the aged 3xTG-AD mouse is more vulnerable to cerebral ischemia. However, the characteristic AD pathologies did not show such differences.

It has been reported that cerebral hypoperfusion, an early characteristic of the AD brain, is associated with degenerative processes and cognitive decline. This study explored such potential mechanistic associations and studied the relationship between changes in amyloid precursor protein and Tau protein expression and neuropathology in the brains of 3xTG-AD mice. We generated neuropathological changes by inducing transient brain ischemia, which led to cognitive decline and increased levels of neuronal cell death, microglia, and astrocytes in mouse brains of the ischemia TG group. In addition,

morphological evidence of cellular atrophy was observed in the ischemia TG group compared to the control TG group.

Neuron cells were observed by merging of the double staining for amyloid precursor protein Y188, which proved that amyloid precursor protein Y188 is a neuronal amyloid.

Tau protein expression was observed in cells that were also identified as microglia. However, when amyloid beta was observed in the control / ischemia TG group, amyloid beta was not observed. Amyloid beta was observed, however, in 18-month-old TG positive controls.

Cerebral ischemia induces oxidative stress and inflammation in the brain, resulting in the breakdown of the blood–brain barrier (BBB). Consequently, microglia and astrocytes are activated and cause neuronal damage (Kahlson and Colodner 2016). Neuronal cell death occurs in the hippocampal CA1 region, which is vulnerable to ischemia. In addition, cerebral ischemia causes a decrease in CBF, resulting in impaired memory function and exacerbation of dementia. Amyloid beta precursor protein, amyloidogenic fragments, tau-like pathology, and other AD factors can be induced at the site of injury in brain ischemia (Kalaria 2000). Neurodegenerative diseases are observed as signs of cerebrovascular disease. Thus, according to our findings, cognitive impairment in AD is associated with cerebral ischemia and subsequent neuronal pathological changes.

In AD, the characteristic neurological changes displayed are often accompanied by additional vascular pathology and features of other neurodegenerative diseases (Kelly, He et al. 2017). According to clinical studies, cerebral vascular occlusion contributes to the pathogenesis of AD, and cerebrovascular disease in AD is more detrimental than cerebral vascular disease in the elderly (Kisler, Nelson et al. 2017). Ischemic stroke has recently been implicated as a factor in the deterioration of AD (Lee, Im et al. 2011).

The cognitive impairment resulting from the combination of AD and ischemic stroke is exacerbated compared to ischemic stroke-related or AD-only injury (Santos, Snyder et al. 2017). The complex relationship between AD and ischemic stroke includes cerebral hypoperfusion, energy deficiency, inflammation, dysfunction of capillaries, immune wasting, oxidative stress, and the accumulation of key proteins, including amyloid beta and Tau protein (Pendlebury 2012, Dong, Maniar et al. 2018).

Tauopathies, aggregations of Tau protein, are characterized by a fibrous inclusion found in neurons and glial cells. Activated microglia and astrocytes and increased levels of proinflammatory molecules are also pathological features of brain regions affected by Tau pathology (Leyns and Holtzman 2017).The occurrence of tauopathy, a major pathologic feature, appears to play a role in nerve inflammation and in aggravation of tau pathology and neurodegeneration (Qiu, Ng et al. 2016). In addition to toxic

protein aggregates, activated astrocytes and macrophages, as well as elevated inflammatory marker levels, are other pathological features characteristic of tauopathies (Kovacs 2015). Chronic glial activation may promote the formation of tangles and the reduction of connective compliance, leading to neurodegenerative diseases (Wyss-Coray and Mucke 2002, Ransohoff 2016).

Therefore, our results show that as tau-positive cells increase, astrocytes significantly increase, and tauopathy is affected by astrocytes (Verkhratsky, Olabarria et al.

2010), which is consequently associated with gliosis and neuroinflammation.

Taken together, the results of this study show that ischemia contributes to the cognitive dysfunction of AD and AD mice, who are more vulnerable to ischemic injury, such as apoptosis, necrosis, and neuronal cell death, and exacerbates cognitive deficits associated with this condition.

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