Selective neurodegeneration of the hippocampus caused by chronic cerebral hypoperfusion: F-18 FDG PET study in rats.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) is known to induce Alzheimer's disease (AD) pathology, but its mechanism remains unclear. The purpose of this study was to identify the cerebral regions that are affected by CCH, and to evaluate the development of AD pathology in a rat model of CCH.
METHODS: A rat model of CCH was established by bilaterally ligating the common carotid arteries in adult male rats (CCH group). The identical operations were performed on sham rats without arteries ligation (control group). Regional cerebral glucose metabolism was evaluated at 1 and 3 months after bilateral CCA ligation using positron emission tomography with F-18 fluorodeoxyglucose. The expression levels of amyloid β40 (Aβ40), amyloid β42 (Aβ42), and hyperphosphorylated tau were evaluated using western blots at 3 months after the ligation. Cognitive function was evaluated using the Y-maze test at 3 months after the ligation.
RESULTS: At 1 month after the ligation, cerebral glucose metabolism in the entorhinal, frontal association, motor, and somatosensory cortices were significantly decreased in the CCH group compared with those in the control group. At 3 months after the ligation, cerebral glucose metabolism was normalized in all regions except for the anterodorsal hippocampus, which was significantly decreased compared with that of the control group. The expression of Aβ42 and the Aβ42/40 ratio were significantly higher in the CCH group than those in the control group. The phosphorylated-tau levels of the hippocampus in the CCH group were significantly lower than those in the control group. Cognitive function was more impaired in the CCH group than that in the control group.
CONCLUSION: Our findings suggest that CCH causes selective neurodegeneration of the anterodorsal hippocampus, which may be a trigger point for the development of AD pathology.

Introduction

Alzheimer’s disease (AD) accounts for 60% to 70% of cases of dementia, which according to current estimates, will affect up to 81.1 million patients worldwide by 2040 [1]. Deposition of amyloid beta (Aβ) and neurofibrillary tangles are the main pathological hallmarks of AD, which are respectively caused by aggregation of Aβ peptides and hyperphosphorylated tau (p-tau) in the brain [2, 3]. Although the cause of AD pathology has not been clearly identified, previous studies have reported the relationship between cerebral hypoperfusion and AD pathology [4]; therefore, cerebral hypoperfusion may be a predisposing factor in the pathological progression of AD. This is supported by the fact that vascular risk factors that cause cerebral hypoperfusion, such as hypertension, diabetes, hypercholesterolemia, and smoking, are also risk factors of AD [5]. In addition, cerebral hypoperfusion is known to potentiate other processes associated with AD such as mitochondrial failure, oxidative stress, and neuroinflammation [6, 7].

Cerebral hypoperfusion can occur in two patterns, acute or chronic, depending on the rate at which cerebral vessels narrow; both cause cognitive deficits in various degrees [8]. While acute cerebral hypoperfusion leads to an infarction within approximately 3 h via necrosis of neuronal cells [9], chronic cerebral hypoperfusion (CCH) causes neurodegeneration over a period of months to years through neuronal apoptosis without infarction [10]. The important role of CCH in AD has already emerged at the vanguard of neurology research [11]. Recent in vivo studies have revealed that CCH accelerates AD pathology, including Aβ aggregation and cognitive dysfunction [12, 13]. In turn, Aβ aggregation has been shown to exacerbate inefficient microcirculation and cause blood brain barrier disruption, indicating a vicious cycle between CCH and AD pathology [14]. However, it is not clear which cerebral regions are affected by CCH to cause AD pathology.

Positron emission tomography (PET) with F-18 fluorodeoxyglucose (FDG) is a minimally invasive diagnostic brain imaging procedure that is used to assess regional cerebral glucose metabolism. F-18 FDG uptake on PET reflects regional glucose consumption and synaptic function in the brain [15, 16]. It has been reported that decreased glucose metabolism in specific cerebral regions is an important indicator for the detection of early AD [17]. In a previous study, F-18 FDG PET of a CCH rat model revealed that CCH results in decreased glucose metabolism in the hippocampus, suggesting that CCH can induce AD pathology [18]. However, the previous study did not provide results on cerebral glucose metabolism during the early stage of CCH, and it could not be established whether the decreased glucose metabolism in the hippocampus was due to an acute change similar to vascular dementia or a chronic change similar to AD development. In addition, since the previous study assessed cerebral blood flow (CBF) for up to 1 month after CCH, it was difficult to determine how the hippocampus was affected by the improvement in CBF after 3 months. Since serial evaluations of regional cerebral glucose metabolism with F-18 FDG PET and serial measurements of CBF can reveal sequential changes in cerebral glucose metabolism by CCH, they may facilitate a better understanding of the pathophysiology of CCH during AD development. Thus, the purpose of this study was to identify the cerebral regions that are affected by CCH using F-18 FDG PET, and to evaluate the development of AD pathology in a rat model of CCH.

Materials and methods

Animals

Eight-week-old male Wistar rats (250–300 g body weight) purchased from Central Lab Animal Korea, Inc. were used for this study. The rats were kept in standard cages with 12 h light/12 h dark cycles (8:00 lights on) and humidity that is kept under control at a temperature of 22–24°C (55–60 percent). Food and drink were available at all times. The surgery, CBF, 2,3,5-triphenyltetrazolium chloride (TTC) assay, F-18 FDG PET, western blot analysis, and Y-maze tests were performed according to the schedule illustrated in Fig 1. All experiments in this study were performed in accordance with the guidelines for animal research from the National Institutes of Health. The Keimyung University Institutional Ethics Committee (Daegu, Korea) approved all the animal experiments (KM-2019-13R1).

Fig 1

Experimental design.

CCH and control groups were performed in 11-week-old Wistar rats. To confirm that there was no cerebral infarction after surgery, TTC assay performed on 7 rats in each group randomly, immediately after surgery. F-18 FDG PET imaging was performed 1 and 3 months after surgery. The Y-maze test and western blot analysis were performed 3 months after surgery. CCH: chronic cerebral hypoperfusion; TTC: 2,3,5-triphenyltetrazolium chloride; F-18 FDG: F-18 fluorodeoxyglucose; PET: positron emission tomography.

Bilateral common carotid artery ligation surgery

The rat model of CCH was established as previously described [19]. In 22 Wistar rats, the bilateral common carotid artery (CCA) was doubly ligated (CCH group), whereas 22 sham-operated rats were treated to the same procedures without CCA ligation (control group). The rats were sedated with 4.0% isoflurane in N2O/O2 (70:30) and maintained with 2.0% isoflurane in N2O/O2 (70:30). (70:30). Throughout the duration of the process, the core temperature was maintained between 37 and 38°C. To enable the separation of the bilateral CCA from the surrounding tissues, a tiny incision was carefully made. The CCA stump was wrapped with polylysine-coated nylon (Nylon Monofilament Suture, Fine Science Tools Inc., Foster City, USA). Only anesthesia and vascular dissection were performed in the control group. Rats were separated into cages for post-surgery care.

Laser-Doppler flowmetry

The CBF was measured in 15 rats in each group (CCH and control) using laser-Doppler flowmetry (OMEGA FLOW FLO-C1 BV, OMEGAWABE, Tokyo, Japan), as previously reported [19, 20]. An incision was made with surgical scissors, and the connective tissue was scraped off to expose the skull under deep anesthesia with 4.0% isoflurane in N2O/O2 (70:30). To position the laser-Doppler flowmetry (LDF) probe within the same area of the skull for longitudinal measurements, a ruler was used to identify the area 1-mm posterior and 2.5-mm lateral to the bregma. The (LDF) probe was applied to the skull, and the CBF output was read. If the signal was good, the LDF probe was fixed using dental resin. The dental resin was easily removed from the skull together with the LDF probe, and the residual resin was carefully removed with sandpaper to prevent damage to the skull. After the measurement of CBF, the incision was closed using Vetbond. CBF measurements were taken right before surgery (baseline), as well as 30 minutes, 3 days, 14 days, 1 month, and 3 months later. The CBF ratio (%) was calculated as a percentage of the baseline CBF before the surgery.

TTC assay

A TTC assay was completed 1 day after bilateral CCA ligation to assess cerebral infarction. A total of seven rats from each group (control and CCH groups) were sacrificed. Their brains were extracted from them and sliced into five 2 mm thick coronal pieces. These sections were submerged in 2% TTC (Sigma-Aldrich, Steinheim, Germany) in saline for 15 minutes before being fixed overnight in 4% paraformaldehyde. TTC showed normal tissue parts red, while white portions of the brain indicated cerebral infarct sites.

F-18 FDG PET

To evaluate cerebral glucose metabolism, 9 rats from each group (control group and CCH group) underwent an F-18 FDG PET at 1 month after bilateral CCA ligation, and 14 rats from each group underwent an F-18 FDG PET at 3 months using the Triumph II PET/CT system (Lab-PET8; Gamma Medica-Ideas, Waukesha, WI, USA). Before the PET scan, the rats were fasted for 12 hours. They were sedated with 2.0% isoflurane in N2O/O2 (70:30) and injected with roughly 37 MBq of F-18 FDG into the tail vein. The rats underwent PET scanning approximately 30 min after F-18 FDG injection to acquire whole-brain images; the PET scan lasted for 20 min. The acquired data were assumed to indicate cerebral glucose metabolism. For spatiotemporal quantification of the cerebral glucose metabolism, a volume-of interest (VOI) analysis was performed for each scan with the use of the PMOD software package (PMOD Technologies, Ltd., Zurich, Switzerland) in conjunction with the W. Schiffer rat brain template and atlas, as stated previously [19, 21]. PMOD was used to transform each of the rat brain PET datasets to the appropriate space, and the W. Schiffer VOI brain atlas was automatically applied to measure the F-18 FDG uptake to acquire standardized F-18 FDG uptake values within defined subregions of the rat brain. The W. Schiffer brain VOI atlas was used in an iterative fashion with the standard brain model to further optimize the fusion of the experimental data. The regional standardized F-18 FDG uptake values ratio (SUVR) was calculated by dividing the standardized F-18 FDG uptake value for the individual target region by that for the bilateral cerebellum.

Western blot analysis

At 3 months following bilateral CCA ligation, Western blots were used to assess the expression of Bax, TNF-α, p-tau (Thr231), soluble Aβ, Aβ40, and Aβ42. T-PERTM Tissue Protein Extraction Reagent (78510; Thermo Fisher Scientific, Waltham, MA, USA) was used to homogenize brain tissue (left hippocampus) from 15 rats in each experimental group, which was then incubated at 4°C for 30 minutes with proteinase inhibitor cocktail tablet 1 (cOmplete Mini, EDTA-free; Roche Applied Science, Germany) and PhosSTOP EASY (Roche Applied Science, Germany). Samples were then centrifuged at 15,000 rpm for 15 min at 4°C. The amount of protein (10 μg) was measured using a bicinchoninic acid assay protein assay (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Proteins separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes, and immunoreactive bands were visualized using a chemiluminescent reagent (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific, Waltham, MA, USA). The signals of the bands were quantified with the scion image software using a FUSIONSOLO5 (KOREA BIOMICS, Korea). The following antibodies were used: anti-bax (1:1000) (ab182733, Abcam, Cambridge, MA, USA), anti-TNF-α (1:1000) (ab6671, Abcam, Cambridge, MA, USA), anti-hyperphosphorylated tau (1:1000) (ab151559, Abcam, Cambridge, MA, USA), anti- Amyloid β (ab216436, Abcam, Cambridge, MA, USA), anti-Amyloid β1–40 (1:1000) (ab17295, Abcam, Cambridge, MA, USA), anti-Amyloid β1–42 (1:1000) (ab201061, Abcam, Cambridge, MA, USA), anti-GAPDH (1:2000) (2118, Cell Signaling Technology, Danvers, MA, USA), anti-mouse IgG HRP-linked antibody (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-rabbit IgG HRP-linked antibody (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Y-maze test

The Y-maze test was performed to assess the spatial working memory of the 7 rats in each experimental group as described previously [22]. The apparatus for the Y-maze comprised three arms (500 mm long, 150 mm high, and 100 mm wide; labeled A, B, and C) diverging at a 120° angle. The Y-maze test was performed in a dimly illuminated testing room 3 months after surgery. Rats moved into the room and allowed them to adapt for 1 h. Each rat was gently placed at the end of start arm and allowed to freely move through the Y-maze for 10 min without reinforcers, such as food, water, or electric shock. During the test, the operator was blinded to the group allocation of each animal and the behaviors of the rats were recorded using a video camera. The floor of the maze was cleaned with 70% ethanol after each rat was tested to avoid olfactory cues. An actual alternation was defined as consecutive entries into all three arms [23]. The maximum number of alternations was then calculated by subtracting two units from the total number of arm entries, and the percentage of spontaneous alternation was calculated as follows: spontaneous alternation (%) = (actual number of alternations/maximum number of alternations) × 100. The total number of arms entered during the test was also recorded.

Statistical analysis

The results are expressed as the mean ± standard deviation (SD). Statistical Package for the Social Sciences (SPSS) software version 26.0 (IBM corporation, Armonk, NY, USA) was used for the statistical analyses. Differences in CBF; infarct volume; regional SUVR; and the recognition index between the CCH and control groups were evaluated using the Student’s two-tailed t-test. The expression levels for Bax, TNF-α, p-tau, Aβ, Aβ40 and Aβ42 in the CCH group were normalized to the average of the control group and were compared with those of the control group using one-sample t-test. A value of p < 0.05 was considered statistically significant.

Results

Chronic cerebral hypoperfusion

Using laser-Doppler flowmetry, the CBF was measured just before and at 30 min, 3 days, 14 days, 1 month, and 3 months after the bilateral CCA ligation (Fig 2). At 30 min and 3 days after the surgery, there was a significant decrease in the CBF ratio in the CCH group compared with that in the control group (43.2 ± 7.3% vs. 97.1 ± 9.6%, p < 0.001 and 32.4 ± 9.2% vs. 98.1 ± 5.1%, p < 0.001, respectively). At 14 days, the CBF ratio of the CCH group began to recover but remained significantly lower than that of the control group (71.3 ± 14.3% vs. 98.9 ± 2.9%, p < 0.001). At 3 months, the CBF ratio of the CCH group was slightly increased compared to that of the control group (106.2 ± 5.8% vs. 99.0 ± 3.4%, p = 0.012).

Fig 2

Comparison of CBF between the CCH and control groups.

The CBF was measured before and at 30 min, 3 days, 14 days, 1 month, and 3 months after surgery, using laser-Doppler flowmetry. At both 30 min and 3 days after surgery, there was a significant decrease in the CBF ratio in the CCH group compared with that in the control group. At 14 days, the CBF ratio in the CCH group began to recover but remained significantly lower than that in the control group. In contrast, the CBF ratio in the CCH group was slightly but significantly increased compared with that of the control group at 3 months after surgery. Asterisks indicate statistical significance: **p < 0.01 *p < 0.05. CBF: cerebral blood flow; CCH: chronic cerebral hypoperfusion.

The TTC assay showed that there was no cerebral infarct in the bilateral cerebral hemisphere in both the control and CCH groups (Fig 3).

Fig 3

Comparison of cerebral infarct volume between the CCH and control groups.

Cerebral infarct volume was evaluated by the TTC assay. (A) TTC assay showing that none of the seven rats in the control group exhibited cerebral infarction. (B) In the CCH group, none of the seven rats exhibited a cerebral infarct. CCH: chronic cerebral hypoperfusion; TTC: 2,3,5-triphenyltetrazolium chloride.

Cerebral glucose metabolism

F-18 FDG PET was performed to evaluate cerebral glucose metabolism in the control and CCH groups 1 month and 3 months after ligation. Regional SUVRs were determined using VOI analysis (Fig 4A). At 1 month after the ligation, the SUVRs of the left entorhinal cortex, bilateral frontal association cortex, right motor cortex, and left somatosensory cortex were significantly lower in the CCH group than those in the control group (p = 0.036, p = 0.023, p = 0.005, and p = 0.043, respectively) (Fig 4B–4C and 4E, Table 1). However, there were no significant differences in the SUVR in other regions of the brain at 1 month. At 3 months after the ligation, only the SUVR of the right anterodorsal hippocampus was significantly lower in the CCH group compared with that of the control group (p = 0.014) (Fig 4D and 4F, Table 1). There were no significant differences in any other regions between the control and the CCH groups at 3 months after the ligation.

Fig 4

Cerebral glucose metabolism measured by F-18 FDG PET.

(A) Regional SUVRs were obtained from the W. Schiffer rat brain VOI analysis using the PMOD software package (see Methods). (B) No abnormal glucose metabolism of the right anterodorsal hippocampus (arrow) and left entorhinal cortex (arrow head) is seen in a control group. (C, E, F) Decreased glucose metabolism of the left entorhinal cortex but not the right anterodorsal hippocampus is seen in a rat with bilateral CCA ligation after 1 month (CCH group). (D, F) 3 months after bilateral common CCA (CCH group), decreased glucose metabolism is only seen in the right anterodorsal hippocampus. F-18 FDG: F-18 fluorodeoxyglucose; PET: positron emission tomography. SUVR: standardized F-18 FDG uptake values ratio; VOI: volume-of interest; CCA: common carotid artery; CCH: chronic cerebral hypoperfusion.

Table 1

Comparison of regional cerebral glucose metabolism at 1 and 3 months after surgery.

Regions	Side	1 Month		p value	3 Months		p value
		Control	CCH		Control	CCH
Accumbens	left	1.02 (0.09)	0.99 (0.08)	0.589	1.03 (0.11)	0.99 (0.08)	0.312
	right	1.03 (0.08)	0.98 (0.09)	0.206	1.02(0.08)	1.02(0.10)	0.939
Amygdala	left	0.89 (0.05)	0.84 (0.06)	0.070	0.87(0.07)	0.83(0.07)	0.128
	right	0.88 (0.06)	0.87 (0.08)	0.945	0.86(0.06)	0.83(0.05)	0.248
Auditory cortex	left	0.89 (0.09)	0.82 (0.08)	0.084	0.90(0.07)	0.89(0.08)	0.616
	right	0.86 (0.08)	0.79 (0.11)	0.111	0.89(0.05)	0.86(0.07)	0.207
Cingulate cortex	left	1.07 (0.15)	0.95 (0.09)	0.050	1.03(0.11)	1.00(0.12)	0.465
	right	1.05 (0.15)	0.94 (0.10)	0.081	1.04(0.10)	1.01(0.15)	0.503
Entorhinal cortex	left	0.89 (0.06)	0.83 (0.07)	0.036*	0.88(0.06)	0.87(0.08)	0.742
	right	0.89 (0.08)	0.83 (0.07)	0.081	0.87(0.08)	0.88(0.08)	0.752
Frontal association cortex	left	0.95 (0.09)	0.84 (0.09)	0.023*	0.88(0.11)	0.87(0.09)	0.852
	right	0.91 (0.08)	0.78 (0.09)	0.005*	0.87(0.11)	0.86(0.08)	0.655
Insular cortex	left	0.97 (0.10)	0.89 (0.08)	0.059	0.96(0.06)	0.96(0.06)	0.990
	right	0.95 (0.09)	0.89 (0.08)	0.119	0.95(0.08)	0.97(0.09)	0.600
Medial prefrontal cortex	left	1.14 (0.16)	1.04 (0.09)	0.102	1.15(0.14)	1.11(0.17)	0.513
	right	1.10 (0.14)	1.02 (0.10)	0.181	1.14(0.13)	1.13(0.19)	0.970
Motor cortex	left	0.94 (0.10)	0.86 (0.07)	0.088	0.93(0.09)	0.93(0.08)	0.969
	right	0.94 (0.08)	0.85 (0.10)	0.047	0.91(0.07)	0.91(0.09)	0.946
Orbitofrontal cortex	left	1.04 (0.10)	0.96 (0.09)	0.083	1.03(0.09)	1.00(0.09)	0.328
	right	1.01 (0.10)	0.94 (0.08)	0.099	1.02(0.08)	0.98(0.09)	0.246
Para cortex	left	0.85 (0.08)	0.80 (0.04)	0.108	0.88(0.08)	0.88(0.09)	0.944
	right	0.85 (0.08)	0.78 (0.12)	0.151	0.84(0.07)	0.82(0.08)	0.449
Retrosplenial cortex	left	0.93 (0.09)	0.89 (0.08)	0.327	0.94(0.07)	0.92(0.12)	0.674
	right	0.93 (0.08)	0.87 (0.06)	0.118	0.94(0.08)	0.90(0.09)	0.293
Somatosensory cortex	left	0.92 (0.08)	0.85 (0.06)	0.043*	0.92(0.06)	0.93(0.07)	0.535
	right	0.91 (0.06)	0.84 (0.11)	0.095	0.89(0.05)	0.90(0.07)	0.717
Visual cortex	left	0.87 (0.08)	0.81 (0.06)	0.152	0.89(0.09)	0.89(0.09)	0.895
	right	0.86 (0.08)	0.79 (0.10)	0.139	0.85(0.06)	0.83(0.08)	0.641
Anterodorsal hippocampus	left	1.03 (0.07)	0.98 (0.07)	0.160	1.02(0.06)	1.02(0.08)	0.949
	right	1.03 (0.07)	0.99 (0.09)	0.233	1.02(0.07)	0.96(0.09)	0.039*
Posterior hippocampus	left	0.96 (0.06)	0.96 (0.05)	0.831	0.93(0.05)	0.92(0.07)	0.650
	right	0.94 (0.06)	0.96 (0.11)	0.632	0.95(0.06)	0.95(0.09)	0.980

Asterisks indicate statistical significance

*p < 0.05; CCH: chronic cerebral hypoperfusion. All values are presented as mean (standard deviation).

Development of AD pathology

Western blot analysis was conducted 3 months after the ligation surgery to determine the protein expression levels of Bax, TNF-α, p-tau, Aβ, Aβ40, Aβ42, and GAPDH in the hippocampus. The expression of Aβ42 was significantly increased in the CCH group compared with that in the control group (p = 0.048) (Fig 5). Moreover, the Aβ42/40 ratio in the hippocampus of the CCH group was significantly elevated in comparison to that of the control group (p = 0.028). The p-tau levels of the hippocampus in the CCH group were significantly lower as compared with those of the control group (p = 0.014). There were no significant differences in the expression levels of Bax, TNF-α, Aβ, and Aβ40 in the hippocampus between the two groups.

Fig 5

Comparison of Alzheimer’s disease pathology in the hippocampus between the CCH and control groups.

(A) The expression levels of Bax, TNF-α, hyperphosphorylated tau (p-tau), amyloid β (Aβ), amyloid β40 (Aβ40), and amyloid β42 (Aβ42) were evaluated in the hippocampus at 3 months after bilateral CCH using western blot analysis. (B-H) The relative ratio of Bax, TNF-α, p-tau, Aβ, Aβ40, and Aβ42 were plotted based on the quantification of band intensity using scion image software. Values are expressed as the mean ± SD. Asterisks indicate statistical significance: *p < 0.05; **p < 0.05. CCH: chronic cerebral hypoperfusion.

Spatial working memory

The Y-maze test was performed 3 months after surgery to assess spatial working memory. There was no difference in the number of arm entries between the control and the CCH groups. In contrast, there was significantly lower spontaneous alternation in the CCH group compared with the control group at 3 (69.25 ± 10.01% vs. 56.58 ± 7.54%, p = 0.020; Fig 6).

Fig 6

Comparison of spatial working memory assessed by the Y-maze test between the CCH and control groups.

The number of arm entries (A) and alternation behaviors (B) measured after 3 months in the bilateral common carotid artery ligation (CCH group) and control group are shown. Asterisks indicate statistical significance: *p < 0.05. CCH: chronic cerebral hypoperfusion.

Discussion

The present study showed that CCH decreases the neuronal activity of the hippocampus, causing cognitive decline in a rat model. The results also showed that CCH induces cerebral Aβ accumulation. These results suggest that CCH causes selective hippocampal degeneration, which induces AD pathology. The present study helps to understand the mechanism by which cerebrovascular disease contributes to AD development.

CCH by permanent bilateral CCA ligation offers several advantages compared to other approaches. It can induce CCH in a more clinically relevant manner, without ischemic lesions in the brain [24, 25]. The present study induced CCH by CCA ligation in the rat brain using real time CBF monitoring, and there was no ischemic lesion in the brain. The CBF monitoring revealed a sharp decrease in the early stages after surgery, resulting in a 68% and 29% decrease in CBF at 3 and 14 days after the surgery, respectively. The CBF gradually recovered from the 14th day after surgery and reached a normal level at 1 month. In agreement with our results, a previous study with rats reported that bilateral occlusion of the CCA led to a dramatic initial drop in CBF, which subsequently returned to 30–45% CBF in the cortex and a 20% reduction in the hippocampus 1 week after surgery [26]. The gradual recovery of the CBF could be caused by supply from the collateral blood vessels, such as through the posterior communicating artery [20]. In addition, compensation mechanisms for the recovery of CBF may involve biomedical regulation of CBF, recruitment of non-perfused capillaries, and angiogenesis [27]. Unexpectedly, in the present study, the CBF in the CCH group, relative to the control group, increased significantly 3 months after bilateral CCA ligation. This may have been observed because the recruitment of capillaries and angiogenesis can be more pronounced in the cerebral cortex, where the capillary is more developed than in the deep structure, in the CCH rat model [28], and the LDF measured the CBF for a specific region in the cerebral cortex rather than the entire brain [29]. This limitation of LDF is associated with the lack of knowledge of the depth of CBF measurement [29]. In support of this finding, a previous study showed that the percentage and areas of capillaries were increased 6 weeks after bilateral CCA occlusion in the CCH rat model relative to the control group [27]. This was consistent with the findings of another study showing an increase in the capillary density of the cerebral cortex in the chronic cerebral hypoxia mouse model [28]. Another possible reason is that LDF could not accurately evaluate CBF at each time point after bilateral CCA ligation. The LDF may not be suitable for the longitudinal measurement of CBF due to the limitations of LDF, such as the effect of the optical properties of the tissues on the perfusion signal, motion artifact noise, lack of quantitative units for perfusion, and the biological zero signal [29]. To overcome this limitation, we attempted to place the LDF probe in the same position on the skull at each time point.

Several studies of patients with late stage of AD have reported decreased cerebral glucose metabolism in the temporal and parietal cortices, posterior cingulate, and precuneus [30]. Moreover, in the early stage of AD, decreased cerebral glucose metabolism has been observed in the hippocampus, and hippocampal dysfunction has been shown to be associated with that of the parietal and temporal cortices, suggesting that hippocampal degeneration could be an important trigger for the onset of AD [31]. A recent study using high-resolution F-18 FDG PET/MRI reported decreased glucose metabolism in the bilateral hippocampus in patients with early-stage AD [32]. Previous studies using animal models have shown that hippocampal degeneration, observed during the early stage of AD can be caused by CCH [33]. A previous F-18 FDG PET study of a CCH rat model revealed that CCH decreased the glucose metabolism in the hippocampus 3 months after bilateral CCA ligation, inducing AD pathology [18]. Following the previous study, the present study revealed that glucose metabolism in the anterodorsal hippocampus was decreased by CCH 3 months after the ligation. In addition to the previous study, the present study also showed that the decreased glucose metabolism in the cortex 1 month after the ligation improved after 3 months with the recovery of cerebral blood flow; the decrease in glucose metabolism in the hippocampus was observed 3 months after the ligation. These findings support the ischemic hypothesis for AD development related to CCH, showing that the hippocampus is more vulnerable to ischemia than the cortex [34]. It is known that the hippocampus, which is involved in memory formation, is susceptible to ischemia [35]. Another previous F-18 FDG PET study in a CCH mouse model revealed that glucose metabolism in the hippocampus, as well as in the cortex, decreased for up to 6 months after applying microcoils to bilateral CCA, but selective vulnerability to ischemia was not detected in the hippocampus [33]. This may be because the compensation mechanisms for the recovery of CBF were not more extensive in mice than in rats, and the sustained decrease in CBF in the cortex affected the decrease in cerebral glucose metabolism [36, 37].

A previous study of ischemic animal models has revealed that CCH can induce AD pathology [38]. A study using specific enzyme-linked immunosorbent assays reported that white matter hyperintensities were significantly associated with plasma Aβ40 and Aβ42 levels in an AD and mild cognitive impairment population [39]. In agreement with previous studies, the present study showed that the expression levels of Aβ42 and the Aβ42/40 ratio were increased in the hippocampus 3 months after bilateral CCA ligation. Thus, chronic ischemia could contribute to the development of AD through alteration of Aβ metabolism was postulated. In mutant APP transgenic mice, it has been shown that long-term hypoxia contributes to increased Aβ deposition and neuritic plaque formation, potentiating memory deficit by increasing the transcription and expression of the β-site APP cleaving enzyme 1 (BACE1) gene, which is primarily mediated by the binding of hypoxia-inducible factor-1α to the BACE1 promoter [40, 41]. Furthermore, following energy deficiency mediated by pharmacological drugs (e.g. insulin, 2-deoxyglucose, 3-nitropropionic acid, or kainic acid), BACE1 activation and subsent Aβ40 overproduction have been seen in Tg2576 animals [42]. These findings collectively suggest that a lack of energy/oxygen promotes AD pathogenesis by increasing BACE1 expression and Aβ overproduction. Chronic cerebral hypoperfusion has been known to induce the hyperphosphorylation of tau [43, 44]. However, in the present study, the expression of p-tau decreased 3 months after surgery. This discordant result is attributed to the differences in the level of p-tau associated with the clinical features of AD, severity, and the location of the affected brain [45]. Furthermore, the level of p-tau can be measured differently depending on the specific site where phosphorylation occurs, such as at Th205, Th212, Th23, Ser262, Ser396, and Ser404 [43], but only tau phosphorylation at Th231 was measured in the present study. One study involving a mouse model of CCH showed that the level of p-tau increased at Th212 and Ser262 of the cortex, but was not significantly changed at Th231, S262, S396, and S404 [43]. Another study involving an oligemic mouse model of cerebral hypoperfusion revealed a significant decrease in the level of p-tau at Ser199, 202, and Th181, but no significant changes at Th214 [46]. These reductions in the level of p-tau also could be due to the overall reduction in the level of tau rather than specific changes in tau phosphorylation and dephosphorylation [46]. Further longitudinal experiments are needed for the measurement of site-specific tau phosphorylation after CCH.

The Y-maze test can be used to specifically assess short-term memory in rats. Spontaneous alternation, which is a measure of spatial working memory, is especially impaired by AD [47, 48]. The present study revealed that CCH with decreased metabolism in the hippocampus aggravates memory impairment on the Y-maze test. A Rotterdam Study of 1,730 participants suggested that CCH precedes and possibly contributes to the onset of clinical dementia [49]. In an animal study of APPswe/PS1 mice, CCH induced by single vessel occlusion has been shown to exacerbate memory deficits [50, 51]. Another study using the J20/APP AD mouse model showed that CCH induced by bilateral carotid artery stenosis exacerbated learning impairment [13]. The memory impairment induced by CCH is presumed to be caused by hippocampal degeneration. In the early stages of AD, the hippocampus is one of the cerebral structures that undergoes neurodegenerative changes [52, 53]. Hippocampal formation is involved in the learning process and is crucial in in processing and remembering spatial and contextual information [54]. In patients with AD, progressive hippocampal dysfunction has been reported to cause memory impairment [55], which was consistent with our results.

The current study has some limitations. First, the mechanism by which selective hippocampal degeneration causes AD pathology has not been identified. Further, it is still unclear whether CCH generates AD pathology directly or in combination with other causes. Second, our animal model did not have persistent CCH, because the CBF recovered to normal values 1 month after the bilateral CCA ligation surgery. Persistent CCH would likely reveal the effect on AD development more clearly, and would be more consistent with the clinical situation. Animal models of diabetes with a high-fat diet or type-I interferon injection have been shown cause small vessel disease or multivessel atherosclerosis resulting in CCH [56]. Third, this study did not consider the effects of age and gender as only young male Wistar rats were used to evaluate the effects of CCH on AD development. Old age is the strongest risk factor for AD, and the mechanisms for AD development can differ with sex; therefore, evaluating the difference in the effects of chronic cerebral ischemia according to sex in older rats may help elucidate the mechanism of AD development more clearly [57]. Fourth, cognitive function was evaluated by only one behavior test without control tests measuring anxiety-like behavior or locomotor activity. Thus, further studies involving physiological animal models with a complete battery of behavior tests suitable for the clinical situation are needed to establish the effect of CCH on AD development. Finally, the mechanism by which CCH induce AD development, as well as other complicated factors, and how to prevent or slow AD progression by inhibiting this process should be focused on future research.

Conclusions

Our findings suggest that CCH induces the AD pathology with selectively degeneration of the hippocampus in rats. CCH may play a significant role in the development of AD and the selective neurodegeneration of the hippocampus may be a trigger point for the development of AD pathology. Further studies are needed to elucidate the mechanisms by which CCH aggravates AD pathology.

(XLSX)

Click here for additional data file.

(PDF)

Click here for additional data file.

27 Aug 2021

PONE-D-21-25820

Selective Neurodegeneration of the Hippocampus Caused by Chronic Cerebral Hypoperfusion: F-18 FDG PET Study in Rats

PLOS ONE

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS ONE. After preliminary editorial evaluation, I found that this study has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands.

Specifically, per PLOS ONE policies submitted manuscripts should include quantitative primary data in a format allowing for their re-evaluation (such as in MS Excel spreadsheet). Your can provide these data in a supplemental file.  Reviewers should also be able to inspect the primary, unedited images of your Western blots, which have been used for preparing Fig. 5.  These can be included as a supplemental PDF file containing uncropped, high resolution WB scans.

Therefore, I invite you to submit a revised version of the manuscript that addresses the points raised during the preliminary review process.  As soon as I have the requested additional information, your manuscript will be forwarded to experts in the field for peer review.

Please submit your revised manuscript as soon as possible but not late than by Oct 03 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:  http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at  https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Sep 2021

Dear Editor:

Thank you for your careful consideration of our manuscript. We appreciate all your valuable comments. As per your recommendation, we additionally uploaded unedited images of the Western blots and supplemental PDF files containing uncropped, high resolution WB scans. We hope that the revised manuscript will be considered suitable for publication.

Sincerely

Hae Won Kim, MD, PhD

Department of Nuclear Medicine, Keimyung University Dongsan Medical Center

56 Dalseong-ro, Jung-gu, Daegu 41931, Republic of Korea

Tel.: 82-53-250-7068, Fax: 82-53-250-8695, E-mail: hwkim.nm@gmail.com

Submitted filename: Response to Editor1.docx

Click here for additional data file.

24 Sep 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors tried to identify the cerebral regions that are affected by chronic cerebral hypoperfusion (CCH), and to evaluate the development of Alzheimer pathology in a rat model of CCH. The findings are interesting, but there are some methodological and interpretational flaws that should be amended.

In the structured Abstract, the authors referred to hyperphosphorylated tau (p-tau) in the Methods but not in the Results. The p-tau (Thr 231) was significantly reduced in the CCH group. Please discuss this potentially interesting result in the Abstract and text.

The authors found significant increase of CBF in the CCH group at 3 months. Longitudinal measurement of CBF is thought not to be accurate at 1 month and 3 months after surgery. Please discuss this.

The authors stated that the gradual recovery of the CBF could be caused by supply from the collateral blood vessels, such as through the posterior communicating artery. This is a plausible explanation of the result shown in Figure 2. However, do collateral blood vessels induce significant increase of the CBF at 3 months? Please discuss the underlying mechanism of the overshoot of compensatory responses.

An important reference is not included. FDG-PET study in a mouse model of CCH was previously reported in PMID: 20448204. This should be included in the references and should be thoroughly discussed regarding similarities and differences in the findings between mice and rats.

Reviewer #2: The study is well powered and offers an assessment of cerebral glucose metabolism and cerebral blood flow at multiple time points. Unfortunately, novelty is lacking. The blood flow time course has already been published, by this group and others. Further, they have already published the cerebral glucose metabolism and neuropathology data at the 3 month time point. Thus, very little new information is presented.

Comments:

• It is unclear how this study is sufficiently different from the previously published work, except for addition of the 1 month time point of glucose metabolism (https://www.nature.com/articles/s41598-019-50681-4#Sec10).

• Sex and age are not appropriately considered ask key biological variables. Only young males are used. Dementia is a disease of aging, so use of young animals should be discussed as a limitation. Further, use of only one sex shoudl be discussed as a major limitation.

• Behavior test results are inaccurately described. Y maze is described as an assessment of learning and recognition memory. However, as tested, the Y maze protocol used assesses working memory. The test should be revised.

• Use of only 1 behavior tests with no control tests that assessed anxiety-like behavior or locomotor activity is a limitation.

• Use of laser Doppler probe for blood flow measurement should be discussed as a limitation. This method allows assessment of only one very small regional of interest and may not reflect a lack of cerebral hypoperfsion in the entire brain at this time point.

• Graphs could be improved. Some are pixelated and none include the individual data points.

• Please mention whether you are assessing soluble or insoluble amyloid beta.

• Please add lot numbers for primary antibodies.

• Please reference table 1 (in addition to 4A-B) when describing differences in cerebral metabolism.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Oct 2021

To Reviewer #1

Thank you for your careful comments. The corrections are indicated by red-font text in the revised manuscript.

1) In the structured Abstract, the authors referred to hyperphosphorylated tau (p-tau) in the Methods but not in the Results. The p-tau (Thr 231) was significantly reduced in the CCH group. Please discuss this potentially interesting result in the Abstract and text.

- As you recommended, we have provided the results of p-tau in the Abstract. Possible reasons for the discordant results for the p-tau level have also been provided in the Discussion section (page 14, 1st paragraph).

2) The authors found significant increase of CBF in the CCH group at 3 months. Longitudinal measurement of CBF is thought not to be accurate at 1 month and 3 months after surgery. Please discuss this. The authors stated that the gradual recovery of the CBF could be caused by supply from the collateral blood vessels, such as through the posterior communicating artery. This is a plausible explanation of the result shown in Figure 2. However, do collateral blood vessels induce significant increase of the CBF at 3 months? Please discuss the underlying mechanism of the overshoot of compensatory responses.

- We agree with your comment. The Laser Doppler flowmetry (LDF) may not suitable for the longitudinal measurement of CBF due to the limitations of LDF, such as the influence of the optical properties of tissues on the perfusion signal, motion artifact noise, lack of quantitative units for perfusion, and the biological zero signal. In addition to these limitations of LDF, the possible underlying mechanisms of the overshoot of compensatory responses are described in the Discussion section (page 12, 2nd paragraph).

3) An important reference is not included. FDG-PET study in a mouse model of CCH was previously reported in PMID: 20448204. This should be included in the references and should be thoroughly discussed regarding similarities and differences in the findings between mice and rats.

- We agree with your insightful comment. A previous study involving the CCH mouse model, which you mentioned, reported that glucose metabolism in the hippocampus, as well as in the cortex, decreased for up to 6 months after bilateral CCA ligation, failing to prove the selective vulnerability to ischemia in the hippocampus. This differs from our results showing that the hippocampus is more vulnerable to ischemia than the cortex. The following may account for the difference in results: the compensation mechanisms for recovery of CBF did not occur more extensively in mice than in rats, and the decreased CBF in the cortex affected the decrease in cerebral glucose metabolism. The Discussion section has been updated accordingly (page 13, 2nd paragraph).

To Reviewer #2

Thank you for your careful comments. The corrections are indicated by red-font in the revised manuscript.

1) It is unclear how this study is sufficiently different from the previously published work, except for addition of the 1 month time point of glucose metabolism.

- We agree with your comment. It is important to note the differences between the results of previous studies and the present study. A previous F-18 FDG PET study of a CCH rat model revealed that CCH decreased the glucose metabolism in the hippocampus 3 months after bilateral CCA ligation, inducing AD pathology. Following the previous study, the present study revealed that glucose metabolism in the anterodorsal hippocampus was decreased by CCH 3 months after the ligation. In addition to the previous study, the present study also showed that the decreased glucose metabolism in the cortex 1 month after the ligation improved after 3 months with the recovery of cerebral blood flow; the decrease in glucose metabolism in the hippocampus was observed 3 months after the ligation. These findings support the ischemic hypothesis for AD development related to CCH, showing that the hippocampus is more vulnerable to ischemia than the cortex. The Introduction and Discussion sections have been updated accordingly (page 3, last paragraph and page 13, 2nd paragraph).

2) Sex and age are not appropriately considered ask key biological variables. Only young males are used. Dementia is a disease of aging, so use of young animals should be discussed as a limitation. Further, use of only one sex should be discussed as a major limitation.

- Old age is the strongest risk factor for AD, and the mechanisms for AD development can differ with sex; therefore, evaluating the differences in the effects of chronic cerebral ischemia according to sex in older rats may help elucidate the mechanism of AD development more clearly. This limitation has been added to the Discussion section (page 15, last paragraph).

3) Behavior test results are inaccurately described. Y maze is described as an assessment of learning and recognition memory. However, as tested, the Y maze protocol used assesses working memory. The test should be revised.

- As you mentioned, the Y-maze test can be used to assess short-term working memory in rats. We have revised the manuscript to highlight the characteristics of the Y-maze test in the Methods and Discussion sections (page 14, last paragraph).

4) Use of only 1 behavior tests with no control tests that assessed anxiety-like behavior or locomotor activity is a limitation.

- We agree that is a limitation of the present study. We have added it to limitation in the Discussion section (page 15, last paragraph).

5) Use of laser Doppler probe for blood flow measurement should be discussed as a limitation. This method allows assessment of only one very small regional of interest and may not reflect a lack of cerebral hypoperfsion in the entire brain at this time point.

- We agree with your comment. The CBF, which was measured by Laser Doppler flowmetry, was for a specific region in the cerebral cortex rather than the entire brain. This is related to the limitation of Laser Doppler flowmetry, with lack of the knowledge of the depth of CBF measurement. We have highlighted this limitation in the Discussion section (page 13, 1st paragraph).

6) Graphs could be improved. Some are pixelated and none include the individual data points.

- Figures 4, 5, and 6 have been modified to include individual data points.

7) Please mention whether you are assessing soluble or insoluble amyloid beta.

- Western blots were used to assess the expressions of soluble Aβ, Aβ40, and Aβ42. We have described this in the Methods section (page 6, last paragraph).

8) Please add lot numbers for primary antibodies.

- We agree with your comment. It is important to record the lot number, because the same primary antibody may yield different results depending on the factory, date of manufacture, and raw material. Unfortunately, we did not record the lot number. We are sorry for not providing the lot number.

9) Please reference table 1 (in addition to 4A-B) when describing differences in cerebral metabolism.

- As you suggested, we have added Table 1 and Figure 4A-B to provide further details on the differences in cerebral metabolism in the Results section (page 9, 1st paragraph).

Submitted filename: Response_to_Reviewers_2.1.docx

Click here for additional data file.

23 Nov 2021

[1] Reviewer 1 has a valid point about the effects of bilateral CCA ligation in MICE, which are misquoted in your manuscript on p. 13.  Unlike rats, many strains of mice have low patency of the posterior communicators.  The original paper, which tested the effects of bilateral CCA ligations in mice (K Kitawa et al, JCBFM, 1998), identified robust forebrain/hippocampal damage after only 15 min of bilateral CCA ligation, particularly in C56BL/6.  Indeed, I think that the manuscript by Kitawa et al. showing incomplete and variable patency of posterior communicator arteries in mice strains has a place in your discussion.  It highlights the difference in perfusion patterns between species with pathological implications.

[2] Reviewer 2 requested additional clarity on statistical analysis.  Specifically, you need to clarify which test was used for analyzing  differences in western blot immunoreactivities.  These cannot be analyzed using two populations t-test, but rather one-population t-test where values are compared to the unity.  If this is not the case, please re-analyze the data and make appropriate corrections in the text.

Assuming these two requirements are met, your manuscript will be accepted.

Please submit your revised manuscript by Jan 07 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

15 Dec 2021

To Reviewer #1

Thank you for your careful comments. The corrections are indicated by red-font text in the revised manuscript.

1) The authors stated that ‘Another previous F-18 FDG PET study involving a CCH mouse model revealed that glucose metabolism in the hippocampus, as well as in the cortex, decreased for up to 6 months after bilateral CCA ligation, failing to prove the selective vulnerability to the ischemia in the hippocampus [33].’ This sentence is not accurate because a CCH mouse model is generated by bilateral CCA stenosis with microcoils instead of ligation. Therefore, please correct it accordingly. In addition, it would be better to replace ‘failing to prove’ by ‘but did not show’.

Response: We agree with your insightful comment. Nishio et al. reported the effects of chronic cerebral hypoperfusion in mice with bilateral common carotid artery stenosis using microcoils. That sentence has been modified as you commented (page 13, 2nd paragraph).

To Reviewer #2

Thank you for your careful comments. The corrections are indicated by red-font in the revised manuscript.

1) In adding individual data points to the graph, I now see additional issues with rigor. The analysis in Figure 5 is inappropriate. There are no data points for the control group because it was all normalized as 1. There should be normalization to the average of the control group so that there is still an ability to plot and see the variation in individual data points within the controls. Please correct this.

Response: We agree with your insightful comment. The expression levels for Bax, TNF-α, p-tau (Thr231), Aβ, Aβ40 and Aβ42 in the CCH group were normalized to the average of the control group, and were compared with those of the control group using one-sample t-test. The results of western blotting using one-sample t-test did not differ from the previous results, so the p value was slightly modified. The statistical analysis method used for the western blotting results was modified in the statistical analysis section (page 7, last paragraph), and Figure 5 was also modified accordingly.

Submitted filename: Response_to_Reviewers_3.0.docx

Click here for additional data file.

20 Dec 2021

Selective Neurodegeneration of the Hippocampus Caused by Chronic Cerebral Hypoperfusion: F-18 FDG PET Study in Rats

PONE-D-21-25820R3

Dear Dr. Kim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Feb 2022

PONE-D-21-25820R3

Selective Neurodegeneration of the Hippocampus Caused by Chronic Cerebral Hypoperfusion: F-18 FDG PET Study in Rats

Dear Dr. Kim:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alexander A. Mongin

Academic Editor

PLOS ONE