Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease

Abstract Previously, we showed that sodium/glucose cotransporter 1 (SGLT1) participates in vascular cognitive impairment in small vessel disease. We hypothesized that SGLT1 inhibitors can improve the small vessel disease induced‐vascular cognitive impairment. We examined the effects of mizagliflozin, a selective SGLT1 inhibitor, and phlorizin, a non‐selective SGLT inhibitor, on vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created using a mouse model of asymmetric common carotid artery surgery (ACAS). Two and/or 4 weeks after ACAS, all experiments were performed. Cerebral blood flow (CBF) was decreased in ACAS compared with sham‐operated mice. Phlorizin but not mizagliflozin reversed the decreased CBF of ACAS mice. Both mizagliflozin and phlorizin reversed the ACAS‐induced decrease in the latency to fall in a wire hang test of ACAS mice. Moreover, they reversed the ACAS‐induced longer escape latencies in the Morris water maze test of ACAS mice. ACAS increased SGLT1 and proinflammatory cytokine gene expressions in mouse brains and phlorizin but not mizagliflozin normalized all gene expressions in ACAS mice. Hematoxylin/eosin staining demonstrated that they inhibited pyknotic cell death in the ACAS mouse hippocampus. In PC12HS cells, IL‐1β increased SGLT1 expression and decreased survival rates of cells. Both mizagliflozin and phlorizin increased the survival rates of IL‐1β‐treated PC12HS cells. These results suggest that mizagliflozin and phlorizin can improve vascular cognitive impairment through the inhibition of neural SGLT1 and phlorizin also does so through the improvement of CBF in a mouse model of small vessel disease.


| INTRODUC TI ON
One of the major public health concerns is aging-related cognitive impairment associated with alteration in cerebral blood vessels (i.e., vascular cognitive impairment, VCI) in recent years. 1 Several types of vascular dementia, such as post-stroke dementia, subcortical ischemic vascular dementia, multi-infarcted cortical dementia, and mixed dementia, have been reported. 2,3 At present, VCI is mainly prevented by treating vascular diseases and other risk factors for VCI. In fact, the management of cardiovascular risk factors, such as hypertension, diabetes, dyslipidemia, obesity, and smoking, greatly reduces the development of VCI in later life. The effective control of cardiovascular risk factors prevents VCI-related disease and may be more effective than current pharmacological treatment for VCI. 4,5 However, currently available therapeutic options are limited for directly improving vascular dementia and cognitive impairment through protection against neural injury.
Several recent studies showed that mid-cerebral artery occlusion induced cerebral neuronal damage through sodium-glucose cotransporter 1 (SGLT1) activation in mice. 6,7 Recently, a new mouse model of small vessel disease with cognitive impairment, generated by surgical implantation of an ameroid constrictor in the right common carotid artery (CCA) and placement of a microcoil in the left CCA, was developed. 8 Our previous study demonstrated that SGLT1 participated in the development of VCI and neural injury in the mouse model described above. 9 These results suggest that blockade of SGLT1 can improve vascular dementia and cognitive impairment through protection against neural injury. However, whether SGLT1 inhibitors are effective for inhibiting neural injury, VCI, and dementia during small vessel disease is still unknown. Mizagliflozin is a new selective SGLT1 blocker and is known to exhibit potential in the amelioration of chronic constipation. 10 Moreover, mizagliflozin shows favorable efficacy and safety for patients with functional constipation and whose risk of hypoglycemia is low. 11 As several glucose transporters (GLUTs) such as GLUT 1-6 exist and function in the brain, the possibility of GLUTs different from SGLT1 participating in the development of VCI and ischemic neuronal injury cannot be denied. Phlorizin is a non-selective SGLT1 blocker that improves mid-cerebral artery occlusion-induced cerebral neuronal damage in mice. 6,7 Here, we examined the effects of mizagliflozin on ischemiainduced VCI and neural injury in a mouse model of small vessel disease and compared them with those of phlorizin.

| Experimental animals and asymmetric common carotid artery stenosis surgery (ACAS)
C57BL/6J male mice (10 weeks old, 25-30-g body weight) were purchased from Japan SLC. To produce VCI, stenosis of both common carotid arteries (CCAs) was surgically created using a microcoil and an ameroid constrictor (ACAS), as described elsewhere. 8,9 Briefly, after mice were anesthetized with isoflurane (2-4%), both CCAs were exposed and freed from their sheaths. A spiral microcoil was placed around the left CCA just below the carotid bifurcation. An ameroid constrictor 12 was also placed around the right CCA ( Figure 1A). The same procedure was performed except for ACAS in sham-operated mice. Finally, we closed the midline cervical incision using 7-0 nylon suture. After the operation, mice were maintained in an environment with day/night reversal throughout the experiments. Sodium pentobarbital (30 mg/kg) application into abdominal cavity and/or isoflurane inhalation was performed to anesthetize all animals before the brain tissue was excised. The degree of motion of the sternum and movement of the extremities was monitored to confirm the adequacy of anesthesia. The appropriate depth of anesthesia was checked by a toe pinch reflex test. An animal glucometer (LAB Gluco, Fore Care Tokyo Co., Ltd.) was used to measure the blood glucose (BG) level.

| Wire hang test
The wire hang test was performed at two weeks after ACAS or sham surgery, as described previously. 9 Briefly, to avoid vibration, the wire was tightly fixed to the top of the box because this might interfere with the performance of the mouse. We placed both upper limbs of a mouse on the metallic wire, and latency to fall was recorded. We repeated the procedure five times with an interval of 5 min between attempts and the mean latency to fall was calculated from the average time of five trials.

| Y-maze test
The Y-maze test was performed to assess spatial working memory and spontaneous activity, as described elsewhere. 9,13 Briefly, the Y-maze test was performed at 2 and 4 weeks after ACAS or sham surgery ( Figure 1B). After introduction to the end of the start arm, mice were allowed to freely explore the three arms during an 8-min session. The numbers of arm entries and trials were recorded using a video camera (Logicool HD Webcam C615, Logicool). A mouse was considered to have entered an arm when all four limbs were within the arm. Spatial working memory was elucidated by the percentage of alternation behavior. Actual alternation behavior was defined as entry into all three arms on consecutive occasions. The maximum alternation behavior was calculated as the total number of arm entries minus two, and the percentage of alternation was calculated as the number of actual alternations divided by that of maximum alternations multiplied by 100. Spontaneous activity was elucidated by the total number of arms entered during a session.

| Morris water maze test
The Morris water maze test was performed at 29-34 days after ACAS or sham surgery, as previously described. 9,14 Briefly, to learn the platform position, a visible platform task was performed using a transparent circular platform (i.e., escape platform) with a diameter of 8 cm three times per day from the first to sixth day. After the visible platform task, we immersed the circular platform 1 cm below the water surface in the center of a quadrant of the pool to perform a hidden platform task. The platform was kept in the same position throughout the experiment. The hidden platform task was performed five times with a 5-min interval between attempts each F I G U R E 1 Panel A, Asymmetric common carotid artery surgery (ACAS) was performed in four of the five groups. An image showing an ameroid constrictor (AC) around the right common carotid artery (CCA) and placement of a microcoil around the left CCA. Panel B, The schema of experimental protocols for each of the seven groups (saline-pretreated sham operation mice, saline-pretreated ACAS mice, low or high dose of mizagliflozinpretreated ACAS mice, phlorizinpretreated ACAS mice, saline-posttreated sham operation mice, and high dose of mizagliflozin-posttreated ACAS mice). Saline, mizagliflozin (100 or 500 μg/μl), or phlorizin (460 μg/μl) was administered for 42 or 30 days using an osmotic mini pump (Alzet 2006 [0.15 µl/h, 200 µl]) implanted subcutaneously in all mice one week before or five days after sham operation and ACAS, respectively day. We released mice into the water at the starting position in three other quadrants. We monitored each mouse tested and recorded the time required until it arrived at the platform. When a mouse arrived at the platform in 90 s, we allowed it to stay there for 15 s. When a mouse did not arrive at the platform in 90 s, we guided it to the platform and stayed it there for 15 s. In each mouse, we calculated latency to reach the platform (escape latency) from the average time of 5 times of the hidden platform task performed each day.

| Cerebral blood flow (CBF) measurement
We measured CBF at 35 days after ACAS or sham surgery using a 2D laser blood flow imager (OMEGAZONE, Omegawave Inc.), as previously described. 8,9 Briefly, laser speckle flowmetry with the average of highspeed 2D imaging was used to measure relative CBF. After anesthesia (1-2% isoflurane), we surgically removed the mouse scalp to expose the skull. The periosteum, which adheres to the skull, was widely removed with fine-tip forceps. The skull surface was wiped with saline-soaked gauze to prevent drying of the surface. Cerebral blood flow recordings were performed through the skull. Circular regions in identically sized ROIs (900 pixels), located 1 mm posterior and 2 mm lateral to the bregma were defined on the image for quantitative measurement (Figure 2A).

| Quantification of mRNA by real-time PCR
The amount of mRNA by real-time reverse transcriptionpolymerase chain reaction (RT-PCR) was performed as previously described. 15 Briefly, after the brain tissue was resected, total RNA was prepared from the brain tissue of the four groups of mice ( Figure 1B Table 1 showed the sequences of oligonucleotide primers. As the expression of GAPDH mRNA was constant between groups the expression of each gene was normalized to that of GAPDH mRNA.

| Histology
Hematoxylin/eosin staining for histopathological analysis was performed as previously described. 9 Briefly, after anesthesia with isoflurane (2%), and sodium pentobarbital (15 mg/kg i.p.) we transcardially perfused and fixed mouse brain with a 4% solution of paraformaldehyde in 0.01 mol/L PBS for more than 5-8 min in the four different groups of mice (shown in Figure 1B, n = 6 for each).
After brain was fixated, we quickly removed each brain and divided it coronally at the bregma, the bregma 1 mm, and bregma 2 mm through the hippocampus. were We embedded the brain samples in paraffin and then cut them into 3μm-thick coronal sections. For histopathological analysis, six sections stained with hematoxylin/eosin were performed were prepared. After HE staining, the shrunken neurons with pyknotic nuclei were counted to assess neuropathy.

| Cell Culture, Hypoxia, and IL-1β Stimulation
Rat PC12HS cells (JCRB0266, Japanese Cancer Research Bank, National Institutes of Biomedical Innovation) were seeded on collagen type I-coated 6-well plates (1 × 10 6 cells/well) and maintained in "complete medium" that consisted of RPMI1640 medium supplemented with 5% fetal bovine serum (FBS) and 10% horse serum) in a humidified incubator at 37℃, 5% CO 2 , for 24 h. Then, the cells were To examine effects of IL-1β stimulation on PC12HS cells, the cells were exposed to 100 ng/ml rat IL-1β (CYT-395, ProSpec) for 48 h.
Mizagliflozin or phlorizin at a concentration of 1 or 10 μM was added to the culture 24 h before or 6 h after IL-1β stimulation started. After hypoxia or the IL-1β stimulation, the cells were harvested and ho- F I G U R E 2 Cerebral blood flow (CBF) and blood sugar (BS) levels in the five different groups of mice at 35 days after ACAS or the sham operation. Saline, mizagliflozin or phlorizin was stared to administer one week before ACAS using an osmotic mini pump. Panel A, Representative CBF images of laser speckle flowmetry. Panel B-C, Mean local CBFs in circular regions of ROI1 and ROI2. Panel D, Blood sugar (BS) in the five different groups of mice. Data are the mean ± SE obtained from 8 mice for each group. Sham-operated + saline, saline-pretreated sham operation mice; ACAS + saline, saline-pretreated ACAS mice; ACAS + mizagliflozin (100 μg/μl), low dose (100 μg/ μl) of mizagliflozin-pretreated ACAS mice; ACAS + mizagliflozin (500 μg/μl), high dose (500 μg/μl) of mizagliflozin-pretreated ACAS mice; ACAS + phlorizin (460 μg/μl), phlorizin (460 μg/μl)-pretreated ACAS mice

| Drugs
propionamide) is a selective SGLT1 blocker, which was kindly provided by Kissei Pharmaceutical Co., Ltd. Phlorizin dihydrate was purchased from Sigma-Aldrich Co.

| Statistical analysis
All data are shown as the mean ± SE. We used an analysis of variance (ANOVA) with Dunnett's test for the statistical analysis of multiple comparisons of data. Two-way and one-way ANOVA with Dunnett's test were used for statistical analysis of the Morris water maze test and other experiments, respectively. p < .05 was considered significant.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 17 and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. 18

| Mizagliflozin did not reverse ACAS-induced decreases in CBF
To examine the effects of mizagliflozin on ACAS-induced decreases in CBF, CBF was measured and local CBFs were calculated in circular regions of ROI1 and ROI2 (Figure 2A). Representative images of laser speckle flowmetry revealed that CBF was decreased in saline-treated ACAS compared with sham operation mice (Figure 2A). CBF was similar between saline-treated and mizagliflozin-treated ACAS mice. Interestingly, CBF was similar between sham-operated and phlorizin-treated ACAS mice.
Quantitative analysis demonstrated that ACAS decreased local CBF in both circular regions of saline-treated ACAS compared with sham operation mice ( Figure 2B and C). Mizagliflozin did not reverse the decreased local CBF in ACAS mice regardless of the infusion concentration. In contrast, phlorizin significantly reversed the decreased local CBF in both circular regions of ACAS mice ( Figure 2B and C).

| Mizagliflozin and phlorizin did not affect blood glucose levels
Blood glucose levels were similar among the five different groups of mice ( Figure 2D).

| Mizagliflozin reversed ACAS-induced shortening of latency to fall measured by wire hang test
The wire hang test showed that the latency to fall was shorter in saline-treated ACAS compared with sham-operated mice ( Figure 3A). Moreover, ACAS significantly shortened the latency to fall in saline-treated ACAS mice compared with mizagliflozintreated or phlorizin-treated ACAS mice. ( Figure 3A). Interestingly, the latency to fall was similar among sham-operated mice, low or high concentrations of mizagliflozin-treated ACAS mice, and phlorizin-treated ACAS mice, indicating that mizagliflozin and phlorizin can improve the shortened latency to fall to the normal level in ACAS mice ( Figure 3A).

| Mizagliflozin did not affect spatial working memory or spontaneous activity measured by Ymaze test
The Y-maze test showed that the percentage of alternation behaviors was similar among the five different groups of mice at two and four weeks after ACAS ( Figure 3B). Moreover, the rates of arm entries were also similar among the five different groups of mice at two and four weeks after ACAS ( Figure 3C).

| Mizagliflozin reversed ACAS-induced prolongation of escape latency measured by Morris water maze test
The Morris water maze test showed that the escape latency was sig-

| Mizagliflozin ameliorated increase in number of shrunken neurons in ACAS mice
To examine the effects of mizagliflozin on ischemia-induced neuronal damage, shrunken neurons with pyknotic nuclei in the hippocampal area were observed. Representative images of the hippocampal area revealed pyknotic cell death (white arrows) in saline-treated ACAS mice but not among the three other groups of mice ( Figure 4A). Quantitative analysis of shrunken neurons with pyknotic nuclei in the C1 area of the hippocampus showed an increased number of shrunken neurons in saline-treated ACAS mice compared with the three other groups of mice ( Figure 4B).

| Mizagliflozin did not reduce IL-1β and TNFα mRNA expressions in ACAS mice
mRNA expressions of IL-1β and TNFα genes were significantly

| Post treatment with mizagliflozin induced prolongation of escape latency measured by Morris water maze test in ACAS mice
We examined the effects of post-treatment with mizagliflozin (5 days after ACAS) on behavioral performance in ACAS mice. CBF was decreased in ACAS mice receiving post-treatment with mizagliflozin compared with sham-operated mice ( Figure 6A). The latency to fall measured by the wire hang test was similar between mizagliflozintreated ACAS and sham-operated mice ( Figure 6B). The Y-maze test showed that the percentage of alternation behaviors and rates of arm entries were similar between mizagliflozin-treated ACAS and sham-operated mice ( Figure 6C). In contrast, the Morris water maze test showed that the escape latency was significantly longer in mizagliflozin-treated ACAS mice than in sham-operated mice during the 5-days acquisition phase ( Figure 6D).

| Both mizagliflozin and phlorizin prevented and improved IL-1β-induced cell injury in PC12HS cells
To examine the mechanism of mizagliflozin-and phlorizin-induced  Figure 8B and B).

| DISCUSS ION
We demonstrated that: 1) mizagliflozin did not ameliorate the de- Cerebral small vessel disease causes blood-brain barrier (BBB) functional damage, chronic inflammation such as the increased IL-1β and TNFα expression, and leukocyte infiltration, leading to the development of neuron and oligodendrocyte injury. 19 A study demonstrated that increased IL-1β and TNFα expressions were observed in the hippocampus of a rat cerebral small vessel disease model. 20 In addition, cerebral hypoperfusion induced a longer escape latency in the Morris water maze test in rats. 21 Moreover, hypoxia-inducible factor-1 (HIF-1) mediated transcriptional activation of IL-1β in astrocyte cultures. 22 Interestingly, SGLT1 existed in neuron and brain small vessel endothelial cells but not in glial cells, 23,24 and cerebral ischemia increased SGLT1 expression in mouse brains. 6 Our previous study demonstrated that ACAS- gested that SGLT1 causes ischemic brain injury, especially simultaneously developed hyperglycemia 6 via Na + influx through SGLT1. 7 In fact, SGLT1 allowed sodium ions to flow into the cells, leading to induce cell membrane depolarization and calcium overload. 28,29 Those changes in neurons were associated with the development of cerebral ischemic neuronal damage. 30,31 Therefore, cerebral SGLT1 inhibition by mizagliflozin might improve cerebral ischemic neuronal damage through the inhibition of membrane depolarization and calcium overload induced by Na + influx.
In this study, cerebral gene expression of MCP-1 was increased in ACAS mice. Cerebral ischemia increased MCP-1 expression in astrocytes, neurons, and endothelial cells. 32,33 In addition, chronic cerebral hypoperfusion induced MCP-1-mediated activation of microglia, white matter lesions, and cognitive deficits in mice. 34 Moreover, MCP-1 is likely to increase IL-1β and TNFα expressions in microglia. 35 Therefore, the cerebral ischemia increases MCP-1 expression in endothelial cells and/or neurons, which causes the activation of microglia, leading to inflammatory response such as increases in IL-1β and TNFα expression, and VCI in small vessel disease. Also, MCP-1 can regulate the migration and infiltration of monocytes/macrophages into the brain. and the activated macrophages release several cytokines such as IL-1β and TNFα, leading to the exacerbation of inflammation and neuronal injury. Moreover, MCP-1 increased brain endothelial permeability through its receptors present on brain endothelial cells. 36 This study demonstrated that even after mizagliflozin treatment, We examined the effects of pre-and post-treatment with mizagliflozin on behavioral performance of ACAS mice in wire hang,  We demonstrated that phlorizin reversed the decreases in cerebral blood flow in ACAS mice, and that the cerebral blood flow was similar between phlorizin-treated ACAS mice and shamoperated mice. Moreover, all of the factors tested were similar between phlorizin-treated ACAS mice and sham-operated mice, suggesting that phlorizin ameliorates neural injury and VCI through the improvement of cerebral blood flow. However, the mechanism of phlorizin-induced improvement of cerebral blood flow and in ACAS mice is unknown. The present study demonstrated that phlorizin increased GLUT1 and 3 gene expressions in ACAS compared with sham-operated mice. It is known that GLUT1 is enriched in central nervous system endothelial cells (Harik et al., 1990). 38 Moreover, GLUT1 haploinsufficiency in mice was reported to decrease brain glucose uptake, reduce cerebral blood flow, and increase BBB permeability. 39 Although phlorizin inhibits GLUT1, phlorizin-induced increases in GLUT1 gene expression in brain endothelial cells may contribute to the improvement of cerebral blood flow in ACAS mice. In contrast to GLUT1 haploinsufficiency, GLUT3 haploinsufficiency did not affect glucose uptake into brains, 40 suggesting that changes in GLUT3 expression do not affect brain function. Phlorizin did not reduce IL-1β-induced increases in SGLT1 and MCP-1 gene expressions,

MCP
whereas it ameliorated IL-1β-induced cell death in PC12HS cells.
These results suggest that phlorizin improves hippocampal neu- This study has study limitations. SGLT1 existed in neurons and brain small vessel endothelial cells but not in glial cells. 23,24 However, recent studies showed that SGLT1 may be expressed in glial cells because SGLT1 mRNA was observed in rat astrocytes, 41 and SGLT1related immunoreactivity was reported in glial cells of the rat ventromedial hypothalamic nucleus. 42 Nevertheless, whether microglia or astrocytes are involved in the protective effects of mizagliflozin is still unclear because different SGLT1 expressions in various organs of mice and rats have been confirmed using quantitative RT-PCR. 43 Therefore, the effects of mizagliflozin on microglia and astrocytes would be examined in future project.
In conclusion, we first demonstrated the possibility of pharmacological therapy using an SGLT1 blocker for the prevention of VCI and SGLT1 blocker-induced improvement of VCI through the inhibition of neural SGLT1 in a mouse model of small vessel disease.

E TH I C S A PPROVA L S TATE M E NT
The study was performed in strict accordance with the recommen- and all efforts were made to minimize suffering.

ACK N OWLED G EM ENTS
We are grateful to Ms. Takahashi for secretarial assistance.
Mizagliflozin was kindly provided by Kissei Pharmaceutical Co., Ltd.

D I SCLOS U R E
No author has an actual or perceived conflict of interest with the contents of this article. Wrote or contributed to the writing of the manuscript: Nanae

AUTH O R S CO NTR I B UTI O N S
Ishida, Masamichi Hirose, Maki Saito.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.