Post Stroke Cognitive Impairment and Hippocampal Neurovascular

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1 1 2 Post Stroke Cognitive Impairment and Hippocampal Neurovascular Remodeling: The Impact of and Sex Rebecca Ward 1, John Paul Valenzuela 2, Weiguo Li 2,4, Guangkuo Dong 2,4, Susan C. Fagan 3,4, Adviye Ergul 2, Departments of 1 Neuroscience and Regenerative Medicine and 2 Physiology, Augusta University, 3 Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, and 4 Charlie Norwood Veterans Administration Medical Center, Augusta, GA Corresponding Author: Adviye Ergul, MD, PhD Department of Physiology Augusta University th Street CA-294 Augusta, GA 3912 aergul@augusta.edu Tel: Fax: Running Title: Hippocampal Neurovascular Remodeling Impact of & Sex Number of Pages: 34 1

2 Number of Figures: 7 Grant Numbers: BX347, R1NS83559, PO1HL12827, BX891, R21NS63965, 17PRE33664 Key Words: Cognition, type 2 diabetes, blood vessels, memory, neurodegeneration Abstract increases the risk and severity of cognitive impairment, especially after ischemic stroke. Pathological remodeling of the cerebrovasculature has been postulated to contribute to poor neuronal repair and worsened cognitive deficits in diabetes. However, little is known about the effect of diabetes on the vascularization of hippocampus, a domain critical to memory and learning. Therefore we had two aims for this study: 1) to determine the impact of diabetes on hippocampal neurovascular remodeling and the resulting cognitive impairment after stroke using two models with varying disease severity, and 2) to compare the effects of ischemia on hippocampal neurovascular injury in diabetic males and females. Stroke was induced by middle cerebral artery occlusion (MCAO) by either the suture or embolic method in control and diabetic age-matched male and female Wistar rats. Hippocampal neuronal density, vascular architecture, and microglial activation as well as cognitive outcomes were measured. Embolic MCAO induced greater neuronal degeneration, pathological vascularization, microglial activation and cognitive impairment in diabetes as compared to control animals or 6 min MCAO. While diabetic males had lower neuronal density at 2

3 baseline, diabetic females had more neurodegeneration after stroke. Control animals recovered cognitive function by D14 after stroke, diabetic animals showed deficits regardless of sex. These results suggest that mechanisms underlying cognitive decline in diabetes may differ in males and females and provide further insight to the impact of diabetes on stroke severity and poststroke cognitive impairment New and Noteworthy The present study is the first to provide comparative information on the effects of diabetes and ischemia on cognitive outcomes in both sexes while also evaluating the neurovascular structure in the hippocampus, a critical region for cognitive and memory-related tasks

4 Introduction Stroke is a leading cause of disability in the U.S. due to sensorimotor and cognitive deficits, which arise as a result of ischemic injury. Comorbid diseases such as diabetes, hypertension and chronic kidney disease can increase the risk of stroke and is often associated with worsened outcome (6). Post-stroke cognitive impairment is a major cause of disability and is worsened in diabetic patients but underlying reasons are not fully understood (42). While ischemic stroke and cognitive impairment are considered diseases of the elderly, an alarming increase in the number of younger patients diagnosed with diabetes also brings the increased risk for stroke and cognitive impairment at relatively earlier ages (6, 11, 13, 26). Acute ischemic stroke is considered a heterogeneous disease in part due to the occlusion of different size vessels by atherosclerotic and embolic mechanisms. Thus, better modeling and understanding of the effect(s) of ischemic stroke on cognitive domains in diabetes can unravel novel mechanisms with strategic and therapeutic potential. The hippocampus is an important contributor to learning and memory. Formation of new episodic memories, spatial memory and spontaneous exploration of novel environments are some of the critical functions of the hippocampus. The hippocampus formation is comprised of the hippocampus proper (CA1-4), subiculum and the dentate gyrus (DG) (24). The hippocampus is extremely sensitive to remote ischemic injury. Global ischemia, which models cardiac arrest, induces delayed neuronal death 2-4 days after ischemia is induced (41, 47, 55). While the middle cerebral artery (MCA) does not directly 4

5 supply the hippocampus, neurodegeneration occurs after MCA occlusion (MCAO), particularly in the CA1 region (49). The effect of diabetes on hippocampal neurodegeneration has been investigated using different models of diabetes and ischemia with varying disease severity in independent studies (19, 22, 3, 38, 51, 53). alone has mediated reductions in hippocampalbased cognition and neuronal density (3, 4, 43, 45). Given the differences in the severity of diabetes and ischemic injury induced by various approaches, and the fact that the MCA is the most commonly occluded vessel leading to ischemic stroke, the first goal of this translational study was to compare the impact of the method of MCAO on hippocampal neurovascular remodeling and the resulting cognitive impairment in a clinically relevant diet-enhanced model of type 2 diabetes. Females with diabetes appear to be particularly vulnerable to ischemic stroke (31). Furthermore, older women with diabetes have higher risk of stroke recurrence than men (39). Following stroke, females are more likely to enter a nursing home due to worsened cognitive impairment (7, 8, 16). Although this can be attributed to a more advanced age at onset, the possibility of differing pathologies has been considered. Experimental studies that investigated the impact of diabetes and stroke on hippocampal structure and function have mostly used male animals. Therefore, the second goal of the study was to determine the effects of ischemia on hippocampal neurovascular remodeling in both sexes under control and diabetic conditions. 16 5

6 Methods Animal Model The animals were housed in Augusta University animal care facilities, which are approved by the American Association for Accreditation of Laboratory Animal Care. This study was conducted in accordance with the National Institute of Health guidelines for the care and use of animals in research. The institutional animal care and use committee approved all protocols used in these studies. was induced using a high-fat diet plus streptozotocin (HFD/STZ) in male and female Wistar rats. Rats were started on a 45 kcal% fat (Research Diets Inc., New Brunswick, NJ) diet at 4 weeks of age and received a single dose STZ injection (3 mg/kg) at 6 weeks of age. Control animals received regular chow. Although STZ used in this diet-enhanced model is typically associated with type 1 diabetes, the presence of insulin resistance, and positive response to metformin treatments supports a type 2 diabetic model (9, 12, 44). Blood glucose and body weight were measured twice a week until euthanasia. Embolic or mechanical suture occlusion was induced at 12-weeks of age. Diabetic animals continued to receive a HFD after stroke induction. Average blood glucose levels were 79 ± 5, 271 ± 36, 75 ± 3 and 261 ± 34 in control male, diabetic male, control female and diabetic female groups, respectively. Hemoglobin A1c (%) levels in male and female diabetic animals were 11.1 ±.7 and 11.4 ±.4, respectively Preparation of Blood Clot 6

7 Clot preparation was modified from earlier reports and improved to increase clot stability as reported previously (2, 52). Briefly, arterial blood from a donor Wistar rat was supplemented with human fibrinogen and immediately withdrawn into polyethylene (PE)-5 tubing (2 cm with an inner diameter of.25 mm) to clot for 6 hr at room temperature, followed by 18 hr at 4 C. Tubing containing the clot was cut into pieces 5 cm long and transferred to a petri dish containing sterile saline for further retraction for 4 hr at room temperature. A single 4±.5 cm long clot was transferred to a PTFE Sub-lite catheter (Braintree Scientific Inc., Braintree, MA) for surgery Stroke Surgery Focal cerebral ischemia was induced by suture or embolic MCAO as described previously (2, 28). Age matched male and female control and diabetic rats were randomly assigned to three different procedures: sham surgery, suture MCAO or embolic MCAO. Sham animals were used for male x female comparisons only (Figs 6-8). Sham groups included n=7 per group with the exception of n=6 in diabetic male group in which 1 animal died before surgery. In the 6 min MCAO study Day animal numbers were 7, 8, 7 and 9 in control male, diabetic male, control female and diabetic female, respectively. Diabetic groups started with extra animals based on our experience with more mortality in this group. Respective animal numbers at Day 14 were 6, 5, 7 and 6 in control male, diabetic male, control female and diabetic female, respectively. Hence, mortality rate was 14, 37.5, and 33.3% in control male, diabetic male, control 7

8 female and diabetic female, respectively. In the embolic stroke study, Day animal numbers were 7, 11, 8 and 12 in control male, diabetic male, control female and diabetic female, respectively. Respective Day 14 animal numbers were 7, 7, 8 and 8 in control male, diabetic male, control female and diabetic female. Mortality rate was, 36, and 33.3% in control male, diabetic male, control female and diabetic female, respectively. If the animals did not survive until Day 14, they were not included in analyses at any time point. Animals were anesthetized with 5% and maintained with 2% isoflurane in 7% N 2 and O 2 using a facemask. In the suture groups, a nylon suture was inserted through the external carotid artery (ECA) to occlude the MCA for 6 min followed by reperfusion. In the embolic groups, the catheter containing the blood clot was inserted up to the origin of the MCA and injected with 1 µl of sterile saline. The catheter was removed directly following embolization. In the sham groups, surgery was stopped once ECA was identified and then neck incision was sutured as in stroked animals. To confirm successful occlusion in both stroke models, CBF reductions were examined using laser Doppler imaging and compared to CBF prior to occlusion (Fig 1). The CBF percentage drop was calculated on the ipsilateral side by subtracting the baseline CBF from the contralateral side. We measure blood flow using a scanning laser Doppler which measures flow in the entire hemisphere without a need to thin the skull or glue the Doppler probe. We have reported that we achieve reproducible infarcts with a 4% drop in flow (18, 28). Previous studies in our lab have shown a gradual reperfusion following embolic MCAO beginning around 2 h after occlusion and 8

9 recovering to baseline levels by 6 h after ischemia in control male rats (28). We did not measure this in the current study due to neuroprotection which may occur by repeated exposure to anesthesia. Female rats underwent surgery during the diestrus phase after careful monitoring of the estrus cycle by vaginal swab for 2 weeks prior to the surgery. While we did not measure blood pressure in this particular study, we have measured blood pressure using the tail cuff method in HFD/STZ diabetic rats. MAP was 13 ± 4 in diabetic and 11 ± 3 in the control groups (n=6/group) Evaluation of Sensorimotor and Neurobehavioral Outcomes Sensorimotor and cognitive tests were recorded and scored in a blinded fashion. Animals were handled and trained in the room where tests were to be carried out for 5-7 days prior to surgery. Sensorimotor function was examined through the adhesive removal test (ART) as described previously (28). Briefly, animals were trained to remove an adhesive paper dot for 5 days prior to baseline recording. Contact and removal latency was reported at baseline, day 1, 7 and 14 after MCAO. For each day, 3 trials were averaged with the maximum removal latency at 18 seconds per trial. This sensitive test was used to ensure a stroke was induced and to compare sensorimotor deficits/recovery between control and diabetic animals. Cognition was measured through novel object recognition (NOR) task as previously described (4). Animals were habituated to test apparatus for 4 days prior to baseline testing for 1 min. On the day of testing, the animal had 3 9

10 phases: acclimation, familiarization and novel. The acclimation phase allowed the animal to habituate to the box each day without any objects present for 5 min. During the familiarization phase, or A/A session, animals were allowed to explore two identical objects for 5 min. The rats were placed back into their home cages for a 15 min delay, followed by a 5 min novel phase (A/B session) in which the familiar object from the A/A session was paired with a novel object. Objects were placed equidistant from the walls, with 2 cm between the two objects. Rats were placed in the center of the box for each session. Between each phase, objects and the testing apparatus were cleaned with 2% ethanol. The time spent exploring each object was recorded and either recognition index (RI; total time (sec) spent exploring novel object (T N ) divided by the total time spent exploring both novel and familiar (T F ) objects: RI=T N /(T N +T F )) or discrimination index (DI; T N minus T F divided by total time spent exploring both objects: DI=(T N - T F )/(T N +T F )) was calculated for both the A/A and A/B sessions. NOR test was administered at baseline and day 14 after stroke surgery. The following inclusion criteria were used: RI must be between.4 and.6 in the A/A session and the animals must explore objects a minimum of 3 seconds within the 5 min trial Assessment of Neurovascular Remodeling Vascular Indices Vascular networks in the hippocampus were visualized with the space filling FITC-dextran injection method, as we reported previously (1, 4). Briefly, rats were anesthetized and injected through the jugular vein with high molecular 1

11 weight FITC-dextran (molecular weight 2,,; Sigma-Aldrich, St. Louis, MO) 1 min prior to euthanasia. Brains were isolated on day 14 post-surgery and fixed in 4% paraformaldehyde for 48 hr, followed by cryoprotection in 3% sucrose at 4 C. Serial images were collected from 1-µm thick sections to produce a 3D image on a Zeiss Upright Confocal 72. The mean value of three 1 µm apart sections were averaged together in the CA1 or DG hippocampal regions on both the ipsilateral and contralateral sides for each animal. Images were processed through Volocity software and reconstructed three dimensionally. Vascular volume refers to ratio of the volume of the vasculature to the total volume. Total volume refers to the total volume of the Z-stacked image. Surface area was calculated by absolute surface area of the vasculature over total volume. Fiji software was used to measure branch density. Branch density was calculated by creating a binary image, skeletonizing this image and averaging the number of branches over the longest/shortest path multiplied by 1%. For each image, 8-1 measurements were averaged after being sorted by longest/shortest path. Identical threshold parameters were used across all the sections. The number of animals per group differed than that reported in behavioral tests because in some animals FITC injection was not successful or brain tissue was too damaged to be processed resulting in n=5-7 per group Neuronal Indices Identical threshold parameters were used across all the sections for all the following measurements. Cryostat sections 2 µm in thickness (2 µm apart, 3 11

12 sections/rat) were incubated in NeuN (1:5; Abcam) antibody at 4ºC overnight. This was followed by incubation with a fluorescent conjugated (1:1; Thermofisher Alexa Fluor 647) for 1 hr at room temperature. The same regions were taken from each animal. Number of NeuN-positive neurons in the entire CA1 or DG was counted in a 2x objective area using the multi-point tool in Fiji by a blinded investigator. Neuron within one section was counted, rather than the entire hippocampus. Cell death was examined using a TUNEL assay (GeneCopoeia, MD) counterstained with DAPI in 2 µm sections. Fluorescent images were captured from the CA1 and DG of the hippocampus on both the ipsilateral and contralateral hemispheres using a Zeiss LSM 78 upright confocal microscope (Carl Zeiss Micro-Imaging, Thornwood, NY). Images were captured at 1x magnification for TUNEL staining in 2-3 sections per animal. In each section, the whole CA1 and DG regions were captured. Total TUNEL-positive cells per section were counted by unbiased investigator. Blood Brain Barrier (BBB) Integrity BBB permeability was calculated using immunohistochemistry for IgG (1:25; BD Bioscience) with an HRP-conjugated secondary (1:1; Vector) in the CA1 and DG regions of the hippocampus using a Zeiss Axioplan 2 Imaging (Carl Zeiss Micro-Imaging, Thornwood, NY). Briefly, free-floating sections had exogenous peroxidases quenched with 3% H 2 O 2 in methanol for 3 min. After blocking for an hour, the primary IgG antibody in blocking solution was incubated overnight at 4ºC. Sections were rocked at room temperature for 3 min the next day. After 3 washes, sections were incubated in the biotinylated secondary 12

13 antibody provided in the Vectastain ABC kit for 1 hr. DAB was used as the peroxidase substrate solution. For each rat, three 2 µm sections that were 2 µm apart were averaged together. Images were captured with the 2x objective and staining was measured using MetaMorph Image Analysis Software (Molecular Devices, LLC; San Jose, CA) by determining the % threshold area. Identical threshold parameters were used across all sections. Threshold was averaged for each animal ipsilaterally. Results were expressed as IgG threshold. Microglial Indices Cryostat sections 2 µm in thickness (2 µm apart, 3 brain sections/rat) were incubated in Iba-1 (1:5; Wako) antibody at 4ºC overnight to examine microglia and macrophages. This was followed by incubation with a HRPconjugated secondary antibody (1:1; Vector) for 1 hr at room temperature. Identical threshold parameters were used across all the sections. Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT) was used to capture and stitch together images of whole hippocampal section stained with Iba-1 at 1x objective. MATLAB was used to produce heat map images from one Iba-1 stained section per animal. The same sections were imaged on a Zeiss Axiovert 2 microscope (Carl Zeiss Micro-Imaging, Thornwood, NY) in the CA1 and DG regions of the hippocampus at 4x objective. These images were then converted to binary and skeletonized using Fiji software. The AnalyzeSkeleton plugin was used to analyze number of microglia process endpoints/cell and summed microglia process length/cell as shown on Fig 5A (34). All data that had 2 or fewer endpoints were discarded and the sum of all endpoints was used for 13

14 the number of microglia process endpoint/cells. To determine the summed microglia process length/cell, the sum of all branch lengths was used after branch lengths less than.1 was removed. Furthermore, cell swelling and number of protrusions per cell were reported for each image. Cell swelling was measured using the freehand selection tool to draw around the cell soma. A blinded investigator counted the number of protrusions from the soma for each microglia Data Analysis All data points are expressed by mean ± SEM. Prism7 was used for all analyses. The effect of ischemia on neurovascular remodeling and functional outcome in control versus diabetic male animals was compared using Student s t-test (Figs 1-3, 5). The interaction of diabetes and stroke on cognitive deficits (RI Day 14) and sensorimotor deficits (ART, Baseline to Day 14) was analyzed by regular and repeated measures of two-way ANOVA, respectively (Fig 4). For figures 6-8, multiple comparisons were made. First, the interaction of diabetes and sex on neurovascular injury and neurological deficits was analyzed by twoway ANOVA [(control vs diabetes) x (females vs males)] for each intervention (sham, 6-min suture or embolic MCAO) separately. Then, the effect of stroke on neurovascular remodeling and functional outcomes were analyzed in a 2 (control vs diabetes) x 2 (sham vs stroke) design for females or males separately. If an interaction was identified, only interaction was marked on graphs rather than 14

15 marking main effects. Bonferroni s post-hoc test was used to compare means from significant ANOVAs. Statistical significance was determined at alpha< Results Comparison of stroke methods on neurovascular remodeling and behavioral deficits in male control and diabetic rats There was a trend for decreased number of neurons in the DG in diabetic animals 14 days after 6 min MCAO (Fig 2A). Embolic stroke in which spontaneous reperfusion gradually starts 2 h after the insertion of the clot (28) significantly reduced the number of NeuN positive neurons in both the CA1 and 322 DG regions in diabetes (Fig 2B). Both 6 min and embolic MCAO induced greater levels of TUNEL-positive cells in the CA1 and DG of the hippocampus in diabetes (Fig 2C-D). Since blood flow is critical to neuronal function and survival, we assessed changes to the vasculature in the hippocampus. 6 min MCAO induced no significant changes in vascular volume (Fig 3A-B), yet vascular volume was higher in both the DG and CA1 region of the hippocampus after embolic MCAO in diabetes (Fig 3C-D). IgG-stained area was greater in diabetic animals compared to control after 6 min MCAO, suggesting increased BBB permeability in these animals (Fig 3E-F). Longer ischemia duration induced by embolic MCAO increased IgG extravasation in the hippocampus to a greater extent in diabetic rats compared to controls (Fig 3H-G). 15

16 Cognitive deficits were evident even at baseline (BL) in diabetic animals (p diabetes =.4; Fig 4). There was a small reduction in recognition index after 6 min MCAO in both groups (p stroke =.411; Fig 4A). This appeared to be more prominent in the diabetic group but it was not significantly different compared to control animals. Sensorimotor deficits were comparable in both control and diabetic animals, which were recovered by day 14 (Fig 4B). In the embolic stroke groups, there was an interaction (F(1,24)=4.951, p=.357) such that cognitive deficits were further reduced in the diabetic but not in the control group after stroke (Fig 4C). There was a trend (p=.75) for greater sensorimotor deficits in diabetic animals as compared to controls over the recovery period, which became significant at Day 14 (Fig 4D). Since inflammation plays an essential role in ischemic injury and outcome, we next examined microglial expression and morphology in the hippocampus. Heatmap analysis was used to visualize Iba-1 positive microglia in both 6 min and embolic MCAO animals (Fig 5A-B). Higher intensity of Iba-1 was expressed in diabetic rats after embolic MCAO ipsilateral to the injury. increased cell body swelling, but reduced the number of microglial protrusions in both 6 min and embolic MCAO compared to control animals, indicating increases in activated microglia. Furthermore, the number of endpoints per microglia and summed process length of microglia was reduced after ischemic injury in both the CA1 and DG in diabetic rats (Fig 5C-F)

17 Comparison of the effect of ischemia and diabetes on hippocampal neurovascular remodeling in female versus male animals Since there was increased vascular remodeling, or change to vascular structure, associated with greater BBB permeability, decreased neuronal density and accompanying functional deficits in male diabetic animals, we next asked the questions whether 1) these changes occur in females and 2) if there is an interaction between diabetes and ischemia. To address these questions, we included female and sham-operated animals in our data analyses. We first compared neuronal density in the DG in sham groups to determine the effect of diabetes alone, which showed reduced number of neurons in diabetic male but not female animals compared with control Wistar rats (interaction F(1,23)=4.946, p=.363). We next compared neuronal density within these four groups of male and female control and diabetic rats after suture or embolic MCAO. Neuron number was less in both diabetic male and female animals compared to controls in 6 min MCAO or embolic stroke group. Given the baseline difference in neuron counts in male animals, we performed additional statistical analyses in which the effect of ischemia was compared with respect to sham surgery in either sex. While ischemia did not cause a further decline in males, both suture and embolic stroke reduced neuron counts in female diabetic animals (Fig 6). Vascular surface area and branch density were analyzed in the DG of both male and female rats in a similar manner to neuronal density assessment (Fig 7). Effect of disease and sex was first examined in each surgery group (sham, 6 min MCAO, or embolic MCAO). Further comparisons were made to 17

18 determine effect of stroke vs sham in both male and female control and diabetic animals. Among sham surgery groups, branch density was greater in both male and female diabetic animals than in control groups. Among 6 min MCAO groups, there was a disease and sex interaction (F(1,14)=3.3) such that branch density was higher in diabetic females but not males. Within the embolic groups, branch density was lower in female rats (p sex =.99) On the other hand, ischemic injury induced by 6 min MCAO (F(1,19)=11.23, p=.34) or embolic stroke (F(1,23)=7.9, p=.139) reduced branch density only in the male diabetic animals (Fig 7B, p interaction =.312 or.139). Surface area, another index of vascularization, suggested a differential effect on diabetes in males versus females. Significant interactions between diabetes (control and diabetes) and sex (male and female) were found (Fig 7C). In the sham group, diabetes decreased surface area in males but increased in females (F(1,28)=12.71, p=.13). Among 6 min MCAO groups, surface area was higher only in the diabetic female rats (F(1,11)=2.35, p<.1). Within the embolic stroke groups, diabetes had a differential effect with surface area being higher in males and lower in females (F(1,2)=21.77, p<.1). 6 min MCAO did not cause any changes in surface area compared to shams in either male or female rats. Embolic stroke, however, increased surface area in male diabetic rats as compared to sham (F(1,21)=55.73, p<.1), while decreased it in females (F(1,27)=9.468, p=.48). In both cohorts of 6 min suture and embolic MCAO, cognitive deficits were evident in the diabetic groups in both sexes at baseline as determined by lower 18

19 discrimination index determined by NOR test. Control animals recovered almost completely. In the 6 min MCAO groups, diabetic animals showed persistent cognitive deficits. While there was no further decline in cognition, the diabetes effect became more prominent (p=.24 baseline vs p=.4). In the embolic stroke groups, D14 cognitive deficits were greater compared to baseline (p=.53) (Fig 8A and B) Discussion Cognitive impairment is an increasingly recognized complication of diabetes that is further amplified by greater incidence of stroke in this population. To the best of our knowledge, the current study is the first to provide comparative information on the effects of diabetes and ischemia on cognitive outcomes in both sexes while also evaluating the neurovascular structure in the hippocampus, a critical region for cognitive and memory-related tasks. Specifically, our findings show 1) that the extent of neurovascular injury, neuroinflammation and cognitive decline in diabetes was more pronounced after embolic stroke as compared to stroke induced by a shorter suture occlusion of MCA, 2) diabetes alone caused hippocampal neuronal loss in male but not female animals even before stroke, 3) diabetic females were more sensitive to ischemic injury as seen by greater neurodegeneration after 6 min and embolic MCAO, 4) hippocampal vascularization patterns at baseline and after ischemic injury differed in males and females, and 5) despite sex differences in the extent and patterns of 19

20 hippocampal neurovascular injury, diabetes worsened cognitive outcomes in both sexes. The hippocampus is an important domain for cognition and memory (24). We wanted to investigate the impact of a remote stroke and compare/contrast strokes of different severities since stroke is very heterogeneous in the clinic. Severe vascular injury following ischemic stroke and the development of secondary hemorrhage in the brain has been observed depending on the duration of occlusion in diabetic animals (28). Therefore, we chose to use a 6 min occlusion to induce less vascular injury and a larger, full blown vascular injury using the embolic MCAO model. The present study showed changes in neuronal density in both males and females after stroke using two different methods of ischemic injury. We showed males have a reduction of neuron density after 1 weeks of diabetes, which supports clinical studies showing up to a 15% reduction in hippocampal volume, with greater loss in the DG (33). Although neuron density was reduced in shams, there was no greater loss after ischemic injury in diabetic males. Contrary to the males, females showed no changes in density at baseline but a decrease 14 days after an ischemic stroke. While there are differences in neuronal density between male and female animals, both have reduced cognition at baseline. Although neuron density was decreased after stroke in females, functional outcome did not further decrease. This discrepancy between neuron counts and functional outcome may be due to the fact that some neurons may not be dead, but are not functioning properly. It should be noted that the number of TUNEL-positive cells is only a small fraction 2

21 of total cells. The present study measured TUNEL at day 14 post-stroke and may have missed the peak time of neurodegeneration. Future studies should investigate neuronal cell death at earlier time points. Studies in male animals have shown impairment of hippocampal synaptic plasticity, dendritic spine loss and lower rates of neurogenesis in both type 1 (19, 46, 54) and type 2 diabetes (29, 45, 5, 51). Perhaps changes in synaptic function in diabetes may differ in males and females, accounting for the differences we observed in functional outcome after stroke. Although the behavioral tasks used in this study implicate hippocampal function, they are not exclusive to the hippocampus. Other domains, such as the prefrontal cortex (a critical domain for executive functioning), is affected by diabetes (25), but not examined in the present study. Neurons rely on constant blood flow for nutrients, which means proper vascular function is essential for proper neuronal function. While blood is supplied to the hippocampus primarily through the posterior communicating artery and a small percentage originating from the anterior choroidal artery (1, 14), it has been shown that MCAO and a short 1 min global ischemia can cause delayed cell death and neurodegeneration, suggesting that the hippocampus is extremely sensitive to ischemic injury (15, 23). This sensitivity to ischemia also implies that any change in perfusion caused by occlusion of a remote artery can have robust effects of hippocampal neuronal survival and in both sexes under control and diabetic conditions. While we did not evaluate post-stroke infarct and edema in the current study, we have previously reported no difference in infarct size between control and diabetic males. It should be noted that in this study, we 21

22 did not observe infarction within the hippocampus. However, hemorrhagic transformation was greater in the diabetic group (28). Contrary to the male cohorts, infarct size, edema and hemorrhagic transformation were all greater in diabetic females compared to controls after stroke (28). The use of the sham group allowed us to assess the impact of diabetes alone on hippocampal vascularization. Vascular architecture was investigated through vascular volume, branch density and surface area calculations. Branch density was increased in both female and male diabetic animals. On the other hand, surface area was reduced in male diabetic rats but increased in female diabetes group. While this finding in male animals is different than a previous study showing increased vascularization in the hippocampus of STZ-induced diabetic mice (46), it is comparable to another study by Beauquis et al. which showed decreased area with von Willebrand Factor immunohistochemistry in the DG of GK rats, a lean and spontaneously diabetic model of type 2 diabetes (5). Various indices of vascularization have also been previously explored in young and old mice fed a high fat diet. Similar to our findings in male diabetic rats, aged mice fed a high fat diet showed less vasculature in the hippocampus (48). While these animals had elevated blood glucose compared to control (155mg/dl), they were not as high as our diabetic rats. This study suggests that perhaps high fat diet alone affects vascularization. Previous studies in our lab have shown that while diabetic male rats develop vasoregression in the cortex and striatum after ischemic injury, control animals show an increase in vasculature after stroke (4). Here we investigated 22

23 the effect of diabetes on hippocampal vasculature after remote ischemic injury. In male diabetic rats, branch density, an index of remodeling and vascular drop out, was reduced after 6 min MCAO, but not after embolic MCAO. Embolic stroke did increase surface area compared to diabetic shams. These results differ from our previous study in which we reported reduced branch density after a 9 min MCAO in the cortex and striatum of male GK rats (4), suggesting local differences in regulation of vascularization. Unlike males, females were not affected after 6 min MCAO but showed reduced vasculature after embolic stroke. Taken together, these results suggest diabetes effects vascular architecture differentially in male and females after stroke and that vascularization pattern may differ depending on the models of stroke and diabetes used in the study. Inflammation is a key mediator of secondary injury after ischemia. While it can play an essential role in protecting the brain from further injury, over activation of inflammatory cells can cause further damage to neurons and endothelial cells (2). is a chronic inflammatory state (32). Activated microglia are more prominent in GK rats and it is thought to contribute to neuronal dysfunction and cognitive impairment (21). A secondary insult, such as a stroke, exacerbates inflammation within the brain. As resident immune cells of the central nervous system, microglia are essential to detecting signals from damaged tissue after ischemic injury. Ramified microglia monitor the healthy brain and are characterized as having a small cell body with extensive branching off the soma (34, 36). On the contrary, activated microglia have a more 23

24 phagocytic phenotype with enlarged somas and retraction of processes (2, 35, 37). Based on these previous studies, we measured cell body swelling, number of endpoints/cell and total length of processes using Iba-1 as a marker, which stains not only resident microglia but also infiltrating macrophages. Using these measurements, we showed increased microglia reactivity in the hippocampus after a short 6 min MCAO and even greater activation after the longer duration embolic MCAO in male diabetic rats. It is important to note, Iba-1 can detect both the M1 and M2 microglial phenotype (56). Therefore, measurements in the current study measure M1 and M2 activation together. Patients with cerebral infarction and diabetes have higher levels of microglial proliferation and activation as seen by ferritin-stained post-mortem sections (27). These results suggest that diabetes amplifies the immune response to stroke, even in remote areas of the brain, and may be a contributing factor to worsened stroke outcome. There are several limitations to the current study. First, this study was focused on determining the impact of diabetes and stroke interaction on the hippocampus in both sexes and hence does not include any mechanistic studies. Secondly, we only investigated microglial/macrophage density and morphology as a measure of inflammation. Further studies are needed to determine the contribution of inflammation to neuronal and vascular dysfunction after diabetic stroke. Additional use of markers that identify resident microglia such as TMEM119 will strengthen our findings. Third, these studies were done in young animals. Stroke is considered an aging disease and although diabetes does increase stroke incidence in younger patients, little is known about neurovascular 24

25 changes in aged animals. Additionally, this study does not include ovariectomized animals to investigate the effect of sex hormones. It has been well documented that 17β-estradiol (E2) is neuroprotective in premenopausal states (17). On the other hand, our previous study showed that young diabetic female animals lose this protection (28). While we did not measure hormone levels, estrous cycle monitoring with vaginal swabs showed regular cycles in diabetic animals suggesting hormone levels are not affected. Nevertheless, further studies are needed to assess the role of sex hormones after diabetic stroke. Finally, we followed animals only up to day 14 after stroke due to increased mortality in the diabetic groups and longer term studies with multiple cognitive/memory tasks are definitely needed. Nevertheless, the present study is the first to investigate the effect of different methods of ischemia on hippocampal neurovascular structure and cognitive function in both sexes a clinically relevant diet-enhanced model of type 2 diabetes. Our findings provide further insight to the design of future studies on cognitive decline in diabetes and diabetic stroke Acknowledgements Adviye Ergul is a Research Career Scientist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, Georgia. This work was supported in part by VA Merit Award (BX347), VA Research Career Scientist Award, and NIH awards (R1NS83559, PO1HL12827) to Adviye Ergul; VA Merit Award (BX891) and NIH award (R21NS63965) to Susan C. Fagan, and American Heart Association Predoctoral Fellowship (17PRE33664) to Rebecca Ward. 25

26 We would like to thank Drs. Stephen Tomlinson, Ali Alawie, and Lin Mei as well as Heath Robinson for their assistance with the microglia heatmap analysis. The contents do not represent the views of the Department of Veterans Affairs or the United States Government Figure Legends Figure 1: Relative cerebral blood flow (rcbf as % of pre MCAO) change 5 min after emboli insertion. Representative images captured with a scanning laser Doppler system from control and diabetic male and female animals and the cumulative data shown in the bar graph demonstrate that there is no difference in the magnitude of CBF drop caused by MCAO among the group. n=4-6/group. A representative image from a sham operated animal given on top shows perfusion in both hemispheres Figure 2: Embolic stroke induces greater neurodegeneration in diabetes. Representative images of the ipsilateral CA1 and DG regions stained with NeuN in control and diabetic rats (A-B). While only embolic MCAO reduced number of neurons (B), both a shorter 6 min suture and longer embolic MCAO increased TUNEL-positive cells (C-D). (n=5-7; Student s t-test control vs diabetes; * p<.5, **p<.1, ***p<.1 vs control) Figure 3: Vascular integrity is compromised after diabetic stroke. FITC- dextran injected vasculature was used to measure vascularization status at 14 26

27 days after stroke. No significant changes were induced in % vascular volume ipsilaterally after 6 min MCAO (A-B). % vascular volume increased after embolic stroke in diabetic rats in the hippocampus (C-D). Increased IgG extravasation (% threshold area) was seen in diabetic animals after both a 6 min and embolic MCAO (E-H). (n=5-7, Student s t-test control vs diabetes, **p<.1, ***p<.1 vs control) Figure 4: induces greater cognitive and sensorimotor deficits after stroke. In the 6 min MCAO study, cognitive deficits were evident even at baseline in diabetic animals. After stroke, there was a small decline from baseline in both groups (A; ANOVA table: interaction p=2.515, diabetes effect p=.4, stroke effect p=.411). Sensorimotor deficits measured by removal latency in ART showed similar deficits in both groups and were recovered by Day 14 (B; ANOVA table: interaction p=.343, diabetes effect p=.92, stroke effect p=.9). Embolic stroke caused a further decline in cognitive deficits in diabetic but not control animals as indicated by a significant interaction (C; ANOVA table: interaction p=.357, diabetes effect p<.1, stroke effect p=.16). There was a trend for greater sensorimotor deficits in diabetic animals that became significant at Day 14 (D; ANOVA table: interaction p=.26, diabetes effect p=.75, stroke effect p<.1). (n=5-7; 2-way ANOVA for RI and 2-way repeated measures of ANOVA for ART). Significant outcomes from ANOVA tables are shown on top (p diabetes = control vs diabetes; p stroke = BL vs D14; p interaction = (control vs diabetes) x (BL vs D14)). 27

28 Figure 5: increases inflammation in the hippocampus after a remote ischemic injury. Heatmaps generated by Iba-1 staining after 6 min (A) and embolic (B) MCAO show increased microglia in the hippocampus. Cell swelling (µm 2 ), number of protrusions from microglia cell body (red lines), number of endpoints at the tips of microglia processes (blue circles) and process length (grey lines) depicted on panel A were calculated from 4x images. Cell swelling increased while protrusions decreased in diabetes after 6 min (C-D) and embolic (E-F) MCAO. (n=4-5; Student s t-test control v diabetes, *p<.5, **p<.1, ***p<.1 vs control) Figure 6: Diabetic females have greater neurodegeneration after stroke. Representative images of NeuN stained DG sections on the ipsilateral hemisphere in sham, 6 min MCAO and embolic stroke groups. Multiple comparisons were made. 2-way ANOVA analysis within each intervention group indicated that diabetes reduced number of neurons in the DG in male but not female sham animals as indicated by a significant interaction (ANOVA table for sham groups: interaction p=.363, diabetes effect p=.26, sex effect p=.12). Within 6 min MCAO (ANOVA table for 6 min MCAO groups: interaction p=.954, diabetes effect p=.358, sex effect p=.4995) and embolic MCAO groups (ANOVA table for embolic MCAO groups: interaction p=.998, diabetes effect p<.1, sex effect p=.7441), number of neurons was lower in diabetic animals as compared to control male and female animals. 2-way 28

29 ANOVA analysis comparing sham control or diabetic animals to stroked animals within each sex showed that no further reduction was observed after stroke in diabetic males (ANOVA table for male sham v 6 min MCAO groups: interaction p=.2219, diabetes effect p=.2, stroke effect p=.4942; ANOVA table for male sham v embolic MCAO groups: interaction p=.814, diabetes effect p=.5, stroke effect p=.791). Female diabetic animals have reduced number of neurons after 6 min (ANOVA table for female sham v 6 min MCAO groups: interaction p=.6713, diabetes effect p=.452, stroke effect p=.17) and embolic MCAO (ANOVA table for female sham v embolic MCAO groups: interaction p=.1558, diabetes effect p=.37, stroke effect p=.18) as compared to sham. n=4-7; 2-way ANOVA tables for disease (control vs diabetes) X sex (male vs female) for each intervention (sham, 6 min MCAO or embolic MCAO) are shown on top. Significant outcomes from 2-way ANOVA results for disease (control vs diabetes) X intervention (sham vs stroke) for each sex were indicated below ANOVA tables if there is significance Figure 7: Vascularization differs after stroke in male and female diabetic rats. (A) Representative images of ipsilateral FITC-injected vasculature in the DG of male and female rats. (B) alone increased branch density in both sexes (ANOVA table for sham groups: interaction p=.1666, diabetes effect p=.2, sex effect p=.9696). Comparison of groups after 6 min MCAO study demonstrated that there is a sex and disease interaction such that branch density is higher only in the diabetic females but not males (ANOVA table for 6 min 29

30 MCAO groups: interaction p<.1, diabetes effect p=.9, sex effect p=.4). On the other hand, after embolic MCAO, branch density was lower in diabetic females as compared to males (ANOVA table for embolic MCAO groups: interaction p=.4376, diabetes effect p=.2912, sex effect p=.99). When stroked groups were compared to sham, additional disease and intervention interactions were noted in male animals (ANOVA table male sham v 6 min MCAO groups: interaction p=.34, diabetes effect p=.181, stroke effect p=.11; ANOVA table female sham v 6 min MCAO groups: interaction p=.2126, diabetes effect p=.4, stroke effect p=.36; ANOVA table male sham v embolic MCAO groups: interaction p=.139, diabetes effect p=.6, stroke effect p=.8451; ANOVA table female sham v embolic MCAO groups: interaction p=.1388, diabetes effect p=.962, stroke effect p=.986). 6 min MCAO caused a decrease, whereas embolic stroke caused an increase in branch density in male diabetic animals. (C) At baseline (sham), surface area was reduced in diabetic males, but increased in diabetic females (ANOVA table for sham groups: interaction p=.13, diabetes effect p=.7195, sex effect p=.5). While surface area was not affected by suture MCAO (ANOVA table for 6 min MCAO groups: interaction p=.9, diabetes effect p=.29, sex effect p=.37), embolic stroke mediated a differential pattern in diabetic animals with males showing greater and females showing less surface area (ANOVA table for embolic MCAO groups: interaction p<.1, diabetes effect p=.2369, sex effect p=.248). When embolic stroke groups were compared to sham, additional disease and intervention interactions were noted. 3

31 caused a decrease in surface area under sham surgery conditions but an increase in the same parameter after embolic stroke (ANOVA table male sham v 6 min MCAO groups: interaction p=.243, diabetes effect p=.2, stroke effect p=.3581; ANOVA table male sham v embolic MCAO groups: interaction p<.1, diabetes effect p=.1567, stroke effect p=.6). On the other hand, in females, diabetes increased surface area at baseline but lowered it after stroke (ANOVA table female sham v 6 min MCAO groups: interaction p=.4111, diabetes effect p=.17, stroke effect p=.3645; ANOVA table female sham v embolic MCAO groups: interaction p=.48, diabetes effect p=.6553, stroke effect p=.586). n=4-8, Significant outcomes from 2-way ANOVA tables for disease (control vs diabetes) X sex (male vs female) for each intervention (sham, 6 min MCAO or embolic MCAO) are shown on top. Significant outcomes from 2- way ANOVA results for disease (control vs diabetes) X intervention (sham vs stroke) for each sex were indicated below ANOVA tables if there is significance Figure 8: Diabetic male and female rats both have stroke-induced cognitive decline. (A) Discrimination index, which shows the difference between time spent exploring novel and familiar objects, is lower in diabetic animals at baseline (ANOVA table for groups at BL: interaction p=.743, diabetes effect p=.24, sex effect p=.145) and remain lower at D14 after 6 min MCAO (ANOVA table groups at D14: interaction p=.293, diabetes effect p=.4, sex effect p=.634) while control animals recovered back to baseline (ANOVA table for male groups: interaction p=.1998, diabetes effect p=.59, stroke effect 31

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42 Sham rcbf drop (% of premcao) M ale F e male Control

43 A. 6 min MCAO B. Embolic MCAO CA1 DG Control Control Control Control CA1 DG Number of Neurons C. D. Control CA1 Control CA1 Control p=.722 DG DG Number of Neurons CA1 CA1 Control * ** DG DG Control Mean TUNEL Positive Neurons ** *** Mean TUNEL Positive Neurons *** ** CA1 DG CA1 DG

44 Ig G S ta in in g T h r e s h o ld Ig G S ta in in g T h r e s h o ld Ig G S ta in in g T h r e s h o ld Ig G S ta in in g T h r e s h o ld V a s c u la r V o lu m e V a s c u la r V o lu m e V a s c u la r V o lu m e V a s c u la r V o lu m e 6 min MCAO Embolic MCAO B. D. A. CA1 DG C. CA1 Control Control Control Control DG p = ** 3 2 ** E.. C o n t r o l D i a b e t e s CA1 Control F.. C o n t r o l DG Control D i a b e t e s H.. C o n tro l CA1 D ia b e te s Control G. C o n t r o l D i a b e t e s DG Control *** *** 4 2 C o n tro l ** D ia b e te s 2 C o n tro l ** D ia b e te s 2 C o n tro l D ia b e te s 2 C o n tro l D ia b e te s

45 6 min MCAO Embolic MCAO A. C. B. Recognition Index Removal Latency (s) Control.4.2. BL 2 ^ 15 1 * *p diabetes =.4 ^p stroke =.411 ^ D14 ^p stroke =.9 ^ ^ 5 BL D1 D7 D14 * D. Recognition Index Removal Latency (s) #p interaction =.357 BL ^ # D14 *p diabetes <.5 ^p stroke <.1 BL D1 D7 D14 ^ ^ *

46 Control A. Contralateral 6 min MCAO Control Ipsilateral Contralateral Embolic MCAO Control Ipsilateral B. Iba-1 Intensity x min MCAO Embolic MCAO Protrusion Endpoints Processes C. Control CA1 D. Control DG E. Control CA1 F. Control DG Cell Body Size (um 2 ) 6 * 4 2 Control Number of Protrusions Control ** Cell Body Size (um 2 ) ** Number of Protrusions Control * Cell Body Size (um 2 ) 6 * 4 2 Control Number of Protrusions Control ** Cell Body Size (um 2 ) Control ** Number of Protrusions Control *** Number of microglia process endpoints/cell Control ** S um m ed m icrog lia process length/cell Control ** Number of microglia process endpoints/cell Control * Summed microglia process length/cell Control * Number of microglia process endpoints/cell Control *** Summed microglia process length/cell Control *** Number of microglia process endpoints/cell Control * Summed microglia process length/cell Control **

47 Male Female Control Control Embolic MCAO 6 min MCAO Sham 5 #p in teractio n =.363 *p diabetes =.358 *p diabetes <.1 Mean Number of Neurons # * &p<.5 vs sham fem ale diabetes * & * Sham 6 min MCAO Embolic MCAO * & Male Control Male Female Control Female

48 B. A. 15 *p diabetes =.2 #p in te rac tio n <.1 ^p sex =.99 Sham Control Male Female Control Branch Density 1 5 * * # p in te rac tio n.34 sham p in te rac tio n.139 sham ^ C. Sham 6 min MCAO Embolic MCAO 6 min MCAO Embolic MCAO % Surface Area #p in te rac tio n =.13 #p in te rac tio n =.9 #p in te rac tio n <.1 p in te rac tio n p in te rac tio n <.1.48 # # sham sham # Sham 6 min MCAO Embolic MCAO Male Control Male Female Control Female