Post-stroke neovascularization and functional outcomes differ in diabetes depending on severity of injury and sex: Potential link to hemorrhagic transformation

Weiguo Li; John Paul Valenzuela; Rebecca Ward; Mahmoud Abdelbary; Guangkuo Dong; Susan C. Fagan; Adviye Ergul

doi:10.1016/j.expneurol.2018.09.013

Post-stroke neovascularization and functional outcomes differ in diabetes depending on severity of injury and sex: Potential link to hemorrhagic transformation

Weiguo Li, John Paul Valenzuela, Rebecca Ward, Mahmoud Abdelbary, Guangkuo Dong, Susan C. Fagan, Adviye Ergul

Research output: Contribution to journal › Article › peer-review

16 Scopus citations

Abstract

Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.

Original language	English (US)
Pages (from-to)	106-114
Number of pages	9
Journal	Experimental Neurology
Volume	311
DOIs	https://doi.org/10.1016/j.expneurol.2018.09.013
State	Published - Jan 2019
Externally published	Yes

Keywords

Diabetes
Female
Hemorrhagic Transformation
Ischemic Stroke
Neurovascular Injury
Vascular Cognitive Impairment/Dementia

ASJC Scopus subject areas

Neurology
Developmental Neuroscience

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10.1016/j.expneurol.2018.09.013

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@article{6cc90a6bf06a4975baf9c27d5d5e6e02,

title = "Post-stroke neovascularization and functional outcomes differ in diabetes depending on severity of injury and sex: Potential link to hemorrhagic transformation",

abstract = "Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.",

keywords = "Diabetes, Female, Hemorrhagic Transformation, Ischemic Stroke, Neurovascular Injury, Vascular Cognitive Impairment/Dementia",

author = "Weiguo Li and Valenzuela, {John Paul} and Rebecca Ward and Mahmoud Abdelbary and Guangkuo Dong and Fagan, {Susan C.} and Adviye Ergul",

note = "Funding Information: As the search for acute neuroprotective strategies and treatments is still continuing after years of failed trials, significant effort has shifted towards better understanding of the neuroreparative and restorative mechanisms after ischemic brain injury in recent years. Numerous studies conducted using male animals provided evidence that angiogenesis and cerebrovascular remodeling are critical physiological responses for neurovascular repair and comorbid conditions such as diabetes and hypertension can hamper these important repair mechanisms ( Abdelsaid et al., 2015 ; Elgebaly et al., 2011 ; Kelly-Cobbs et al., 2012 ). The current study expands this line of research and provides evidence that diet-induced diabetes negates neurovascular protection typically seen in female animals. Furthermore, post-stroke vascularization pattern is influenced by the degree of acute vascular injury and correlates with functional recovery in both sexes. While diabetes is an endocrine disease due to the lack or insufficiency of insulin action, it is also a vascular disease as most, if not all, complications of the disease have a vascular basis ( Forbes and Fotheringham, 2017 ). Stroke is no exception and it is considered as macrovascular complication for long time due to accelerated atherosclerosis in diabetes. Small vessel disease also contributes to increased risk and severity of stroke in diabetes as evidenced by amplified edema and HT, indicating the vulnerability of diabetes-stricken vessels to a second hit by ischemia/reperfusion injury ( Abdelsaid et al., 2015 ; Ergul et al., 2012 ). Thus, it is not surprising that numerous clinical and experimental studies have shown that diabetes worsens cognitive and motor outcomes following stroke ( Arnold et al., 2014 ; Capes et al., 2001 ; Ergul et al., 2012 ; Ning et al., 2012 ; Piernik-Yoder and Ketchum, 2013 ; Prakash et al., 2013b ). By the same token, healthy vasculature is also important for the healing process. We have previously reported that there is loss of the cerebrovasculature after stroke in a lean model of spontaneous type 2 diabetes and this was associated with poor functional recovery ( Prakash et al., 2013b ). Sweetnam et al. showed that in a model of type 1 diabetes, recovery was severely blunted and this was associated with decreased synaptic plasticity ( Sweetnam et al., 2012 ). The same group later showed that branch density is also decreased without a significant change in vascular density ( Reeson et al., 2016 ). Given that 30–40% of ischemic stroke patients have diabetes, diet-induced metabolic derangements are the main cause of the diabetes epidemic and stroke is the leading cause of adult disability, the current study used a clinically relevant and reproducible diet-induced model of diabetes. Several important observations were made. Similar to the GK model of diabetes, vascularization indices were higher in the sham operated diabetic rats than in control animals suggesting that this vascular response is common in different models of diabetes ( Prakash et al., 2013a ; 2012 ). Furthermore, there was a decrease in vascularization indices in diabetic animals 14 days after stroke induced by a 90-min MCAO in both hemispheres. The pattern of decreased vascularization was similar to our previous results in GK rats but the magnitude was less in this diet-induced model of diabetes ( Prakash et al., 2013b ). These results are different from those reported by Ye et al., which showed greater arterial density in the peri-infarct zone in type 1 diabetic mice as compared to nondiabetic mice 14 days after MCAO ( Ye et al., 2011 ). On the other hand, Reeson and colleagues ( Reeson et al., 2015 ) demonstrated there is a reduction in branch points suggesting vascular pruning in type 1 diabetic animals similar to our findings. The greater vasoregression we observed may be due to the differences in the diabetes model used or the extent of the ischemic injury as discussed below. In the US, there has been a decline in the mortality rate for men with diabetes but not in women ( Gregg et al., 2007 ). This coincides with the report that women with type 2 diabetes have a greater chance of recurrent stroke independent of age, a trend not seen in men ( Policardo et al., 2015 ). Despite the growing evidence that sex plays an important role in the stroke pathophysiology, treatment and outcome, there is still a substantial gap in knowledge in the research field. Therefore, in the next set of experiments we wished to compare functional outcomes and the vascular repair response in male and female animals after a 90-min MCAO as in our first set of experiments. However, due to increased mortality in aged matched diabetic females, we changed our protocol to 60 min MCAO. Surprisingly, there were no difference in the behavioral tests between control and diabetic male and female animals. Furthermore, with the exception of branch density measurements, we did not observe any difference in vascularization indices measured. These results suggest that the severity of the initial injury is a critical factor in the vascular and functional recovery response. This fact also explained the variable vasoregression findings in type 1 diabetes studies discussed above. Based on these findings, we compared the early neurovascular injury in the same groups 3 days after 60-min MCAO. First, neuroprotection observed in young female rats was lost when animals were diabetic. One other study reported that ischemic brain damage is much greater in genetically diabetic female KKAy mice as compared to male KKAy mice ( Sakata et al., 2011 ). A possible reason for the loss of neuroprotection in diabetes could be due to the effect of diabetes on estrogen or estrogen receptors. While sexual dimorphism in ischemic stroke cannot be fully attributed to sex hormones, estrogens, especially 17β estradiol (E2), contribute to neuroprotection through activation of estrogen receptor-α (ERα). We did not measure the circulating E2 levels or brain ER expression and distribution in this study. However, when estrus cycle was monitored for stroke surgery timing, there was no notable difference in vaginal swaps between control and diabetic rats. While several clinical studies suggested that diabetes increases E2 levels in postmenopausal women, data on premenopausal women is missing and it is possible that E2 is decreased in our model ( Han et al., 2014 ). ERα in the hypothalamus has been shown to regulate energy expenditure and glucose homeostasis. Interestingly HFD reduces this receptor in male but not female animals ( Morselli et al., 2016 ). Whether ERα expression and distribution are altered in our model remains to be determined. The second important finding was that 60-min MCAO did not cause significant HT. Previous studies reported that 3 h occlusion time that is needed to induce HT in control animals is reduced in diabetic male animals ( Li et al., 2013 ). We recently reported for the first time that 90-min suture or embolic MCAO causes greater bleeding in diabetic female rats ( Li et al., 2017 ). The current study builds upon those findings and show that a shorter occlusion does not cause HT. This finding also raised the possibility that lack of bleeding may be a factor in the lack of vasoregression and differences in functional outcomes as in all our previous studies where we saw these deficits, there was also greater bleeding into the brain. There are limitations in this study. Due to the increased mortality rate in diabetes, number of animals are less in the diabetic, especially female groups. The mortality in diabetes group was higher than expected when the Exp 1 was finished. Hence, the ischemia time was shortened in both Exp 2 and Exp 3. However, the changes we saw are robust so we believe that this did not affect our conclusions. Second, vascularization indices were measured using a space filling method that requires proper perfusion. Changes in cerebral perfusion in diabetes, especially in the diabetic animals, may influence FITC filling. In previous studies, we confirmed that the decrease in vascularization could be detected by immunohistochemistry for endothelial staining as well. While further studies are needed to determine whether the presence or lack of HT is a determinant of vasoregression and poor recovery observed in diabetes, this translational study provides insight that the severity of the initial injury during ischemia is important for early and long-term neurovascular injury and repair. It is also one of the few papers to compare vascular and neuronal injury in male and female control and diabetic animals. Conflict of interests All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare. AE is a Senior Research Career Scientist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, GA, USA. The contents do not represent the views of the Department of Veterans Affairs or the US Government. Funding information This work was supported in part by a Veterans Affairs (VA) Merit Award ( BX000347 ), VA Senior Research Career Scientist Award and National Institutes of Health (NIH) awards ( R01NS083559 , PO1HL134604 ) to AE, and ( R21NS088016 ) to SCF. WL is the recipient of the Feasibility Grant from NIDDK Diabetic Complications Consortium (DiaComp, www.diacomp.org ) grants DK076169 and DK115255 . JPV and RW were recipients of Diversity Enhancement Award ( NS083559-SI ) and American Heart Association Predoctoral Fellowship ( 17PRE33660400 ), respectively. Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",

year = "2019",

month = jan,

doi = "10.1016/j.expneurol.2018.09.013",

language = "English (US)",

volume = "311",

pages = "106--114",

journal = "Neurodegeneration",

issn = "0014-4886",

publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Post-stroke neovascularization and functional outcomes differ in diabetes depending on severity of injury and sex

T2 - Potential link to hemorrhagic transformation

AU - Li, Weiguo

AU - Valenzuela, John Paul

AU - Ward, Rebecca

AU - Abdelbary, Mahmoud

AU - Dong, Guangkuo

AU - Fagan, Susan C.

AU - Ergul, Adviye

N1 - Funding Information: As the search for acute neuroprotective strategies and treatments is still continuing after years of failed trials, significant effort has shifted towards better understanding of the neuroreparative and restorative mechanisms after ischemic brain injury in recent years. Numerous studies conducted using male animals provided evidence that angiogenesis and cerebrovascular remodeling are critical physiological responses for neurovascular repair and comorbid conditions such as diabetes and hypertension can hamper these important repair mechanisms ( Abdelsaid et al., 2015 ; Elgebaly et al., 2011 ; Kelly-Cobbs et al., 2012 ). The current study expands this line of research and provides evidence that diet-induced diabetes negates neurovascular protection typically seen in female animals. Furthermore, post-stroke vascularization pattern is influenced by the degree of acute vascular injury and correlates with functional recovery in both sexes. While diabetes is an endocrine disease due to the lack or insufficiency of insulin action, it is also a vascular disease as most, if not all, complications of the disease have a vascular basis ( Forbes and Fotheringham, 2017 ). Stroke is no exception and it is considered as macrovascular complication for long time due to accelerated atherosclerosis in diabetes. Small vessel disease also contributes to increased risk and severity of stroke in diabetes as evidenced by amplified edema and HT, indicating the vulnerability of diabetes-stricken vessels to a second hit by ischemia/reperfusion injury ( Abdelsaid et al., 2015 ; Ergul et al., 2012 ). Thus, it is not surprising that numerous clinical and experimental studies have shown that diabetes worsens cognitive and motor outcomes following stroke ( Arnold et al., 2014 ; Capes et al., 2001 ; Ergul et al., 2012 ; Ning et al., 2012 ; Piernik-Yoder and Ketchum, 2013 ; Prakash et al., 2013b ). By the same token, healthy vasculature is also important for the healing process. We have previously reported that there is loss of the cerebrovasculature after stroke in a lean model of spontaneous type 2 diabetes and this was associated with poor functional recovery ( Prakash et al., 2013b ). Sweetnam et al. showed that in a model of type 1 diabetes, recovery was severely blunted and this was associated with decreased synaptic plasticity ( Sweetnam et al., 2012 ). The same group later showed that branch density is also decreased without a significant change in vascular density ( Reeson et al., 2016 ). Given that 30–40% of ischemic stroke patients have diabetes, diet-induced metabolic derangements are the main cause of the diabetes epidemic and stroke is the leading cause of adult disability, the current study used a clinically relevant and reproducible diet-induced model of diabetes. Several important observations were made. Similar to the GK model of diabetes, vascularization indices were higher in the sham operated diabetic rats than in control animals suggesting that this vascular response is common in different models of diabetes ( Prakash et al., 2013a ; 2012 ). Furthermore, there was a decrease in vascularization indices in diabetic animals 14 days after stroke induced by a 90-min MCAO in both hemispheres. The pattern of decreased vascularization was similar to our previous results in GK rats but the magnitude was less in this diet-induced model of diabetes ( Prakash et al., 2013b ). These results are different from those reported by Ye et al., which showed greater arterial density in the peri-infarct zone in type 1 diabetic mice as compared to nondiabetic mice 14 days after MCAO ( Ye et al., 2011 ). On the other hand, Reeson and colleagues ( Reeson et al., 2015 ) demonstrated there is a reduction in branch points suggesting vascular pruning in type 1 diabetic animals similar to our findings. The greater vasoregression we observed may be due to the differences in the diabetes model used or the extent of the ischemic injury as discussed below. In the US, there has been a decline in the mortality rate for men with diabetes but not in women ( Gregg et al., 2007 ). This coincides with the report that women with type 2 diabetes have a greater chance of recurrent stroke independent of age, a trend not seen in men ( Policardo et al., 2015 ). Despite the growing evidence that sex plays an important role in the stroke pathophysiology, treatment and outcome, there is still a substantial gap in knowledge in the research field. Therefore, in the next set of experiments we wished to compare functional outcomes and the vascular repair response in male and female animals after a 90-min MCAO as in our first set of experiments. However, due to increased mortality in aged matched diabetic females, we changed our protocol to 60 min MCAO. Surprisingly, there were no difference in the behavioral tests between control and diabetic male and female animals. Furthermore, with the exception of branch density measurements, we did not observe any difference in vascularization indices measured. These results suggest that the severity of the initial injury is a critical factor in the vascular and functional recovery response. This fact also explained the variable vasoregression findings in type 1 diabetes studies discussed above. Based on these findings, we compared the early neurovascular injury in the same groups 3 days after 60-min MCAO. First, neuroprotection observed in young female rats was lost when animals were diabetic. One other study reported that ischemic brain damage is much greater in genetically diabetic female KKAy mice as compared to male KKAy mice ( Sakata et al., 2011 ). A possible reason for the loss of neuroprotection in diabetes could be due to the effect of diabetes on estrogen or estrogen receptors. While sexual dimorphism in ischemic stroke cannot be fully attributed to sex hormones, estrogens, especially 17β estradiol (E2), contribute to neuroprotection through activation of estrogen receptor-α (ERα). We did not measure the circulating E2 levels or brain ER expression and distribution in this study. However, when estrus cycle was monitored for stroke surgery timing, there was no notable difference in vaginal swaps between control and diabetic rats. While several clinical studies suggested that diabetes increases E2 levels in postmenopausal women, data on premenopausal women is missing and it is possible that E2 is decreased in our model ( Han et al., 2014 ). ERα in the hypothalamus has been shown to regulate energy expenditure and glucose homeostasis. Interestingly HFD reduces this receptor in male but not female animals ( Morselli et al., 2016 ). Whether ERα expression and distribution are altered in our model remains to be determined. The second important finding was that 60-min MCAO did not cause significant HT. Previous studies reported that 3 h occlusion time that is needed to induce HT in control animals is reduced in diabetic male animals ( Li et al., 2013 ). We recently reported for the first time that 90-min suture or embolic MCAO causes greater bleeding in diabetic female rats ( Li et al., 2017 ). The current study builds upon those findings and show that a shorter occlusion does not cause HT. This finding also raised the possibility that lack of bleeding may be a factor in the lack of vasoregression and differences in functional outcomes as in all our previous studies where we saw these deficits, there was also greater bleeding into the brain. There are limitations in this study. Due to the increased mortality rate in diabetes, number of animals are less in the diabetic, especially female groups. The mortality in diabetes group was higher than expected when the Exp 1 was finished. Hence, the ischemia time was shortened in both Exp 2 and Exp 3. However, the changes we saw are robust so we believe that this did not affect our conclusions. Second, vascularization indices were measured using a space filling method that requires proper perfusion. Changes in cerebral perfusion in diabetes, especially in the diabetic animals, may influence FITC filling. In previous studies, we confirmed that the decrease in vascularization could be detected by immunohistochemistry for endothelial staining as well. While further studies are needed to determine whether the presence or lack of HT is a determinant of vasoregression and poor recovery observed in diabetes, this translational study provides insight that the severity of the initial injury during ischemia is important for early and long-term neurovascular injury and repair. It is also one of the few papers to compare vascular and neuronal injury in male and female control and diabetic animals. Conflict of interests All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare. AE is a Senior Research Career Scientist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, GA, USA. The contents do not represent the views of the Department of Veterans Affairs or the US Government. Funding information This work was supported in part by a Veterans Affairs (VA) Merit Award ( BX000347 ), VA Senior Research Career Scientist Award and National Institutes of Health (NIH) awards ( R01NS083559 , PO1HL134604 ) to AE, and ( R21NS088016 ) to SCF. WL is the recipient of the Feasibility Grant from NIDDK Diabetic Complications Consortium (DiaComp, www.diacomp.org ) grants DK076169 and DK115255 . JPV and RW were recipients of Diversity Enhancement Award ( NS083559-SI ) and American Heart Association Predoctoral Fellowship ( 17PRE33660400 ), respectively. Publisher Copyright: © 2018 Elsevier Inc.

PY - 2019/1

Y1 - 2019/1

N2 - Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.

AB - Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.

KW - Diabetes

KW - Female

KW - Hemorrhagic Transformation

KW - Ischemic Stroke

KW - Neurovascular Injury

KW - Vascular Cognitive Impairment/Dementia

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Post-stroke neovascularization and functional outcomes differ in diabetes depending on severity of injury and sex: Potential link to hemorrhagic transformation

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