Acute Ischemic Stroke by Middle Cerebral Artery Occlusion in Rat Models of Diabetes: Importance of Pre-op and Post-op Care, Severity of Hyperglycemia, and Sex.

Diabetes mellitus (DM) is associated with poor stroke outcomes, including high mortality and disability rates. Ischemic injury modeling large artery stroke in diabetic animals also results in high mortality and poor acute and long-term outcomes. In this chapter, we describe middle cerebral artery occlusion (MCAO) in a high-fat diet/low-dose streptozotocin (STZ) model of diabetes including details on pre-op and post-op care that improve survival rate for successful completion of the studies.

Cerebral microvascular matrix metalloproteinase-3 (MMP3) contributes to vascular injury after stroke in female diabetic rats.

Diabetes exacerbates hemorrhagic transformation (HT) after stroke and worsens clinical outcomes. Female patients with diabetes are at a greater risk of stroke and worsened recovery. We have shown that activation of matrix metalloprotease 3 (MMP3) in hyperglycemic settings mediates HT in male rats. In light of our recent findings that diabetic female rats develop greater HT, the current study was designed to test the hypotheses that: 1) cerebral microvascular MMP3 activation contributes to poor functional outcomes and increased hemorrhagic transformations (HT) after ischemic stroke, and 2) MMP3 inhibition can improve functional outcomes in female diabetic rats. Female control and diabetic Wistar rats were subjected to 60 min of middle cerebral artery occlusion (MCAO). One cohort of diabetic animals received a single dose of MMP3 inhibitor (UK356618; 15 mg/kg; iv) or vehicle after reperfusion. Neurobehavioral outcomes, brain infarct size, edema, HT, and MMPs were measured in brain tissue. Diabetic rats had significant neurological deficits on Day 3 after stroke. MMP3 expression and enzyme activity were significantly increased in both micro and macro vessels of diabetic animals. MMP3 inhibition improved functional outcomes and reduced brain edema and HT scores. In conclusion, cerebral endothelial MMP3 activation to vascular injury in female diabetic rats. Our findings identify MMP3 as a potential therapeutic target in diabetic stroke.

Deferoxamine prevents poststroke memory impairment in female diabetic rats: potential links to hemorrhagic transformation and ferroptosis.

Diabetes increases the risk of poststroke cognitive impairment (PSCI). Greater hemorrhagic transformation (HT) after stroke is associated with vasoregression and cognitive decline in male diabetic rats. Iron chelator deferoxamine (DFX) prevents vasoregression and improves outcomes. Although diabetic female rats develop greater HT, its impact on poststroke cerebrovascularization and cognitive outcomes remained unknown. We hypothesized that diabetes mediates pathological neovascularization, and DFX attenuates poststroke cerebrovascular remodeling and improves neurological outcomes in female diabetic rats. Female control and diabetic animals were treated with DFX or vehicle for 7 days after stroke. Vascular indices, microglial activation, and blood-brain barrier (BBB) integrity were evaluated on day 14. Results from diabetic female rats were partially compared with our previously published findings in male counterparts. Hemin-induced programmed cell death was studied in male and female brain microvascular endothelial cell lines (BMVEC). There was no vasoregression after stroke in either control or diabetic female animals. DFX prevented diabetes-mediated gliovascular remodeling and compromised BBB integrity while improving memory function in diabetes. Comparisons of female and male rats indicated sex differences in cognitive and vascular outcomes. Hemin mediated ferroptosis in both male and female BMVECs. DFX improved survival but had differential effects on ferroptosis signaling in female and male cells. These results suggest that stroke and associated HT do not affect cerebrovascularization in diabetic female rats, but iron chelation may provide a novel therapeutic strategy in the prevention of poststroke memory impairment in females with diabetes via the preservation of gliovascular integrity and improvement of endothelial cell survival.NEW & NOTEWORTHY The current study shows for the first time that diabetes does not promote aberrant cerebrovascularization in female rats. This contrasts with what we reported in male animals in various diabetes models. Deferoxamine preserved recognition memory function in diabetic female animals after stroke. The effect(s) of stroke and deferoxamine on cerebrovascular density and microglial activation also appear(s) to be different in female diabetic rats. Lastly, deferoxamine exerts detrimental effects on animals and BMVECs under control conditions.

Endothelin A receptors contribute to senescence of brain microvascular endothelial cells.

Cellular senescence plays a pivotal role in the aging and progression of neurodegenerative diseases, including vascular cognitive impairment and dementia (VCID). In postmortem brains from individuals with VCID, endothelin-1 (ET-1) levels closely correlate with blood barrier breakdown and cerebral hypoperfusion. Brain microvascular endothelial cells (BMVECs), previously thought to have exclusively endothelin B receptors, also possess endothelin A (ETA) receptors; however, the functional significance of this receptor in BMVECs is not known. We hypothesize that ETA receptors mediate BMVEC senescence. Serum-starved human BMVECs (HBEC5i) were incubated with ET-1 (1 µmol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20 µmol/L). Cells were collected for Western blot and quantitative real-time PCR analyses. Treatment of ET-1 increased protein expression of ETA receptor, while it was prevented by the ETA receptor antagonist. ET-1 increased p21, p16, p53, LIF1 and cyclin D1 protein levels, and ß-galactosidase accumulation, which were prevented in the presence of ETA blockade. While there was no change in tight junction proteins, ET-1 decreased adherent junction protein vascular endothelial cadherin (VE-cadherin) levels. In conclusion, ET-1 upregulates ETA receptors in BMVECs in an autocrine manner and triggers the activation of senescence. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of endothelial senescence in VCID.

Porphyromonas gingivalis infection upregulates the endothelin (ET) system in brain microvascular endothelial cells.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to Alzheimer's disease and related dementias (ADRD) progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (Coykendall et al.) Shah & Collins to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells. Cells were infected with a multiplicity of infection 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 h. Cell lysates were collected for analysis of endothelin A receptor (ETA)/endothelin B receptor (ETB) receptor as well as senescence markers. ET-1 levels in cell culture media were measured with enzyme-linked immunosorbent assay. P. gingivalis infection increased ET-1 (pg/mL) secretion, as well as the ETA receptor expression, whereas decreased lamin A/C expression compared to control. Tight junction protein claudin-5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

Impact of diabetes and ischemic stroke on the cerebrovasculature: A female perspective.

There is a very complex interaction between the brain and the cerebral vasculature to meet the metabolic demands of the brain for proper function. Preservation of vascular networks and cerebrovascular function ultimately plays a key role in this intricate communication within the brain in health and disease. Experimental evidence showed that diabetes not only affects the architecture of cerebral blood arteries causing adverse remodeling, pathological neovascularization, and vasoregression, but also alters cerebrovascular function resulting in compromised myogenic reactivity and endothelial dysfunction. Coupled with the disruption of blood brain barrier (BBB) integrity, changes in blood flow and microbleeds into the brain can rapidly occur. When an ischemic insult is superimposed on this pathology, not only is the neurovascular injury greater, but repair mechanisms fail, resulting in greater physical and cognitive deficits. While clinically it is known that women suffer disproportionately from diabetes as well as ischemic stroke and post-stroke cognitive impairment, the cerebrovascular architecture, patho/physiology, as well as cerebrovascular contributions to stroke recovery in female and diabetic animal models are inadequately studied and highlighted in this review.

Delayed Administration of Angiotensin Receptor (AT2R) Agonist C21 Improves Survival and Preserves Sensorimotor Outcomes in Female Diabetic Rats Post-Stroke through Modulation of Microglial Activation.

About 70% of stroke victims present with comorbid diseases such as diabetes and hypertension. The integration of comorbidities in pre-clinical experimental design is important in understanding the mechanisms involved in the development of stroke injury and recovery. We recently showed that administration of compound C21, an angiotensin II type 2 receptor agonist, at day 3 post-stroke improved sensorimotor outcomes by lowering neuroinflammation in diabetic male animals. In the current study, we hypothesized that a delayed administration of C21 would also lower chronic inflammation post-stroke in diabetic female animals. Young female diabetic rats were subjected to 1 h of middle cerebral artery occlusion (MCAO). Three days post-stroke, rats were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 4 weeks. The impact of C21 on microglial polarization was analyzed by flow cytometry in vivo and in vitro. Compound 21 treatment improved fine motor skills after MCAO through modulation of the microglia/macrophage inflammatory properties. In addition, C21 increased M2 polarization and reduced the M1:M2 ratio in vitro. In conclusion, delayed administration of C21 downregulates post-stroke inflammation in female diabetic animals. C21 may be a useful therapeutic option to lower neuro-inflammation and improve the post-stroke recovery in diabetes.

Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis.

It is a clinically well-established fact that patients with diabetes have very poor stroke outcomes. Yet, the underlying mechanisms remain largely unknown. Our previous studies showed that male diabetic animals show greater hemorrhagic transformation (HT), profound loss of cerebral vasculature in the recovery period, and poor sensorimotor and cognitive outcomes after ischemic stroke. This study aimed to determine the impact of iron chelation with deferoxamine (DFX) on (1) cerebral vascularization patterns and (2) functional outcomes after stroke in control and diabetic rats. After 8 weeks of type 2 diabetes induced by a combination of high-fat diet and low-dose streptozotocin, male control and diabetic animals were subjected to thromboembolic middle cerebral artery occlusion (MCAO) and randomized to vehicle, DFX, or tPA/DFX and followed for 14 days with behavioral tests. Vascular indices (vascular volume and surface area), neurovascular remodeling (AQP4 polarity), and microglia activation were measured. Brain microvascular endothelial cells (BMVEC) from control and diabetic animals were evaluated for the impact of DFX on ferroptotic cell death. DFX treatment prevented vasoregression and microglia activation while improving AQP4 polarity as well as blood-brain barrier permeability by day 14 in diabetic rats. These pathological changes were associated with improvement of functional outcomes. In control rats, DFX did not have an effect. Iron increased markers of ferroptosis and lipid reactive oxygen species (ROS) to a greater extent in BMVECs from diabetic animals, and this was prevented by DFX. These results strongly suggest that (1) HT impacts post-stroke vascularization patterns and recovery responses in diabetes, (2) treatment of bleeding with iron chelation has differential effects on outcomes in comorbid disease conditions, and (3) iron chelation and possibly inhibition of ferroptosis may provide a novel disease-modifying therapeutic strategy in the prevention of post-stroke cognitive impairment in diabetes.

Novel Targets and Interventions for Cognitive Complications of Diabetes.

Diabetes and cognitive dysfunction, ranging from mild cognitive impairment to dementia, often coexist in individuals over 65 years of age. Vascular contributions to cognitive impairment/dementia (VCID) are the second leading cause of dementias under the umbrella of Alzheimer's disease and related dementias (ADRD). Over half of dementia patients have VCID either as a single pathology or a mixed dementia with AD. While the prevalence of type 2 diabetes in individuals with dementia can be as high as 39% and diabetes increases the risk of cerebrovascular disease and stroke, VCID remains to be one of the less understood and less studied complications of diabetes. We have identified cerebrovascular dysfunction and compromised endothelial integrity leading to decreased cerebral blood flow and iron deposition into the brain, respectively, as targets for intervention for the prevention of VCID in diabetes. This review will focus on targeted therapies that improve endothelial function or remove iron without systemic effects, such as agents delivered intranasally, that may result in actionable and disease-modifying novel treatments in the high-risk diabetic population.

Diabetes-related sex differences in the brain endothelin system following ischemia in vivo and in human brain endothelial cells in vitro.

The endothelin (ET) system has been implicated to contribute to the pathophysiology of cognitive impairment and stroke in experimental diabetes. Our goals were to test the hypotheses that (1) circulating and (or) periinfarct ET-1 levels are elevated after stroke in both sexes and this increase is greater in diabetes, (2) ET receptors are differentially regulated in the diabetic brain, (3) brain microvascular endothelial cells (BMVEC) of female and male origin express the ETA receptor subtype, and (4) diabetes- and stroke-mimicking conditions increase ET-1 levels in BMVECs of both sexes. Control and diabetic rats were randomized to sham or stroke surgery. BMVECs of male (hBEC5i) and female (hCMEC/D3) origin, cultured under normal and diabetes-mimicking conditions, were exposed to normoxia or hypoxia. Circulating ET-1 levels were higher in diabetic animals and this was more pronounced in the male cohort. Stroke did not further increase plasma ET-1. Tissue ET-1 levels were increased after stroke only in males, whereas periinfarct ET-1 increased in both control and diabetic females. Male BMVECs secreted more ET-1 than female cells and hypoxia increased ET-1 levels in both cell types. There was sexually dimorphic regulation of ET receptors in both tissue and cell culture samples. There are sex differences in the stroke- and diabetes-mediated changes in the brain ET system at the endothelial and tissue levels.

Endothelin-1 (ET-1) promotes a proinflammatory microglia phenotype in diabetic conditions.

Diabetes increases the risk and severity of cognitive impairment, especially after ischemic stroke. It is also known that the activation of the endothelin (ET) system is associated with cognitive impairment and microglia around the periinfarct area produce ET-1. However, little is known about the effect of ET-1 on microglial polarization, especially under diabetic conditions. We hypothesized that (i) ET-1 activates microglia to the proinflammatory M-1-like phenotype and (ii) hypoxia/ lipopolysaccharide (LPS) activates the microglial ET system and promotes microglial activation towards the M-1 phenotype in diabetic conditions. Microglial cells (C8B4) cultured under normal-glucose (25 mmol/L) conditions and diabetes-mimicking high-glucose (50 mmol/L) conditions for 48 h were stimulated with ET-1, cobalt chloride (200 µmol/L), or LPS (100 ng/mL) for 24 h. PPET-1, ET receptor subtypes, and M1/M2 marker gene mRNA expression were measured by RT-PCR. Secreted ET-1 was measured by ELISA. A high dose of ET-1 (1 µmol/L) increases the mRNA levels of ET receptors and activates the microglia towards the M1 phenotype. Hypoxia or LPS activates the ET system in microglial cells and shifts the microglia towards the M1 phenotype in diabetic conditions. These in vitro observations warrant further investigation into the role of ET-1-mediated activation of proinflammatory microglia in post-stroke cognitive impairment in diabetes.

Diabetic Stroke Promotes a Sexually Dimorphic Expansion of T Cells.

We recently reported that diabetes negates the cerebrovascular protection typically seen in adult female rats resulting in cognitive impairment, which is worsened by increased parenchymal bleeding and edema after ischemic stroke. Although women experience more severe diabetes and suffer from a higher rate of diabetic complications, including stroke and cognitive impairment, underlying mechanisms contributing to sex differences are limited. Emerging evidence suggests interleukin (IL)-17 contributes to cerebrovascular pathologies: (1) high salt diet-mediated expansion of IL-17-producing T cells (Th17) in the gut microbiome promotes cerebrovascular dysfunction and cognitive impairment in male mice, (2) increased IL-17-producing γδTCR cells exacerbates stroke injury in male mice, and (3) IL-17 promotes rupture of cerebral aneurysms in female mice. Based on these premises, we investigated the potential involvement of IL-17-producing inflammatory cells in cerebrovascular dysfunction and post-stroke vascular injury in diabetes by measuring intestinal, circulating, or cerebral T cell profiles as well as in plasma IL-17 in both sexes. Cell suspensions prepared from naive or stroked (3 days after stroke) diabetic and control rats were analyzed by flow cytometry, and IL-17 levels were measured in plasma using ELISA. Diabetes deferentially promoted the expansion of cerebral Th17 cells in females. In response to stroke, diabetes had a sexually dimorphic effect on the expansion of numerous T cell profiles. These results suggest that a better understanding of the role of IL-17-producing cells in diabetes may identify potential avenues in which the molecular mechanisms contributing to these sex differences can be further elucidated.

NLRP3 inflammasome inhibition with MCC950 improves diabetes-mediated cognitive impairment and vasoneuronal remodeling after ischemia.

Diabetes increases the risk and worsens the progression of cognitive impairment via the greater occurrence of small vessel disease and stroke. Yet, the underlying mechanisms are not fully understood. It is now accepted that cardiovascular health is critical for brain health and any neurorestorative approaches to prevent/delay cognitive deficits should target the conceptual neurovascular unit (NVU) rather than neurons alone. We have recently shown that there is augmented hippocampal NVU remodeling after a remote ischemic injury in diabetes. NLRP3 inflammasome signaling has been implicated in the development of diabetes and neurodegenerative diseases, but little is known about the impact of NLRP3 activation on functional and structural interaction within the NVU of hippocampus, a critical part of the brain that is involved in forming, organizing, and storing memories. Endothelial cells are at the center of the NVU and produce trophic factors such as brain derived neurotrophic factor (BDNF) contributing to neuronal survival, known as vasotrophic coupling. Therefore, the aims of this study focused on two hypotheses: 1) diabetes negatively impacts hippocampal NVU remodeling and worsens cognitive outcome after stroke, and 2) NLRP3 inhibition with MCC950 will improve NVU remodeling and cognitive outcome following stroke via vasotrophic (un)coupling between endothelial cells and hippocampal neurons. Stroke was induced through a 90-min transient middle cerebral artery occlusion (MCAO) in control and high-fat diet/streptozotocin-induced (HFD/STZ) diabetic male Wistar rats. Saline or MCC950 (3 mg/kg), an inhibitor of NLRP3, was injected at 1 and 3 h after reperfusion. Cognition was assessed over time and neuronal density, blood-brain barrier (BBB) permeability as well as NVU remodeling (aquaporin-4 [AQP4] polarity) was measured on day 14 after stroke. BDNF was measured in endothelial and hippocampal neuronal cultures under hypoxic and diabetes-mimicking condition with and without NLRP3 inhibition. Diabetes increased neuronal degeneration and BBB permeability, disrupted AQP4 polarity, impaired cognitive function and amplified NLRP3 activation after ischemia. Inhibition with MCC950 improved cognitive function and vascular integrity after stroke in diabetic animals and prevented hypoxia-mediated decrease in BDNF secretion. These results are the first to provide essential data showing MCC950 has the potential to become a therapeutic to prevent neurovascular remodeling and worsened cognitive decline in diabetic patients following stroke.

Inhibition of Toll-Like Receptor-4 (TLR-4) Improves Neurobehavioral Outcomes After Acute Ischemic Stroke in Diabetic Rats: Possible Role of Vascular Endothelial TLR-4.

Diabetes increases the risk of occurrence and poor functional recovery after ischemic stroke injury. Previously, we have demonstrated greater hemorrhagic transformation (HT), edema, and more severe functional deficits after stroke in diabetic animals that also presented with cerebral vasoregression and endothelial cell death in the recovery period. Given that Toll-like receptor 4 (TLR-4) activation in microvascular endothelial cells triggers a robust inflammatory response, we hypothesized that inhibition of TLR-4 signaling prevents endothelial cell death and improves outcomes after stroke. Animals were treated with vehicle or TLR-4 inhibitor TAK242 (3 mg/kg; i.p.) following middle cerebral artery occlusion (MCAO). Neurobehavioral deficits were measured at baseline and day 3 after ischemic stroke. Primary brain microvascular endothelial cells (BMVECs) from diabetic animals were subjected to oxygen glucose deprivation re-oxygenation (OGDR) and treated with 0.1 mM iron(III)sulfate hydrate (iron) (to mimic the post-stroke bleeding) and TLR-4 inhibitors. Ischemic stroke increased the expression of TLR-4 in both hemispheres and in the microvasculature of diabetic animals. Cerebral infarct, edema, HT, and functional deficits were greater in diabetic compared to control animals. Inhibition of TLR-4 significantly reduced the neurovascular injury and improved functional outcomes. OGDR and iron reduced the cell viability and increased the expression of TLR-4 associated proteins (RIP3, MyD88, phospho-NF-kB, and release of IL-6) in BMVECs from diabetic animals. In conclusion, TLR-4 is highly upregulated in the microvasculature and that beneficial effects of TLR-4 inhibition are more profound in diabetes. This suggests that inhibition of vascular TLR-4 may provide therapeutic benefits for stroke recovery in diabetes.

Impact of p53 arg72pro SNP on Breast Cancer Risk in North Indian Population.

BACKGROUND: Genetic changes in p53 gene contribute to breast cancer susceptibility. OBJECTIVE AND METHODS: A case-control study and a meta-analysis were performed to investigate the role of p53 codon72 SNP with breast cancer susceptibility in Indian women. RESULTS: p53 heterozygous arginine variant was associated with decreased risk of breast cancer in total cohort. In meta-analysis, Allelic and GG vs. CC genetic comparison model were found to be associated with breast cancer risk. Moreover, recessive comparison model indicated a protective correlation with breast cancer occurrence. CONCLUSION: The findings of our case-control study and meta-analysis suggest a significant association between p53 Arg72Pro polymorphism and an increased risk of breast cancer in Indian population.

Impact of Metabolic Diseases on Cerebral Circulation: Structural and Functional Consequences.

Metabolic diseases including obesity, insulin resistance, and diabetes have profound effects on cerebral circulation. These diseases not only affect the architecture of cerebral blood arteries causing adverse remodeling, pathological neovascularization, and vasoregression but also alter the physiology of blood vessels resulting in compromised myogenic reactivity, neurovascular uncoupling, and endothelial dysfunction. Coupled with the disruption of blood brain barrier (BBB) integrity, changes in blood flow and microbleeds into the brain rapidly occur. This overview is organized into sections describing cerebrovascular architecture, physiology, and BBB in these diseases. In each section, we review these properties starting with larger arteries moving into smaller vessels. Where information is available, we review in the order of obesity, insulin resistance, and diabetes. We also tried to include information on biological variables such as the sex of the animal models noted since most of the information summarized was obtained using male animals. © 2018 American Physiological Society. Compr Physiol 8:773-799, 2018.

PI3K/Akt Pathway: A Role in δ-Opioid Receptor-Mediated RGC Neuroprotection.

Purpose: This study examines the role of PI3K/Akt pathway in δ-opioid receptor agonist (SNC-121)-induced RGC neuroprotection in a chronic glaucoma rat model. Methods: Injecting hypertonic saline into the limbal veins of Brown Norway rats elevated IOP. Rats were treated either with 1 mg/kg SNC-121 or 3 mg/kg 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294002; PI3K/Akt inhibitor) plus SNC-121 once daily for 7 days. Pattern ERGs were recorded in response to contrast reversal of patterned visual stimuli. Retinal ganglion cells (RGC) were visualized by Fluorogold retrograde labeling. Optic nerve head (ONH) astrocytes were pretreated with PI3K/Akt inhibitors for 30 minutes followed by 1-µM SNC-121 treatment. Changes in matrix metalloproteinases (MMP-1, -2, and -3) production and PI3K/Akt activation in optic nerve and TNF-α treated ONH astrocytes were measured by immunohistochemistry and Western blotting. Results: SNC-121 activates the PI3K/Akt pathway in ONH astrocytes and the retina. In ONH astrocytes, SNC-121-induced Akt activation was fully inhibited by PI3K/Akt inhibitors. A sustained decline (7-42 days post injury) in Akt activation was seen in the ocular-hypertensive retina and optic nerve. This decline is reversed to normal levels by 1-mg/kg intraperitoneally (i.p.) SNC-121 treatment. Both pattern ERG amplitudes and RGC numbers were reduced in ocular hypertensive eyes, which were significantly increased in SNC-121-treated animals. Interestingly, SNC-121-induced increase in pattern-ERG amplitudes and RGC numbers were inhibited in LY-294002 pretreated animals. Additionally, SNC-121 treatment inhibited MMP-1, -2, and -3 production from the optic nerve of ocular hypertensive rats and TNF-α-treated ONH astrocytes. Conclusions: PI3K/Akt pathway plays a crucial role in SNC-121-mediated RGC neuroprotection against glaucomatous injury.

Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes.

AIMS: The anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral blood flow in diabetic rats through improved insulin-induced cerebrovascular relaxation and reversal of pathological cerebrovascular remodeling that subsequently leads to improvement of cognitive function. MAIN METHODS: Male type-2 diabetic Goto-Kakizaki (GK) and nondiabetic Wistar rats were treated with linagliptin, and ET-1 plasma levels and dose response curves to ET-1 (0.1-100nM) in basilar arteries were assessed. The impact of TLR2 antagonism on ET-1 mediated basilar contraction and endothelium-dependent relaxation to acetylcholine (ACh, 1nM-1M) in diabetic GK rats was examined with antibody directed against the TLR2 receptor (Santa Cruz, 5µg/mL). The expression of TLR2 in middle cerebral arteries (MCAs) from treated rats and in brain microvascular endothelial cells (BMVEC) treated with 100nM linagliptin was assessed. KEY FINDINGS: Linagliptin lowered plasma ET-1 levels in diabetes, and reduced ET-1-induced vascular contraction. TLR2 antagonism in diabetic basilar arteries reduced ET-1-mediated cerebrovascular dysfunction and improved endothelium-dependent vasorelaxation. Linagliptin treatment in the BMVEC was able to reduce TLR2 expression in cells from both diabetic and nondiabetic rats. CONCLUSIONS: These results suggest that inhibition of DPPIV using linagliptin improves the ET-1-mediated cerebrovascular dysfunction observed in diabetes through a reduction in ET-1 plasma levels and reduced cerebrovascular hyperreactivity. This effect is potentially a result of linagliptin causing a decrease in endothelial TLR2 expression and a subsequent increase in NO bioavailability.

Regulation of nitric oxide production by δ-opioid receptors during glaucomatous injury.

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve.

Interferon-gamma (IFN-γ)-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC) death using this model. RGC function was measured by pattern electroretinograms (ERGs) in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03) and RGC numbers (37%, p = 0.0001) were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174 ± 27% vs. HLA-A2: 100%; p = 0.04). The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189 ± 11% vs. HLA-A2: 100%; p = 0.023). Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF) and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer), which was substantially reduced in IFN-γ ((-/-)) knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.