Hao Liang Xu

ATP release and hydrolysis contribute to rat pial arteriolar dilatation elicited by neuronal activation

Owing to their intimate anatomical relationship with cerebral arterioles, astrocytes have been postulated as signal transducers, transferring information from activated neurones to the cerebral microcirculation. These forwarded signals may involve the release of vasoactive factors from the end‐feet of astrocytes. This mechanism is termed ‘neurovascular coupling’ and its anatomical components (i.e. neurone, astrocyte and vascular cells) are termed the ‘neurovascular unit’. The process of neurovascular coupling often involves upstream dilatation. This is necessary during periods of increased metabolic demand, in order to permit more blood to reach dilated downstream vessels, thereby improving nutrient supply to the activated neurones. Without it, that downstream dilatation might be ineffective, placing neurones at risk, especially during episodes of intense neuronal activity, such as seizure. In the brain, pial arterioles represent important ‘upstream’ vascular segments. The pial arterioles overlie a thick layer of astrocytic processes, termed the glia limitans. This essentially isolates pial arterioles, anatomically, from the neurones below. Vasodilating signals that originate in the neurones therefore reach the pial arterioles via indirect pathways, primarily involving astrocytes and the glia limitans. Here we discuss a process whereby purinergic mechanisms play a key and neuronal activity‐dependent role in astrocyte to astrocyte communication, as well as in glia limitans to pial arteriolar signals leading to vasodilatation.

Loss of benefit from estrogen replacement therapy in diabetic ovariectomized female rats subjected to transient forebrain ischemia

In nondiabetic animals, estrogen has been shown to provide significant neuroprotection in focal and transient forebrain ischemia models. However, that neuroprotection may be diminished or lost in the diabetic. In this study, we compared the level of brain damage in intact, ovariectomized (OVX) and 17beta-estradiol (E(2))-treated OVX female rats rendered diabetic and chronically ( approximately 4 weeks) hyperglycemic via streptozotocin (STZ). Rats were subjected to 20 min of unilateral transient forebrain ischemia (reduction in cortical CBF to 20% of baseline). Neurologic function was analyzed daily and brain histopathology (in H&E-stained sections) was evaluated at 72 h of reperfusion. Supplemental histopathologic information was obtained from additional TUNEL-stained sections. When comparing neurologic outcome scores in the three groups, E(2)-treated OVX females displayed the highest degree of dysfunction and intact females the least (OVX rats not treated with E(2) were intermediate), with the difference between the intact and E(2)-treated groups being statistically significant. That same order was often observed with the regional histopathologic analyses of H&E-stained tissue. A significantly higher magnitude of neuronal loss in both OVX groups, when compared to intact females, was observed in the CA4 sector of the hippocampus and in the cortex. In addition, cell loss in the dorsal thalamus of the E(2)-treated group was significantly greater than in the intact females. Those results were generally corroborated by TUNEL-analysis, with 67% of the E(2)-treated, 33% of the control OVX, and only 17% of the intact females displaying TUNEL-positive cells in multiple regions. In conclusion, the present findings strongly suggest that the neuroprotective benefits of estrogen replacement therapy may be lost in the diabetic female rat.

Degeneration of noradrenergic fibres from the locus coeruleus causes tight-junction disorganisation in the rat brain

Although functional studies demonstrate that noradrenaline controls the permeability of the blood–brain barrier, it has never been determined whether this neurotransmitter regulates the tight junction (TJ) assembly that confers the barrier property to brain microvessels. We thus tested in rats the effect of pharmacological depletion of noradrenaline with the noradrenergic toxin DSP4 (5 mg/kg) on the expression of the TJ proteins zonula occludens‐1 (ZO1) and occludin. The effectiveness of the lesion was confirmed by tyrosine hydroxylase immunoreactivity, which showed noradrenergic fibre reduction accompanied by debris and swollen fibres in DSP4‐treated brains. Noradrenergic fibre degeneration caused: (i) gliosis; (ii) disappearance of TJ proteins in vascular cell‐to‐cell contacts (49.9 and 38.3% reductions for occludin and ZO1, respectively); (iii) a 49.2% decrease in total ZO1 protein, measured by Western blot analysis, parallel to a 39.5% decrease in ZO1 mRNA, measured by real‐time PCR; and (iv) a relative increase in the beta occludin isoform (62.9%), with no change in total occludin protein or mRNA. The expression of endothelial brain antigen, a marker of a functionally competent brain endothelium, was also reduced. We conclude that damage to the ascending fibres from the locus coeruleus caused TJ disruption and gliosis, a sign of inflammation. These results imply that the locus coeruleus degeneration reported in Alzheimers and Parkinsons diseases may contribute to these disorders by causing blood–brain barrier dysfunction. Whether the vascular damage is the result of impaired noradrenergic transmission or secondary to the inflammatory reaction remains to be determined.

Estrogen Replacement Treatment in Diabetic Ovariectomized Female Rats Potentiates Postischemic Leukocyte Adhesion in Cerebral Venules

Background and Purpose— Chronic 17β-estradiol (E2) replacement therapy in ovariectomized (OVX) female rats reduces leukocyte adhesion and brain damage after transient forebrain ischemia. Recently, we found that E2 treatment in diabetic OVX females was associated with enhanced postischemic neuropathology. We tested the hypothesis that in chronically hyperglycemic diabetic OVX females, chronic E2 replacement potentiates post-transient forebrain ischemia leukocyte adhesion. Methods— Pial venules were observed through closed cranial windows. Adherence of rhodamine 6G–tagged leukocytes was monitored before and 10 hours after transient forebrain ischemia (20 minutes right common carotid artery occlusion plus hemorrhagic hypotension) in intact, untreated OVX and E2-treated OVX females rendered diabetic via streptozotocin. Leukocyte adhesion was quantitated as the percentage venular area occupied by adherent leukocytes. Results— At 2 hours after transient forebrain ischemia, a similar low level of leukocyte adhesion was seen in the 3 groups (<3% of the venular area). Starting at ≈4 hours after ischemia, leukocyte adhesion in the E2-treated OVX females rose to significantly higher levels compared with the other groups. Relative to the 2-hour value, the level of adhesion at 10 hours was 12.5-fold, 4-fold, and 5-fold greater in the E2-treated OVX, OVX, and intact groups, respectively. Leukocyte extravasation (beginning after 6 hours of reperfusion) was observed in a majority (64%) of the E2-treated animals, with limited or no extravasation seen in the intact or OVX groups. Conclusions— These results suggest that factors associated with diabetes and chronic hyperglycemia convert E2 from a counterinflammatory to a proinflammatory substance in an ischemic setting.

Pharmacologic blockade of vascular adhesion protein-1 lessens neurologic dysfunction in rats subjected to subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) is a potentially devastating clinical problem. Despite advances in the diagnosis and treatment of SAH, outcome remains unfavorable. An increased inflammatory state, one that is characterized by enhanced leukocyte trafficking has been reported to contribute to neuronal injury in association with multiple brain insults, including hemorrhagic and ischemic stroke. This study was designed to investigate, in rats, the neuropathologic consequences of heightened leukocyte trafficking following SAH, induced via endovascular perforation of the anterior cerebral artery. Experiments focused on the initial 48 h post-SAH and sought to establish whether blockade of vascular adhesion protein-1 (VAP-1), with LJP-1586, was able to provide dose-dependent neuroprotection. Treatment with LJP-1586 was initiated at 6h post-SAH. An intravital microscopy and closed cranial window system, that permitted examination of temporal patterns of rhodamine-6G-labeled leukocyte adhesion/extravasation, was used. Effects of LJP-1586 on neurologic outcomes and leukocyte trafficking at 24 h and 48 h post-SAH were examined. In VAP-1-inhibited vs control rats, results revealed a significant attenuation in leukocyte trafficking at both 24 h and 48 h after SAH, along with an improvement in neurologic outcome. In conclusion, our findings support the involvement of an amplified inflammatory state, characterized by enhanced leukocyte trafficking, during the first 48 h after SAH. VAP-1 blockade yielded neuroprotection that was associated with an attenuation of leukocyte trafficking and improved neurologic outcome.

Aldose reductase inhibition ameliorates the detrimental effect of estrogen replacement therapy on neuropathology in diabetic rats subjected to transient forebrain ischemia

Estrogen replacement therapy (ERT) elicits a deleterious, instead of protective, effect on neuropathology in diabetic ovariectomized (OVX) rats subjected to cerebral ischemia. This transformation may be linked to an estrogen-associated increase in function of the receptor for advanced glycation end-products (RAGE). Moreover, under diabetic conditions, advanced glycation end-products (AGEs) are excessively generated through the aldose reductase (AR)-polyol pathway. As such, in diabetic rats given ERT, a RAGE-related exacerbation of post-ischemic brain injury can occur. Thus, in the present study, we evaluated the contribution of AR in estrogens detrimental effect on diabetic animals subjected to transient forebrain ischemia (TFI). Streptozotocin- and 17-beta estradiol-treated OVX female rats were divided into two groups, where AR activity was blocked using epalrestat; or AGEs production was restricted, via administrating the protein glycation crosslink breaker, ALT-711. In all animals, ERT was initiated approximately 10days before TFI. Pial venular leukocyte adhesion was evaluated over 10h post-TFI using a cranial window/intravital microscopy technique. In vehicle-treated control groups, a significant increase in leukocyte adhesion was observed post-TFI. Leukocyte extravasation, starting at approximately 6h post-TFI, was detected in most of the control animals. Chronic administration of either epalrestat or ALT-711 was associated with a marked decrease in post-TFI leukocyte adhesion, and the complete prevention of leukocyte extravasation. Animals receiving either epalrestat or ALT-711 exhibited a significant improvement in neurologic function, at 72h post-ischemia, compared to vehicle-treated controls. Post-ischemic (72h) histopathology was significantly reduced by epalrestat. Compared to the non-diabetic (ND) controls, diabetic OVX rats in the absence or presence of ERT showed a significant 2-fold or 3-fold increase in cortical AR mRNA levels, respectively. In contrast, only a modest increase in AR protein expression, relative to ND control, was detected in the two diabetic groups. The present findings suggest that AR participates in estrogens deleterious action on post-ischemic neuropathology in diabetics by promoting inflammation. Targeting the AR-controlled polyol pathway may be a clinically promising strategy to restore the neuroprotection of ERT in diabetic females.

Protective role of fingolimod (FTY720) in rats subjected to subarachnoid hemorrhage.

BackgroundSubarachnoid hemorrhage (SAH) is a neurological emergency with limited pharmacological treatment options. Inflammation is increasingly recognized as a key pathogenic contributor to brain injury in this condition. In the present study, we examined the neuroprotective effects of the immunomodulatory agent, fingolimod, in rats subjected to SAH.MethodsWe utilized an endovascular rat perforation model of SAH. Animals were divided into four groups: (1) sham-vehicle; (2) sham-fingolimod; (3) SAH-vehicle; and (4) SAH-fingolimod. Rats received either vehicle solution or fingolimod (0.5 mg/kg) intraperitoneally 3 hours after sham surgery or SAH. A closed cranial window and intravital microscope system was used at 48 hours to assess neuroinflammation, which was represented by rhodamine-6G-labeled leukocyte trafficking in pial venules, and pial arteriolar dilating responses to a variety of vasodilators, including hypercapnia, and topically-applied acetylcholine, adenosine, and S-nitroso-N-acetyl penicillamine. In addition, motor-sensory function was evaluated.ResultsCompared to sham-vehicle rats, SAH-vehicle animals displayed a four-times greater increase in pial venular intraluminal leukocyte adhesion. Treatment with fingolimod largely reduced the intravascular leukocyte adhesion. Vehicle-treated SAH animals displayed a significant decrease in pial arteriolar responses to all the vasodilators tested and vascular reactivity was preserved, to a significant degree, in the presence of fingolimod. In addition, neurological scores obtained at 48 hours post-SAH indicated significant neurological deficits in the vehicle-treated group (versus sham-vehicle surgical control). Those deficiencies were partially reduced by fingolimod (P < 0.0001 compared to the vehicle-treated SAH group).ConclusionsTreatment of rats with fingolimod was associated with a marked limitation in the intravascular adhesion of leukocytes to pial venules, preserved pial arteriolar dilating function, and improved neurological outcome in rats subjected to SAH.

Estrogen replacement therapy in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules via a RAGE-related process

In this study, we tested the hypothesis that the documented transformation of 17beta-estradiol (E2) from a counterinflammatory hormone in nondiabetic (ND) rats to a proinflammatory agent in rats with diabetes mellitus (DM) is due to an enhanced contribution from the receptor for advanced glycation end products (RAGE). Rhodamine 6G-labeled leukocytes were observed through a closed cranial window in rats. In vivo pial venular leukocyte adherence and infiltration were measured over 10 h reperfusion after transient forebrain ischemia in DM (streptozotocin) versus ND intact, ovariectomized (OVX), and E2-replaced (for 7-10 days) OVX (OVE) females. The role of RAGE was examined in two ways: 1) RAGE knockdown via topical application of RAGE antisense versus missense oligodeoxynucleotide or 2) intracerebroventricular injection of the RAGE decoy inhibitor, soluble RAGE. Among diabetic rats, the lowest levels of cortical RAGE mRNA and immunoreactivity of the RAGE ligand, AGE, were seen in OVX females, with significantly higher levels exhibited in intact and OVE females. However, results from the analysis of cortical RAGE protein only partially tracked those findings. When comparing ND to DM rats, cortical AGE immunoreactivity was significantly lower in OVE and intact females but similar in OVX rats. In DM rats, the level of postischemic leukocyte adhesion and infiltration (highest to lowest) was OVE>intact>>untreated OVX. In NDs, adhesion was highest in the untreated OVX group. Leukocyte extravasation was observed at >6 h postischemia but only in diabetic OVE and intact females and in ND OVX (untreated) rats. Pretreatment with RAGE antisense-oligodeoxynucleotide or soluble RAGE attenuated postischemic leukocyte adhesion and prevented infiltration but only in the diabetic OVE and intact groups. These results indicate that the exacerbation of postischemic leukocyte adhesion by chronic E2 replacement therapy in diabetic OVX females involves a RAGE-related mechanism. Targeting RAGE may restore the neuroprotective effect of E2 replacement therapy in diabetic females.

Complex modulation of the expression of PKC isoforms in the rat brain during chronic type 1 diabetes mellitus

We previously demonstrated that chronic hyperglycemia has a detrimental influence on neurovascular coupling in the brain-an effect linked to an alteration in the protein kinase C (PKC)-mediated phosphorylation pattern. Moreover, the activity of PKC was increased, in diabetic rat brain, in a tissue fraction composed primarily of the superficial glia limitans and pial vessels, but trended toward a decrease in cerebral cortical gray matter. However, that study did not examine the expression patterns of PKC isoforms in the rat brain. Thus, in a rat model of streptozotocin (STZ)-induced chronic type 1 diabetes mellitus (T1DM), and in non-diabetic (ND) controls, two hypotheses were addressed. First, chronic T1DM is accompanied by changes in the expression of PKC-α, βII, γ, δ, and ε Second, those changes differ when comparing cerebral cortex and glio-pial tissue. In addition, we analyzed the expression of a form of PKC-γ, phosphorylated on threonine 514 (pT514-PKC-γ), as well as the receptor for activated C kinase 1 (RACK1). The expression pattern of different PKC isoforms was altered in a complex and tissue-specific manner during chronic hyperglycemia. Notably, in the gray matter, PKC-α expression significantly decreased, while pT514-PKC-γ expression increased. However, PKC-βII, -γ, -δ, -ε, and RACK1 expressions did not change. Conversely, in glio-pial tissue, PKC-α and RACK1 were upregulated, whereas PKC-γ, pT514-PKC-γ, and PKC-ε were downregulated. PKC-βII, and PKC-δ, were unchanged. These findings suggest that the PKC activity increase previously seen in the glio-pial tissue of diabetic rats may be due to the selective upregulation of PKC-α, and ultimately lead to the impairment of neurovascular coupling.

Changes in the metabolism of sphingolipids after subarachnoid hemorrhage

We previously described how ceramide (Cer), a mediator of cell death, increases in the cerebrospinal fluid (CSF) of subarachnoid hemorrhage (SAH) patients. This study investigates the alterations of biochemical pathways involved in Cer homeostasis in SAH. Cer, dihydroceramide (DHC), sphingosine‐1‐phosphate (S1P), and the activities of acid sphingomyelinase (ASMase), neutral sphingomyelinase (NSMase), sphingomyelinase synthase (SMS), S1P‐lyase, and glucosylceramide synthase (GCS) were determined in the CSF of SAH subjects and in brain homogenate of SAH rats. Compared with controls (n = 8), SAH patients (n = 26) had higher ASMase activity (10.0 ± 3.5 IF/µl· min vs. 15.0 ± 4.6 IF/µl • min; P = 0.009) and elevated levels of Cer (11.4 ± 8.8 pmol/ml vs. 33.3 ± 48.3 pmol/ml; P = 0.001) and DHC (1.3 ± 1.1 pmol/ml vs. 3.8 ± 3.4 pmol/ml; P = 0.001) in the CSF. The activities of GCS, NSMase, and SMS in the CSF were undetectable. Brain homogenates from SAH animals had increased ASMase activity (control: 9.7 ± 1.2 IF/µg • min; SAH: 16.8 ± 1.6 IF/µg • min; P < 0.05) and Cer levels (control: 3,422 ± 26 fmol/nmol of total lipid P; SAH: 7,073 ± 2,467 fmol/nmol of total lipid P; P < 0.05) compared with controls. In addition, SAH was associated with a reduction of 60% in S1P levels, a 40% increase in S1P‐lyase activity, and a twofold increase in the activity of GCS. In comparison, NSMase and SMS activities were similar to controls and SMS activities similar to controls. In conclusion, our results show an activation of ASMase, S1P‐lyase, and GCS resulting in a shift in the production of protective (S1P) in favor of deleterious (Cer) sphingolipids after SAH. Additional studies are needed to determine the effect of modulators of the pathways described here in SAH.