Regulation of Expression of Extracellular Matrix Proteins by Differential Target Multiplexed Spinal Cord Stimulation (SCS) and Traditional Low-Rate SCS in a Rat Nerve Injury Model.

There is limited research on the association between the extracellular matrix (ECM) and chronic neuropathic pain. The objective of this study was twofold. Firstly, we aimed to assess changes in expression levels and the phosphorylation of ECM-related proteins due to the spared nerve injury (SNI) model of neuropathic pain. Secondly, two modalities of spinal cord stimulation (SCS) were compared for their ability to reverse the changes induced by the pain model back toward normal, non-injury levels. We identified 186 proteins as ECM-related and as having significant changes in protein expression among at least one of the four experimental groups. Of the two SCS treatments, the differential target multiplexed programming (DTMP) approach reversed expression levels of 83% of proteins affected by the pain model back to levels seen in uninjured animals, whereas a low-rate (LR-SCS) approach reversed 67%. There were 93 ECM-related proteins identified in the phosphoproteomic dataset, having a combined 883 phosphorylated isoforms. DTMP back-regulated 76% of phosphoproteins affected by the pain model back toward levels found in uninjured animals, whereas LR-SCS back-regulated 58%. This study expands our knowledge of ECM-related proteins responding to a neuropathic pain model as well as providing a better perspective on the mechanism of action of SCS therapy.

Activation of Neuroinflammation via mTOR Pathway is Disparately Regulated by Differential Target Multiplexed and Traditional Low-Rate Spinal Cord Stimulation in a Neuropathic Pain Model.

Introduction: Spinal cord stimulation (SCS) has been used for decades to treat neuropathic pain conditions with limited understanding of its mechanisms of action. The mTOR pathway is a well-known co-factor in chronic pain and has not been previously linked to SCS therapy. Proteomic and phosphorylation analyses allow capturing a broad view of tissue response to an injury model and subsequent therapies such as SCS. Here, we evaluated the effect of differential target multiplexed SCS programming (DTMP) and traditional low-rate spinal cord stimulation (LR-SCS) on the mTOR pathway using proteomic and phosphoproteomic analyses. Methods: The spared nerve injury (SNI) model of neuropathic pain in animals was established followed by continuous treatment with either DTMP or LR-SCS for 48 hours. Control groups included sham-stimulated (No-SCS) and uninjured animals (No-SNI). Proteins were extracted from spinal cord tissue removed post-stimulation and subjected to liquid chromatography/tandem mass spectrometry to assess changes in protein expression and states of phosphorylation. Bioinformatics tools and literature were used to identify mTOR-related proteins in the various groups. Results: Over 7000 proteins were identified and filtered to find 1451 and 705 proteins significantly affected by DTMP and LR-SCS (p < 0.05), respectively, relative to No-SCS. Literature and bioinformatic tools yielded 192 mTOR-related proteins that were cross-referenced to the list of DTMP and LR-SCS affected proteins. Of these proteins, 49 were found in the proteomic dataset. Eight of these proteins showed a significant response to the pain model, 25 were significantly modulated by DTMP, and 8 by LR-SCS. Phosphoproteomic analyses yielded 119 mTOR-related phosphoproteins affected by the injury model with a 66% reversal following DTMP versus a 58% reversal by LR-SCS. Conclusion: Proteomic and phosphoproteomic analyses support the hypothesis that DTMP, and to a lesser extent LR-SCS, reverse injury induced changes of the mTOR pathway while treating neuropathic pain.

Proteomic and Phosphoproteomic Changes of MAPK-Related Inflammatory Response in an Animal Model of Neuropathic Pain by Differential Target Multiplexed SCS and Low-Rate SCS.

Introduction: Neuropathic pain initiates an interplay of pathways, involving MAP kinases and NFκB-signaling, leading to expression of immune response factors and activation and inactivation of proteins via phosphorylation. Neuropathic pain models demonstrated that spinal cord stimulation (SCS) may provide analgesia by modulating gene and protein expression in neuroinflammatory processes. A differential target multiplexed programming (DTMP) approach was more effective than conventional SCS treatments at modulating these. This work investigated the effect of DTMP and low rate SCS (LR-SCS) on proteins associated with MAP kinases and NFκB-signaling relevant to neuroinflammation. Methods: Animals subjected to the spared nerve injury model (SNI) of neuropathic pain were treated continuously (48h) with either DTMP or LR-SCS. No-SNI and No-SCS groups were included as controls. Proteomics and phosphoproteomics of stimulated spinal cord tissues were performed via liquid chromatography/tandem mass spectrometry. Proteins were identified from mass spectra using bioinformatics. Expression levels and fold changes (No-SCS/No-SNI and SCS/No-SCS) were obtained from spectral intensities. Results: Analyses identified 7192 proteins, with 1451 and 705 significantly changed by DTMP and LR-SCS, respectively. Eighty-one proteins, including MAP kinases, facilitating NFκB-signaling as part of inflammatory processes were identified. The pain model significantly increased expression levels of complement pathway-related proteins (LBP, NRG1, APP, CFH, C3, C5), which were significantly reversed by DTMP. Expression levels of other complement pathway-related proteins (HMGB1, S100A8, S100A9, CRP, C4) were decreased by DTMP, although not significantly affected by SNI. Other proteins (ORM1, APOE, NG2, CNTF) involved in NFκB-signaling were increased by SNI and decreased by DTMP. Expression levels of phosphorylated protein kinases involved in NFκB-signaling (including MAP kinases, PKC, MARK1) were affected by the pain model and reverse modulated by DTMP. LR-SCS modulated inflammatory-related proteins although to a lesser extent than DTMP. Conclusion: Proteomic analyses support the profound effect of the DTMP approach on neuroinflammation via MAP kinases and NFκB-mediated signaling to alleviate neuropathic pain.

Differential target multiplexed spinal cord stimulation programming modulates proteins involved in ion regulation in an animal model of neuropathic pain.

The effect of spinal cord stimulation (SCS) using differential target multiplexed programming (DTMP) on proteins involved in the regulation of ion transport in spinal cord (SC) tissue of an animal model of neuropathic pain was evaluated in comparison to low rate (LR) SCS. Rats subjected to the spared nerve injury model (SNI) and implanted with a SCS lead were assigned to DTMP or LR and stimulated for 48 h. A No-SCS group received no stimulation, and a Sham group received no SNI or stimulation. Proteins in the dorsal ipsilateral quadrant of the stimulated SC were identified and quantified using mass spectrometry. Proteins significantly modulated by DTMP or LR relative to No-SCS were identified. Bioinformatic tools were used to identify proteins related to ion transport regulation. DTMP modulated a larger number of proteins than LR. More than 40 proteins significantly involved in the regulation of chloride (Cl-), potassium (K+), sodium (Na+), or calcium (Ca2+) ions were identified. SNI affected proteins that promote the increase of intracellular Ca2+, Na+, and K+ and decrease of intracellular Cl-. DTMP modulated proteins involved in glial response to neural injury that affect Ca2+ signaling. DTMP decreased levels of proteins related to Ca2+ transport that may result in the reduction of intracellular Ca2+. Presynaptic proteins involved in GABA vesicle formation and release were upregulated by DTMP. DTMP also upregulated postsynaptic proteins involved with elevated intracellular Cl-, while modulating proteins, expressed by astrocytes, that regulate postsynaptic Cl- inhibition. DTMP downregulated K+ regulatory proteins affected by SNI that affect neuronal depolarization, and upregulated proteins that are associated with a decrease of intracellular neuronal K+ and astrocyte uptake of extracellular K+. DTMP treatment modulated the expression of proteins with the potential to facilitate a reversal of dysregulation of ion transport and signaling associated with a model of neuropathic pain.

Modulation of microglial activation states by spinal cord stimulation in an animal model of neuropathic pain: Comparing high rate, low rate, and differential target multiplexed programming.

While numerous studies and patient experiences have demonstrated the efficacy of spinal cord stimulation as a treatment for chronic neuropathic pain, the exact mechanism underlying this therapy is still uncertain. Recent studies highlighting the importance of microglial cells in chronic pain and characterizing microglial activation transcriptomes have created a focus on microglia in pain research. Our group has investigated the modulation of gene expression in neurons and glial cells after spinal cord stimulation (SCS), specifically focusing on transcriptomic changes induced by varying SCS stimulation parameters. Previous work showed that, in rodents subjected to the spared nerve injury (SNI) model of neuropathic pain, a differential target multiplexed programming (DTMP) approach provided significantly better relief of pain-like behavior compared to high rate (HRP) and low rate programming (LRP). While these studies demonstrated the importance of transcriptomic changes in SCS mechanism of action, they did not specifically address the role of SCS in microglial activation. The data presented herein utilizes microglia-specific activation transcriptomes to further understand how an SNI model of chronic pain and subsequent continuous SCS treatment with either DTMP, HRP, or LRP affects microglial activation. Genes for each activation transcriptome were identified within our dataset and gene expression levels were compared with that of healthy animals, naïve to injury and interventional procedures. Pearson correlations indicated that DTMP yields the highest significant correlations to expression levels found in the healthy animals across all microglial activation transcriptomes. In contrast, HRP or LRP yielded weak or very weak correlations for these transcriptomes. This work demonstrates that chronic pain and subsequent SCS treatments can modulate microglial activation transcriptomes, supporting previous research on microglia in chronic pain. Furthermore, this study provides evidence that DTMP is more effective than HRP and LRP at modulating microglial transcriptomes, offering potential insight into the therapeutic efficacy of DTMP.

Impairment of neurovascular coupling in Type 1 Diabetes Mellitus in rats is prevented by pancreatic islet transplantation and reversed by a semi-selective PKC inhibitor.

Streptozotocin (STZ)-induced chronic hyperglycemia has a detrimental effect on neurovascular coupling, linked to increased PKC-mediated phosphorylation and PKC isoform expression changes. Here, we sought to determine whether: 1) selective PKC-α/ß/γ inhibitor, GF109203X, could reverse the effects of chronic hyperglycemia on cerebrovascular reactivity; 2) pancreatic islet transplantation could prevent the development of cerebrovascular impairment seen in a rat model of Type 1 Diabetes. We studied the effect of GF109203X in diabetic (DM), non-diabetic (ND), and transplanted (TR) Lewis rats during either sciatic nerve stimulation (SNS) or the topical applications of the large-conductance Ca2+-operated K+(BKCa) channel opener, NS1619, or the K+ inward rectifier (Kir) channel agonist, KCl. Pial arteriole diameter changes were monitored using a closed cranial window in vivo microscopy technique. The pial arteriole dilatory response associated with SNS was decreased by ~45%, when comparing DM vs either ND or TR rats. Also, pial arteriolar dilations to topical KCl and NS1619 were largely attenuated in DM rats, but not in ND or TR animals. These responses were completely restored by the acute application of GF109203X to the brain surface. The PKC inhibitor had no effect on vascular responses in normoglycemic and TR animals. In conclusion, DM-associated chronic impairment of neurovascular coupling may be readily reversed by a PKC-α/ß/γ inhibitor or prevented via pancreatic islet transplantation. We believe that specific PCK isoforms (α/ß/γ) are mechanistically linked to the neurovascular uncoupling seen with hyperglycemia.

Intracerebroventricular application of S100B selectively impairs pial arteriolar dilating function in rats.

S100B is an astrocyte-derived protein that can act through the receptor for advanced glycation endproducts (RAGE) to mediate either "trophic" or "toxic" responses. Its levels increase in many neurological conditions with associated microvascular dysregulation, such as subarachnoid hemorrhage (SAH) and traumatic brain injury. The role of S100B in the pathogenesis of microvasculopathy has not been addressed. This study was designed to examine whether S100B alters pial arteriolar vasodilating function. Rats were randomized to receive (1) artificial cerebrospinal fluid (aCSF), (2) exogenous S100B, and (3) exogenous S100B+the decoy soluble RAGE (sRAGE). S100B was infused intracerebroventricularly (icv) using an osmotic pump and its levels in the CSF were adjusted to achieve a concentration similar to what we observed in SAH. After 48 h of continuous icv infusion, a cranial window/intravital microscopy was applied to animals for evaluation of pial arteriolar dilating responses to sciatic nerve stimulation (SNS), hypercapnia, and topical suffusion of vasodilators including acetylcholine (ACh), s-nitroso-N-acetyl penicillamine (SNAP), or adenosine (ADO). Pial arteriolar dilating responses were calculated as the percentage change of arteriolar diameter in relation to baseline. The continuous S100B infusion for 48 h was associated with reduced responses to the neuronal-dependent vasodilator SNS (p<0.05) and the endothelial-dependent vasodilator ACh (p<0.05), compared to controls. The inhibitory effects of S100B were prevented by sRAGE. On the other hand, S100B did not alter the responses elicited by vascular smooth muscle cell-dependent vasodilators, namely hypercapnia, SNAP, or ADO. These findings indicate that S100B regulates neuronal and endothelial dependent cerebral arteriolar dilation and suggest that this phenomenon is mediated through RAGE-associated pathways.

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.

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, ßII, 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-ßII, -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-ßII, 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.

Post-ischemic vascular adhesion protein-1 inhibition provides neuroprotection in a rat temporary middle cerebral artery occlusion model.

We examined the neuroprotective efficacy associated with post-ischemic vascular adhesion protein-1 (VAP-1) blockade in rats subjected to transient (1 h) middle cerebral artery occlusion (MCAo). We compared saline-treated control rats to rats treated with a highly selective VAP-1 inhibitor, LJP-1586 [Z-3-fluoro-2-(4-methoxybenzyl) allylamine hydrochloride]. Initial intraperitoneal LJP-1586 (or saline control) treatments were delayed until 6 h or 12 h reperfusion. At 72-h reperfusion, LJP-1586-treated rats displayed 51% and 33% smaller infarct volumes, relative to their controls, in the 6- and 12-h treatment groups, respectively. However, only in the 6-h treatment group was the infarct volume reduction significant (p < 0.05). On the other hand, we observed significantly improved neurologic functions in both 6- and 12-h treatment groups, versus their matched controls (p < 0.05). Also, the effect of 6-h LJP-1586 treatment on post-ischemic leukocyte trafficking in pial venules overlying the ischemic cortex was evaluated using intravital microscopy. These experiments revealed that: 1) LJP-1586 did not affect intravascular leukocyte (largely neutrophil) adhesion, at least out to 12-h reperfusion; and 2) the onset of neutrophil extravasation, which occurred between 6-8-h reperfusion in control rats, was prevented by LJP-1586-treatment. In conclusion, in rats subjected to transient MCAo, selective VAP-1 pharmacologic blockade provided neuroprotection, with a prolonged therapeutic window of 6-12-h reperfusion.

Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BK(Ca) and Kir channels.

We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ∼30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats.

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5'-nucleotidase inhibitor α,ß-methylene adenosine 5'-diphosphate (AOPCP), 2) the A(2) receptor blocker ZM 241385, 3) the ADP P2Y(1) receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 µM, but not 100 µM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 µM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A(2) receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y(1) activation.

Purinergic mechanisms in gliovascular coupling.

Regional elevations in cerebral blood flow (CBF) often occur in response to localized increases in cerebral neuronal activity. An ever expanding literature has linked this neurovascular coupling process to specific signaling pathways involving neuronal synapses, astrocytes and cerebral arteries and arterioles. Collectively, these structures are termed the "neurovascular unit" (NVU). Astrocytes are thought to be the cornerstone of the NVU. Thus, not only do astrocytes "detect" increased synaptic activity, they can transmit that information to proximal and remote astrocytic sites often through a Ca(2+)- and ATP-related signaling process. At the vascular end of the NVU, a Ca(2+)-dependent formation and release of vasodilators, or substances linked to vasodilation, can occur. The latter category includes ATP, which upon its appearance in the extracellular compartment, can be rapidly converted to the potent vasodilator, adenosine, via the action of ecto-nucleotidases. In the present review, we give consideration to experimental model-specific variations in purinergic influences on gliovascular signaling mechanisms, focusing on the cerebral cortex. In that discussion, we compare findings obtained using in vitro (rodent brain slice) models and multiple in vivo models (2-photon imaging; somatosensory stimulation-evoked cortical hyperemia; and sciatic nerve stimulation-evoked pial arteriolar dilation). Additional attention is given to the importance of upstream (remote) vasodilation; the key role played by extracellular ATP hydrolysis (via ecto-nucleotidases) in gliovascular coupling; and interactions among multiple signaling pathways.

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation.

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,ß-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.

An excitatory loop with astrocytes contributes to drive neurons to seizure threshold.

Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron-astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures.

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 estrogen's 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 estrogen's 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.

Caffeine and the control of cerebral hemodynamics.

While the influence of caffeine on the regulation of brain perfusion has been the subject of multiple publications, the mechanisms involved in that regulation remain unclear. To some extent, that uncertainty is a function of a complex interplay of processes arising from multiple targets of caffeine located on a variety of different cells, many of which have influence, either directly or indirectly, on cerebral vascular smooth muscle tone. Adding to that complexity are the target-specific functional changes that may occur when comparing acute and chronic caffeine exposure. In the present review, we discuss some of the mechanisms behind caffeine influences on cerebrovascular function. The major effects of caffeine on the cerebral circulation can largely be ascribed to its inhibitory effects on adenosine receptors. Herein, we focus mostly on the A1, A2A, and A2B subtypes located in cells comprising the neurovascular unit (neurons, astrocytes, vascular smooth muscle); their roles in the coupling of increased neuronal (synaptic) activity to vasodilation; how caffeine, through blockade of these receptors, may interfere with the "neurovascular coupling" process; and receptor-linked changes that may occur in cerebrovascular regulation when comparing acute to chronic caffeine intake.

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.

Astrocytes are a key conduit for upstream signaling of vasodilation during cerebral cortical neuronal activation in vivo.

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to "sense" neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator.

Pial arteriolar vasomotion changes during cortical activation in rats.

The oscillatory pattern of pial arterioles, i.e. vasomotion, has been described since early 1980s, but the impact of neural activation on such oscillations has never been formally examined. Sciatic nerve stimulation, a well characterized model for studying neurovascular coupling (NVC), leads to a neural activity-related increase of pial arteriolar diameter in the contralateral hindlimb somatosensory cortex. Exploiting such an experimental model, the aim of the present study was to explore vasomotion and its changes during NVC with a novel analytical approach. Indeed, to characterize oscillations, we evaluated the total spectral power in the range 0.02-2.00 Hz and subdivided this frequency interval into seven 50% overlapping frequency bands. Results indicated that only arterioles overlying the stimulated hindlimb cortex showed a significant increase of total power, unlike arterioles overlaying the whisker barrel cortex, used as control for the vascular response specificity. The total power increase was sustained mainly by marked increments in the low frequency range, with two peaks at 0.03 and 0.08 Hz, and by a wide increase in the high frequency range (0.60-2.00 Hz) in the averaged spectrum. These activity-related spectral changes suggest: (i) that it is possible to assess the vascular responses by using total power; (ii) the existence of at least three distinct mechanisms involved in the control of NVC, two with a feedback frequency loop in the low frequency range and another one in the high range; (iii) a potential involvement of vasomotion in NVC. Moreover, these findings highlight the oscillatory nature of the mechanisms controlling NVC.