ObjectiveIn spite of its apparent promise, bioelectric modulation of the vagal innervation of the stomach has had limited success modifying ingestive behavior. But, importantly, stimulation parameters in previous bioelectrical applications were arbitrarily employed without information as to their stimulus‐response relationships or optimal locations. With this in mind, we established a model of chronic vagal nerve stimulation (VNS) where we record GI motility continuously in unanesthetized, freely moving rats while their intake is monitored as they consume chow and water ad libitum (automated food and liquid intake monitors; BioDAQ, Research Diets Inc.).MethodsA patch electrode (MicroProbes) was attached to the muscle wall of the proximal forestomach of healthy adult male Sprague‐Dawley rats at a site known to contain the highest density of vagal mechanoreceptors. A strain gauge was similarly attached to the muscle wall of the antrum. Rats were maintained in their home cages equipped with BioDAQ feeders, while continuously tethered to overhead commutators. Following recovery, the feeding and drinking behavior as well as antral motility of individual rats was continuously monitored and recorded with and without concomitant VNS (pulse parameters, derived from acute studies: 0.3mA/0.2ms/10Hz; 20s On:40s Off). Subjects were exposed to each of the following four conditions on different days at the onset of lights off: a 2 h fast, a 2 h fast while being stimulated, 2 h ad lib access to chow, and 2 h ad lib access to chow while being stimulated. In a separate study, 2 days of strain gauge activity was continuously recorded: 24 h without stimulation followed by 24 h with stimulation.ResultsRats recovered quickly following surgery. Compared to the 2 h fasted condition, there was a 17% increase in antral peristalsis (change in amplitude and frequency expressed as area under the curve) when subjects were fasted while being stimulated; similarly, there was an 18% increase in antral peristalsis when subjects were stimulated while consuming chow compared to 2 h intake of chow without stimulation. The same pattern was observed in the 2 d study with a 23% increase in antral activity during 24 h of stimulation compared to 24 h pre‐stimulation. Interestingly, a 12% increase in total chow intake occurred when stimulated for 24 h while no difference was noted in water intake between the two conditions.ConclusionsInitial exposure to VNS results in a modest increase in antral activity with and without the presence of food in the stomach. Analyses are underway to tease apart the effect of stimulation on motility in relation to meal size. Finally, these findings confirm that our chronic VNS model has considerable potential for determining optimal stimulation parameters necessary for making meaningful changes in gut motility.Support or Funding InformationSPARC/NIH Office of the Director OT2 OD023847NIDDK/NIH R01 DK027627

Transcutaneous auricular vagus nerve stimulation (taVNS) is a promising, non‐invasive approach to modulate activity in the central nervous system, heart, lungs, stomach and other organs that receive projections from the vagus nerve. The optimal stimulus parameters for modulating stomach function are unknown, preventing further development of taVNS as a device‐based treatment for motility disorders like gastroparesis. We hypothesized that taVNS parameters could be tuned to preferentially modulate antroduodenal motility with fewer off‐target effects on the heart than cervical VNS. Using a custom‐made stimulation and recording system (Autonomous Neural Control, or ANC), we surveyed the taVNS‐mediated effects on cardiac, vagal (ventral gastric branch), antral and duodenal motility in male Sprague Dawley rats under isoflurane anesthesia (250–400 g; N = 14 rats). With custom carbon gel electrodes, we stimulated the left cymba concha at 1 or 10 Hz using 28 combinations of stimulus pulse currents and durations (0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mA pulse currents in combination with 0.1, 0.2, 0.4 and 0.8 ms pulse durations), while measuring the electrocardiogram, antral electrogastrogram, and antral/duodenal motility with implanted strain gauges. The ANC software applied each stimulus parameter combination in a random order in 60 s cycles (20 s ON/40 s OFF). Stimulus pulse durations less than 0.4 ms were not associated with any significant effects on cardiac (measured from the electrocardiogram) or gastric activity (measured from the antral electrogastrogram as well as antral and duodenal strain gauge recordings), but did produce notable increases in nerve activity at the level of the ventral gastric branch (measured with an implanted bipolar cuff electrode). A pulse duration of 0.8 ms produced the most robust and consistent decrease in heart rate, increase in heart rate variability, increase in antral smooth muscle activity, and increase in antroduodenal motility, as is expected from an increase in vagal outflow. In contrast to cervical VNS, where the optimal parameters for increasing antral motility largely overlapped with those that induced severe bradycardia, the optimal taVNS parameters that increased antroduodenal motility at 1 or 10 Hz (Pulse Current: 0.2–0.6 mA | Pulse Duration: 0.4–0.8 ms) required less current, on average, than the stimulus parameters that produced the greatest reduction in heart rate and increase in heart rate variability (Pulse Current: 0.4–1.0 mA | Pulse Duration: 0.4–0.8 ms). Pulse currents greater than 0.6 mA did not have any significant effect on antroduodenal motility, supporting the value of our parameter search approach in sorting the useful from the useless stimulus parameters. These results strongly support taVNS as a viable approach to modulate gastrointestinal activity with greater specificity and control over stomach function than cervical VNS.Support or Funding InformationThis work was supported by NIH SPARC OT2 OD023847

PurposeThe gut communicates with the brain, allowing the gastrointestinal state to influence cognition and emotion and vice versa. In the resting state, gastric electrical activity has been shown to be synchronized with the blood‐oxygen‐level‐dependent (BOLD) signal in the so‐called gastric network in humans [1]. However, the finding has been rarely replicated. Here, we explored the gut‐brain synchrony in rats. Whole‐brain functional magnetic resonance imaging (fMRI) was acquired simultaneously with electrogastrogram (EGG) recording. Cross‐correlation between EGG and BOLD signals was used to map resting state networks influenced by gastric activity.MethodBrain fMRI was performed on three SD rats together with multi‐channel EGG recording. Each rat was trained to consume diet gel enriched with Gadolinium contrast media. Before the experiment, the rat was fed with 5g diet gel. After the feeding, the animal was anesthetized with continuous dexmedetomidine and isoflurane. The multi‐channel EGG signal was recorded using an electrophysiological recording system (Tucker Davis Technologies Inc.) together with fMRI scans using a 7‐tesla small‐animal MRI system (BioSpec 70/30, Bruker). The principal component analysis was applied to EGG signals. The correlation between the voxel‐wise fMRI signal and the component‐wise EGG signal was calculated, yielding multiple gut‐associated brain networks. The voxel‐wise fMRI signal was also modeled as a linear combination of EGG component time series with varying time shifts.ResultsFMRI was acquired simultaneously with the EGG recording ( Fig. 1A). EGG was denoised and cross‐correlated with the BOLD activity in every voxel to map an EGG‐correlated network (or the gastric network) ( Fig. 1C). The BOLD signal was averaged across voxels in the gastric network. The resulting network‐wide signal was found to be phase‐locked to EGG with a time delay ( Fig. 1B). Accounting for the delay variable across voxels, we mapped an even broader network phase‐locked with EGG ( Fig. 2A). Different EGG‐coupled regions were found to lag behind EGG by different times, ranging from 4 to 12 seconds ( Fig. 2B & C). From the EGG‐coupled BOLD response, the visual and auditory cortex had a relatively longer delay, whereas the somatosensory and anterior cingulate cortex had a shorter delay. The cingulate cortex showed a complex response pattern, likely implying multiple functional roles with respect to the gut.ConclusionIn rats, brain activity is intrinsically synchronized with gastric activity at a specific resting state network. In this network, different brain regions are time‐locked to gastric activity with varying time delays. Speculatively, the pattern of time delay may reflect how gastric information propagates through a cascade of brain regions for sequential processing.Support or Funding InformationThis study was funded by National Institutes of Health’s SPARC ‐ Stimulating Peripheral Activity to Relieve Conditions ‐ program (OT2OD023847).Simultaneous EGG and brain fMRI recording. (A) shows the layout of the 32‐channel electrode array for EGG recording. (B) are examples of EGG and fMRI time series. (C) shows an example of the EGG correlation map. The correlation between EGG and fMRI time series is color‐coded.Figure 1The EGG network considering the phase delays between EGG and fMRI signal. (A) is a summary of the EGG network. The color represents the F statistic of the multiple linear regression. (B) selects six voxels in the EGG network. The curve shows the relation between the F statistic and EGG‐fMRI delays for the selected voxels, which are marked with blue box in (A). (C) is the phase delay map that delineates the phase differences between EGG and fMRI time series.Figure 2

BACKGROUNDThe discovery that the stomach secretes the hormone leptin, plus the finding that vagal afferents express receptors for leptin, in addition to the recognized role of adipocytes in the secretion of the adipokine, have initiated extensive investigations into leptin’s possible roles in the control of feeding. The gastric secretion of leptin and its binding sites on vagal afferents suggest the possibility that gastric electrical stimulation (GES) might produce leptin secretion and modulate visceral sensory information arising from the stomach and relayed to the brain. Such a pathway might have therapeutic potential for treating GI disorders (gastroparesis, eating disorders, etc.).AIMSTo explore or map defined gastric regional patterns of leptin release, secretion of the hormone to GES at different sites was measured in terms of both amplitude and time course.METHODSIn fasted (18 hrs.), anesthetized (Isoflurane) SD rats (n = 49), patch electrodes were sutured on ventral stomach wall, a strain gauge was attached to duodenum, and a catheter was inserted into left femoral artery. Stimulation (biphasic, 0.3mA, 0.2ms, 10Hz, 20s‐on‐40s‐off; 5 cycles) was applied from 0 to 5 min. The gastric antrum, corpus and forestomach were each divided into three regions corresponding to the distance between the lower esophageal sphincter (LES) and the greater curvature (GC); each of the three gastric compartments (verified post mortem) was stimulated near the LES, at a mid‐point, and near the GC. Multiple blood samples (0.15ml/each) were collected to measure leptin concentrations.RESULTSStimulation of much of the antrum (mid‐and near‐GC antral regions) evoked fast, robust and long‐lasting leptin secretion. Stimulation of the corpus in a more limited area (mid‐corpus) produced moderate, long‐lasting leptin secretion. Stimulation of a limited forestomach area (mid‐forestomach) yielded a mild, short‐interval leptin secretion. For the effective loci, leptin secretion amplitude changes in post‐stim time points of 5, 15, and 30 min, respectively, compared to control values, were: Mid‐antrum: +58%, +70%, +48%; Near‐GC antrum: +43%, +70%, +33%; Mid‐corpus: +25%. +38%, +38%; and Mid‐forestomach: +38%, +22%, +8%.DISCUSSIONGastric leptin is secreted by both exocrine and endocrine pathways. Antrum sensitivity to stimulation may reflect higher density of leptin‐secretory epithelium, corpus may have moderate density leptin‐related epithelium. Without leptin‐related epithelium in forestomach, leptin release may be caused indirectly.CONCLUSIONOur findings reveal that GES in different stomach regions can produce strikingly different leptin secretion patterns. Selectively modulating leptin secretion with GES applied to different stomach regions could be used as a neuromodulation strategy to treat GI disorders such as gastroparesis, obesity, and various eating disorders.Support or Funding InformationFUNDING: SPARC/NIH OT2 OD023847NIDDK/NIH R01 DK027627

BEFORE STARTING A simple approach to identify the influence of left vagal stimulus pulse parameters on vagal and gastric electrical activity in rat Matthew Ward , Thomas V Nowak , Zhenjun Tan , Bartek Rajwa , Robert Phillips , Terry L Powley Weldon School of Biomedical Engineering (Purdue University, West Lafayette, IN) and Indiana University School of Medicine (Indianapolis, IN), Indiana University School of Medicine (Indianapolis, IN), Dept. of Psychological Sciences (Purdue University, West Lafayette, IN), Bindley Bioscience Center (Purdue University

This protocol describes a process for the measurement of electrical stimulation-induced effects on duodenal motility in young adult Sprague-Dawley rats. Signals recorded from strain gauges attached to the proximal duodenal surface were used to measure the effect of stimulation by patch electrodes implanted at multiple sites across the rat stomach in an acute anesthetized preparation. The effect of stimulation was quantified as the ratio of various motility assessments during and after stimulation vs. before stimulation, and the data was used to create a functional map of duodenal motor response to localized gastric stimulation.

The failed translation of proposed therapeutic agents for ischemic stroke from preclinical to clinical studies has led to increased scrutiny of preclinical studies, namely the model and outcome measures utilized. Preclinical studies routinely use infarct volume as an experimental endpoint or measure in studies employing young-adult, healthy male animals despite the fact that clinically, ischemic stroke is a disease of the elderly and improvements in functional outcome from pre- to post-intervention remains the most widely utilized assessment. The validity of infarct volume as a surrogate measure for functional outcome remains unclear in clinical studies as well as preclinical studies, particularly those utilizing a more clinically relevant aged thromboembolic model. In this work, we will address the relationship between acute and chronic functional outcome and infarct volume using a variety of functional assessments ranging from more simplistic, subjective measurements such as the modified Neurologic Severity Score (mNSS), to more complex, objective measurements such as grip strength and inclined plane.

Ischemic stroke and Alzheimer's disease (AD), despite being distinct disease entities, share numerous pathophysiological mechanisms such as those mediated by inflammation, immune exhaustion, and neurovascular unit compromise. An important shared mechanistic link is acute and chronic changes in protein kinase C (PKC) activity. PKC isoforms have widespread functions important for memory, blood-brain barrier maintenance, and injury repair that change as the body ages. Disease states accelerate PKC functional modifications. Mutated forms of PKC can contribute to neurodegeneration and cognitive decline. In some cases the PKC isoforms are still functional but are not successfully translocated to appropriate locations within the cell. The deficits in proper PKC translocation worsen stroke outcome and amyloid-β toxicity. Cross talk between the innate immune system and PKC pathways contribute to the vascular status within the aging brain. Unfortunately, comorbidities such as diabetes, obesity, and hypertension disrupt normal communication between the two systems. The focus of this review is to highlight what is known about PKC function, how isoforms of PKC change with age, and what additional alterations are consequences of stroke and AD. The goal is to highlight future therapeutic targets that can be applied to both the treatment and prevention of neurologic disease. Although the pathology of ischemic stroke and AD are different, the similarity in PKC responses warrants further investigation, especially as PKC-dependent events may serve as an important connection linking age-related brain injury.

Blood–brain barrier (BBB) disruption and hemorrhagic transformation (HT) following ischemic/reperfusion injury contributes to post-stroke morbidity and mortality. Bryostatin, a potent protein kinase C (PKC) modulator, has shown promise in treating neurological injury. In the present study, we tested the hypothesis that administration of bryostatin would reduce BBB disruption and HT following acute ischemic stroke; thus, prolonging the time window for administering recombinant tissue plasminogen activator (r-tPA). Acute cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery (MCAO) in 18–20-month-old female rats using an autologous blood clot with delayed r-tPA reperfusion. Bryostatin (or vehicle) was administered at 2 h post-MCAO and r-tPA was administered at 6 h post-MCAO. Functional assessment, lesion volume, and hemispheric swelling measurements were performed at 24 h post-MCAO. Assessment of BBB permeability, measurement of hemoglobin, assessment of matrix metalloproteinase (MMP) levels by gel zymography, and measurement of PKCε, PKCα, PKCδ expression by western blot were conducted at 24 h post-MCAO. Rats treated with bryostatin prior to r-tPA administration had decreased mortality and hemispheric swelling when compared with rats treated with r-tPA alone. Administration of bryostatin also limited BBB disruption and HT and down-regulated MMP-9 expression while up-regulating PKCε expression at 24 h post-MCAO. Bryostatin administration ameliorates BBB disruption and reduces the risk of HT by down-regulating MMP-9 activation and up-regulating PKCε. In this proof-of-concept study, bryostatin treatment lengthened the time-to-treatment window and enhanced the efficacy and safety of thrombolytic therapy.

Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5h. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47 ± 5% vs. 29 ± 5%), striatal (50 ± 2%, vs. 40 ± 3%) and total (48 ± 3%vs. 33 ± 4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs. 6.8 ± 0.8) and decreased hemoglobin extravasation in cortical tissue (1.9 ± 0.1mg/dl vs. 1.4 ± 0.1mg/dl) striatal tissue (2.1 ± 0.3mg/dl vs. 1.4 ± 0.1mg/dl) and whole brain tissue (2.0 ± 0.2mg/dl vs. 1.4 ± 0.1mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke.

Recent wars in Iraq and Afghanistan have accounted for an estimated 270,000 blast exposures among military personnel. Blast traumatic brain injury (TBI) is the ‘signature injury’ of modern warfare. Blood brain barrier (BBB) disruption following blast TBI can lead to long-term and diffuse neuroinflammation. In this study, we investigate for the first time the role of bryostatin-1, a specific protein kinase C (PKC) modulator, in ameliorating BBB breakdown. Thirty seven Sprague–Dawley rats were used for this study. We utilized a clinically relevant and validated blast model to expose animals to moderate blast exposure. Groups included: control, single blast exposure, and single blast exposure + bryostatin-1. Bryostatin-1 was administered i.p. 2.5 mg/kg after blast exposure. Evan’s blue, immunohistochemistry, and western blot analysis were performed to assess injury. Evan’s blue binds to albumin and is a marker for BBB disruption. The single blast exposure caused an increase in permeability compared to control ( t = 4.808, p < 0.05), and a reduction back toward control levels when bryostatin-1 was administered ( t = 5.113, p < 0.01). Three important PKC isozymes, PKCα, PKCδ, and PKCε, were co-localized primarily with endothelial cells but not astrocytes. Bryostatin-1 administration reduced toxic PKCα levels back toward control levels ( t = 4.559, p < 0.01) and increased the neuroprotective isozyme PKCε ( t = 6.102, p < 0.01). Bryostatin-1 caused a significant increase in the tight junction proteins VE-cadherin, ZO-1, and occludin through modulation of PKC activity. Bryostatin-1 ultimately decreased BBB breakdown potentially due to modulation of PKC isozymes. Future work will examine the role of bryostatin-1 in preventing chronic neurodegeneration following repetitive neurotrauma.

Studies in both stroke patients and experimental animals indicate an association between ischemic stroke and post stroke peripheral inflammation, but the mechanisms are poorly understood. Our ongoing study indicated that circulating microparticles (MPs), the small vesicles released from the cell membrane upon activation, were significantly elevated in both patients and animals with diabetes, and increased circulating MPs with phosphatidylserine (PS) exposure on their outer membrane played important roles in the development of diabetes‐associated microvascular complications. The objective of this study was to investigate whether local ischemic stroke produced increased circulating MPs and determine their impact on post stroke‐associated peripheral vascular inflammation. Acute cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCAO) in 3‐4 month old rats using an autologous blood clot. Functional assessment, lesion volume, and hemispheric swelling were evaluated at 24h after MCAO. Plasma MPs 24h after stroke induction were quantified and characterized using flow cytometry. The number of PS exposed MPs in stroke plasma, identified by Annexin V binding, significantly increased from 5.0 x 103 (normal rats) to 30 x 103 per µl. Antibodies directly against cell specific antigens were used to differentiate the MP cell origin. Results showed that 67% of the increased stroke plasma MPs were derived from platelets. Perfusion of individually cannulated normal rat mesenteric microvessels with isolated stroke MPs for 30 min followed by 10 min of resumed blood flow induced a 4‐fold increase in leukocyte adhesion compared to baseline. These results indicate that stroke‐induced increased circulating MPs serve as mediators capable of disseminating local inflammation to remote vasculatures, and therefore, promoting post stroke systemic complications.Grant Funding Source: Supported by HL56237, DK097391, NS061954

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

Background and Purpose Bryostatin, a potent protein kinase C (PKC) activator, has demonstrated therapeutic efficacy in preclinical models of associative memory, Alzheimer disease, global ischemia, and traumatic brain injury. In this study, we tested the hypothesis that administration of bryostatin provides a therapeutic benefit in reducing brain injury and improving stroke outcome using a clinically relevant model of cerebral ischemia with tissue plasminogen activator reperfusion in aged rats.Methods Acute cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery (MCAO) in 18- to 20-month-old female Sprague-Dawley rats using an autologous blood clot with tissue plasminogen activator-mediated reperfusion. Bryostatin was administered at 6 hours post-MCAO, then at 3, 6, 9, 12, 15, and 18 days after MCAO. Functional assessment was conducted at 2, 7, 14, and 21 days after MCAO. Lesion volume and hemispheric swelling/atrophy were performed at 2, 7, and 21 days post-MCAO. Histological assessment of PKC isozymes was performed at 24 hours post-MCAO.Results Bryostatin-treated rats showed improved survival post-MCAO, especially during the first 4 days. Repeated administration of bryostatin post-MCAO resulted in reduced infarct volume, hemispheric swelling/atrophy, and improved neurological function at 21 days post-MCAO. Changes in PKC expression and epsilon PKC expression in neurons were noted in bryostatin-treated rats at 24 hours post-MCAO.Conclusions Repeated bryostatin administration post-MCAO protected the brain from severe neurological injury post-MCAO. Bryostatin treatment improved survival rate, reduced lesion volume, salvaged tissue in infarcted hemisphere by reducing necrosis and peri-infarct astrogliosis, and improved functional outcome after MCAO.

This study utilized middle cerebral artery occlusion (MCAO) with tissue plasminogen activator (tPA) to assess inhibition of the NOX2 isoform of NADPH oxidase on brain injury and functional recovery in aged rats. Effects of NOX2 on the degree of brain injury and functional recovery following MCAO and tPA reperfusion was assessed in young adult and aged rats. Rats received apocynin (NOX2 inhibitor; 5 mg/kg) or saline 30 min prior to MCAO. At 24 h following MCAO, blood-brain barrier permeability (BBB), stroke infarct volume, edema formation, and oxidative damage were measured. Apocynin treatment in aged rats increased mortality rate and failed to improve functional outcome, total infarct volume, edema formation, and BBB permeability. Aged rats displayed increased BBB permeability to sucrose in the contralateral hemisphere following MCAO and diminished antioxidant capacity in the brain as compared to young adult rats. We conclude that inhibition of NOX2 in the aged rat exacerbates stroke injury and diminishes functional outcome. These results suggest age is an important factor in stroke damage and more rigorous examination of apocynin as a therapeutic agent for treatment of stroke must be done. (C) 2009 Elsevier B.V. All rights reserved.

Age is a primary risk factor in stroke that is often overlooked in animal studies. We contend that using aged animals yields insight into aspects of stroke injury and recovery that are masked, or not elicited, in younger animals. In this study, we examined effects of co-administration of a plasminogen activator inhibitor type 1 derived peptide, Glu-Glu-Iso-Iso-Met-Asp (EEIIMD), with tissue plasminogen activator (tPA) on infarct volume and functional outcome in aged rats following a transient middle cerebral artery occlusion. Results of our study showed aged (18-20 months) rats treated with EEIIMD along with tPA had reduced cortical infarction volume. However, aged rats showed no improvement in total infarction volume, edema formation, or functional outcome as compared to aged rats administered only tPA. Young adult rats (3-4 months) treated with EEIIMD showed significant improvement in cortical and total infarction volumes, edema formation, and functional outcome. Striatal infarction volume was unaffected by EEIIMD treatment in both young adult and aged rats. These findings emphasize that physiological differences exist between young adult and aged rats and suggest that taking aging processes into account when assessing stroke may improve our ability to discern which therapeutics can be translated from bench to bedside.

GABA-releasing cortical interneurons are crucial for the neural transformations underlying sensory perception, providing "feedforward" inhibition that constrains the temporal window for synaptic integration. To mediate feedforward inhibition, inhibitory interneurons need to fire in response to ascending thalamocortical inputs, and most previous studies concluded that ascending inputs activate mainly or solely proximally targeting, parvalbumin-containing "fast-spiking" interneurons. However, when thalamocortical axons fire at frequencies that are likely to occur during natural exploratory behavior, activation of fast-spiking interneurons is rapidly and strongly depressed, implying the paradoxical conclusion that feedforward inhibition is absent when it is most needed. To address this issue, we took advantage of lines of transgenic mice in which either parvalbumin- or somatostatin-containing interneurons express GFP and recorded the responses of interneurons from both subtypes to thalamocortical stimulation in vitro. We report that during thalamocortical activation at behaviorally expected frequencies, fast-spiking interneurons were indeed activated only transiently because of rapid depression of their thalamocortical inputs, but a subset of layer 5 somatostatin-containing interneurons were robustly and persistently activated after a delay, due to the facilitation and temporal summation of their thalamocortical excitatory postsynaptic potentials. Somatostatin-containing interneurons are considered distally targeting. Thus, they are likely to provide delayed dendritic inhibition during exploratory behavior, contributing to the maintenance of a balance between cortical excitation and inhibition while leaving a wide temporal window open for synaptic integration and plasticity in distal dendrites.