White Matter Cerebrovascular Reactivity: Effects of Microangiopathy and Proximal Occlusions on the Dynamic BOLD Response.

Introduction: Cerebral microangiopathy often manifests as white matter hyperintensities (WMH) on T2-weighted MR images and is associated with elevated stroke risk. Large vessel steno-occlusive disease (SOD) is also independently associated with stroke risk, however, the interaction of microangiopathy and SOD is not well understood. Cerebrovascular reactivity (CVR) describes the capacity of cerebral circulation to adapt to changes in perfusion pressure and neurovascular demand, and its impairment portends future infarctions. CVR can be measured with blood oxygen level dependent (BOLD) imaging following acetazolamide stimulus (ACZ-BOLD). We studied CVR differences between WMH and normal-appearing white matter (NAWM) in patients with chronic SOD, hypothesizing additive influences upon CVR measured by novel, fully dynamic CVR maxima ( CVR max ). Methods: A cross sectional study was conducted to measure per-voxel, per-TR maximal CVR ( CVR max ) using a custom computational pipeline in 23 subjects with angiographically-proven unilateral SOD. WMH and NAWM masks were applied to CVR max maps. White matter was subclassified with respect to the SOD-affected hemisphere, including: i. contralateral NAWM; ii. contralateral WMH iii. ipsilateral NAWM; iv. ipsilateral WMH. CVR max was compared between these groups with a Kruskal-Wallis test followed by a Dunn-Sidak post-hoc test for multiple comparisons. Results: 19 subjects (age 50±12 years, 53% female) undergoing 25 examinations met criteria. WMH volume was asymmetric in 16/19 subjects with 13/16 exhibiting higher volumes ipsilateral to SOD. Pairwise comparisons of CVR max between groups was significant with ipsilateral WMH CVR max lower than contralateral NAWM (p=0.015) and contralateral WMH (p=0.003) when comparing in-subject medians and lower than all groups when comparing pooled voxelwise values across all subjects (p<0.0001). No significant relationship between WMH lesion size and CVR max was detected. Conclusion: Our results suggest additive effects of microvascular and macrovascular disease upon white matter CVR, but with greater overall effects relating to macrovascular SOD than to apparent microangiopathy. Dynamic ACZ-BOLD presents a promising path towards a quantitative stroke risk imaging biomarker. BACKGROUND: Cerebral white matter (WM) microangiopathy manifests as sporadic or sometimes confluent high intensity lesions in MR imaging with T2-weighting, and bears known associations with stroke, cognitive disability, depression and other neurological disorders 1-5 . Deep white matter is particularly susceptible to ischemic injury owing to the deprivation of collateral flow between penetrating arterial territories, and hence deep white matter hyperintensities (WMH) may portend future infarctions 6-8 . The pathophysiology of WMH is variable but commonly includes a cascade of microvascular lipohyalinosis and atherosclerosis together with impaired vascular endothelial and neurogliovascular integrity, leading to blood brain barrier dysfunction, interstitial fluid accumulation, and eventually tissue damage 9-14 . Independent of the microcirculation, cervical and intracranial large vessel steno-occlusive disease (SOD) often results from atheromatous disease and is associated with increased risk of stroke owing to thromboembolic phenomena, hypoperfusion, or combinations thereof 15-17 . White matter disease is more common in the affected hemisphere of patients with asymmetric or unilateral SOD, producing both macroscopic WMH detectable by routine structural MRI, as well as microstructural changes and altered structural connectivity detected by advanced diffusion microstructural imaging 18, 19 . An improved understanding of the interaction of microvascular disease (i.e., WMH) and macrovascular steno-occlusion could better inform stroke risk stratification and guide treatment strategies when coexistent. Cerebrovascular reactivity (CVR) is an autoregulatory adaptation characterized by the capacity of the cerebral circulation to respond to physiological or pharmacological vasodilatory stimuli 20-22 . CVR may be heterogeneous and varies across tissue type and pathological states 1, 16 . Alterations in CVR are associated with elevated stroke risk in SOD patients, although white matter CVR, and in particular the CVR profiles of WMH, are only sparsely studied and not fully understood 1, 23-26 . We have previously employed blood oxygen level dependent (BOLD) imaging following a hemodynamic stimulus with acetazolamide (ACZ) in order to measure CVR (i.e. ACZ-BOLD) 21, 27, 28 . Despite the emergence of ACZ-BOLD as a technique for clinical and experimental use, poor signal-to-noise characteristics of the BOLD effect have generally limited its interpretation to coarse, time-averaged assessment of the terminal ACZ response at arbitrarily prescribed delays following ACZ administration (e.g. 10-20 minutes) 29 . More recently, we have introduced a dedicated computational pipeline to overcome historically intractable signal-to-noise ratio (SNR) limitations of BOLD, enabling fully dynamic characterization of the cerebrovascular response, including identification of previously unreported, unsustained or transient CVR maxima ( CVR max ) following hemodynamic provocation 27, 30 . In this study, we compared such dynamic interrogation of true CVR maxima between WMH and normal appearing white matter (NAWM) among patients with chronic, unilateral SOD in order to quantify their interaction and to assess the hypothesized additive effects of angiographically-evident macrovascular stenoses when intersecting microangiopathic WMH.

Evaluation of Complications and Costs During Overlapping Transsphenoidal Surgery in the Treatment of Pituitary Adenoma.

BACKGROUND: Pituitary adenomas are among the most common primary brain tumors. Recently, overlapping surgery has been curbed in many institutions because of the suggestion there might be more significant adverse events, despite several studies showing that complication rates are equivalent. OBJECTIVE: To assess complications and costs associated with overlapping surgery during the transsphenoidal resection of pituitary adenomas. METHODS: A single-center, retrospective cohort study was performed to evaluate the cases of patients who underwent a transsphenoidal approach for pituitary tumor resection. Patient, surgical, complication, and cost (value-driven outcome) variables were analyzed. RESULTS: A total of 629 patients (302 nonoverlapping, 327 overlapping cases) were identified. No significant differences in age (P = .6), sex (P = .5), tumor type (P = .5), or prior rates of pituitary adenoma resection (P = .5) were seen. Similar presenting symptoms were observed in the 2 groups, and follow-up length was comparable (P = .3). No differences in tumor sizes (P = .5), operative time (P = .4), fat/fascia use (P = .4), or cerebrospinal fluid diversion (P = .8) were seen between groups. The gross total resection rate was not significantly different (P = .9), and no difference in recurrence rate was seen (P = .4). A comparable complication rate was seen between groups (P = .6). No differences in total or subtotal costs were seen either. CONCLUSION: The results of this study offer additional evidence that overlapping surgery does not result in worsened complications, lengthened surgery, or increased patient cost for patients undergoing transsphenoidal resection of pituitary adenomas. Thus, studies and policy aiming to improve patient safety and cost should focus on optimizing other aspects of healthcare delivery.

Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.

Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.

Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse.

Microglia, the resident immune cells of the brain parenchyma, are highly responsive to tissue injury. Following cell damage, microglial processes redirect their motility from randomly scouting the extracellular space to specifically reaching toward the compromised tissue. While the cell morphology aspects of this defense mechanism have been characterized, the intracellular events underlying these responses remain largely unknown. Specifically, the role of intracellular Ca(2+) dynamics has not been systematically investigated in acutely activated microglia due to technical difficulty. Here we used live two-photon imaging of the mouse cortex ubiquitously expressing the genetically encoded Ca(2+) indicator GCaMP5G and fluorescent marker tdTomato in central nervous system microglia. We found that spontaneous Ca(2+) transients in microglial somas and processes were generally low (only 4% of all microglia showing transients within 20 min), but baseline activity increased about 8-fold when the animals were treated with LPS 12 h before imaging. When challenged with focal laser injury, an additional surge in Ca(2+) activity was observed in the somas and protruding processes. Notably, coherent and simultaneous Ca(2+) rises in multiple microglial cells were occasionally detected in LPS-treated animals. We show that Ca(2+) transients were pre-dominantly mediated via purinergic receptors. This work demonstrates the usefulness of genetically encoded Ca(2+) indicators for investigation of microglial physiology.

Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse.

New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

Autoimmune responses to the brain after stroke are associated with worse outcome.

BACKGROUND AND PURPOSE: Immune responses to brain antigens occur after stroke, and experimental studies show that the likelihood of developing a detrimental autoimmune response to these antigens is increased by systemic inflammation at the time of stroke. The aim of this study was to determine if patients who developed infection in the poststroke period would be similarly predisposed to develop autoimmune responses to central nervous system antigens. METHODS: We enrolled 114 patients within 72 hours of ischemic stroke. Clinical and demographic data were obtained, and cellular immune responses to a panel of central nervous system antigens were assessed during the initial week and again at Day 90. Outcome was assessed using the modified Rankin Scale. RESULTS: Patients who developed an infection, especially pneumonia, in the 15 days after stroke were more likely to evidence a Th1(+) response to myelin basic protein and glial fibrillary acidic protein (P=0.019 and P=0.039, respectively) at 90 days after stroke. Further, more robust Th1 responses to myelin basic protein at 90 days were associated with a decreased likelihood of good outcome, even after adjusting for baseline stroke severity and patient age (OR, 0.477; 95% CI, 0.244 to 0.935; P=0.031). CONCLUSIONS: This study demonstrates that immune responses to brain antigens occur after stroke. Although these responses are likely to be an epiphenomenon of ischemic brain injury, the response to myelin basic protein appears to have clinical consequences. The potential role of postischemic autoimmune-mediated brain injury deserves further investigation.

α-MSH: a potential neuroprotective and immunomodulatory agent for the treatment of stroke.

Alpha-melanocyte-stimulating hormone (MSH) is a neuropeptide with profound immunomodulatory properties; we evaluated the effects of α-MSH on stroke outcome and its ability to modulate the postischemic immune response. In Lewis rats subjected to 3 hours of middle cerebral artery occlusion (MCAO), plasma concentrations of α-MSH rapidly decreased and returned to baseline over the course of days. Exogenous administration of α-MSH (100 or 500 µg/kg) improved 24 hour outcome in animals subjected to 2 hours MCAO; α-MSH 500 µg/kg also decreased infarct volume at this time point. Both doses of α-MSH were ineffective in improving outcome or decreasing infarct volume in animals subjected to 3 hours MCAO. The splenocyte response to phytohemagglutin in animals treated with α-MSH was attenuated at 24 hours after MCAO. At 1 month after MCAO, treatment with α-MSH 500 µg/kg at the time of stoke was associated with a decrease in TH1 response to myelin basic protein (MBP) in animals subjected to 2 hours MCAO, although treatment was not associated with improved outcome at this time point. Given the early benefits of α-MSH treatment and its effect on immunologic outcome, further studies to evaluate the utility of α-MSH for the treatment of cerebral ischemia are warranted.

Post-stroke infection: a role for IL-1ra?

BACKGROUND: Infection is common following stroke and is independently associated with worse outcome. Clinical studies suggest that infections occur more frequently in those individuals with stroke-induced immunologic dysfunction. This study sought to explore the contribution of immunomodulatory cytokines and hormones to lymphocyte function and infection risk. METHODS: Patients (N = 112) were enrolled as soon as possible after the onset of ischemic stroke. Blood was drawn to assess plasma cortisol, IL-10, IL-1ra, lymphocyte numbers, and lymphocyte function at 72 h after stroke onset; infections were censored through 21 days after stroke onset. RESULTS: Infection occurred in 25% of patients. Stroke severity was the most important predictor of infection risk. Increased plasma cortisol, IL-10, and IL-1ra, as well as decreased lymphocyte numbers, at 72 h after stroke onset were associated with risk of subsequent infection. After controlling for stroke severity, only IL-1ra was independently associated with infection risk, and the degree of risk was consistent throughout the post-stroke period. Infection, but not IL-1ra itself, was associated with worse outcome at 3 months. CONCLUSIONS: In this study cohort, increased plasma IL-1ra was independently associated with the risk of post-stroke infection. Further studies are needed to validate this finding, which could have important implications for stroke therapy.

CNS immune responses following experimental stroke.

BACKGROUND AND PURPOSE: Animals subjected to an inflammatory insult with lipopolysaccharide (LPS) at the time of stroke are predisposed to develop a detrimental autoimmune response to myelin basic protein (MBP). In this study, we sought to determine whether other inflammatory stimuli could similarly invoke central nervous system (CNS) autoimmunity and whether these detrimental autoimmune responses occurred to antigens other than MBP. METHODS: Male Lewis rats underwent 3 h middle cerebral artery occlusion (MCAO) and received intraperitoneal injections of LPS, staphylococcal enterotoxin B (SEB), lipoteichoic acid (LTA) or saline at the time of reperfusion. Behavioral tests were performed at set time intervals after MCAO and animals were sacrificed at 1 month to analyze the immune response to MBP, neuron specific enolase (NSE) and proteolipid protein (PLP). RESULTS: Lymphocytes from SEB treated animals were highly reactive to all tested CNS antigens, but treatment with LPS was most likely to lead to a TH: 1(+) response. A TH: 1(+) response to MBP, NSE or PLP in spleen was associated with worse outcome, although the response to NSE was most predictive of poor outcome. Animals with a cell mediated autoimmune response to either MBP or NSE in spleen had a concomitant humoral response to these antigens. CONCLUSIONS: These data show that LPS, but not other inflammatory stimuli, increase the likelihood of developing a detrimental autoimmune response to an array of brain antigens.

Long term immunologic consequences of experimental stroke and mucosal tolerance.

BACKGROUND: An inflammatory insult following middle cerebral artery occlusion (MCAO) is associated with a predisposition to develop a deleterious autoimmune response to the brain antigen myelin basic protein (MBP). Induction of immunologic tolerance to brain antigens prior to MCAO prevents this deleterious autoimmune response and is associated with better functional outcome early after stroke. In this study, we sought to determine the long term immunologic consequences of experimental stroke and induction of mucosal tolerance. METHODS: Male Lewis rats were tolerized to MBP or ovalbumin (OVA) by intranasal administration prior to MCAO and administration of lipopolysaccharide (LPS). Neurological outcome was assessed at set points after MCAO and animals sacrificed at 3 months; the immune response to MBP in brain and spleen was determined using ELISPOT assay and degree of cellular inflammatory brain infiltrate assessed by immunocytochemistry. RESULTS: Animals that developed a pro-inflammatory (TH1) response to MBP experienced worse outcome, while those that developed a regulatory response (TREG) experienced better outcome. A TREG response in spleen was also associated with decreased inflammation and an increase in the number of FoxP3 positive cells in brain. In this study, tolerization to MBP prior to MCAO was associated with a tendency to develop a TH1 response to MBP by 3 months after MCAO. CONCLUSION: These data show that induction of immunological tolerance to MBP is associated with improved outcome after stroke. This study, however, raises concern about the potential for inadvertent induction of detrimental autoimmunity through mucosal administration of antigen.

Induction of immunologic tolerance to myelin basic protein prevents central nervous system autoimmunity and improves outcome after stroke.

BACKGROUND AND PURPOSE: Animals subjected to an inflammatory insult at the time of stroke are predisposed to the development of an inflammatory autoimmune response to brain. This response is associated with worse neurological outcome. Because induction of immunologic tolerance to brain antigens before stroke onset is associated with improved outcome, we sought to determine whether this paradigm could prevent the deleterious autoimmune response to brain provoked by an inflammatory stimulus at the time of ischemia. METHODS: Male Lewis rats were tolerized to myelin basic protein (MBP) or ovalbumin by intranasal administration before middle cerebral artery occlusion. At the time of reperfusion, all animals received lipopolysaccharide (1 mg/kg intraperitoneal). Behavioral tests were performed at set time intervals. RESULTS: One month after middle cerebral artery occlusion, lymphocytes from the spleens of MBP-tolerized animals were less likely to evidence an autoimmune response and more likely to evidence a regulatory response (Treg) toward MBP than those from ovalbumin-tolerized animals. Animals that had an inflammatory response toward MBP (a Th1 response) performed worse on behavioral tests than those that did not. Fractalkine, a surrogate marker of inflammation, was elevated in animals with a Th1 response to MBP. CONCLUSIONS: These data extend our previous findings and suggest that deleterious autoimmunity to brain antigens can be prevented by prophylactically inducing regulatory T-cell responses to those antigens.

Lymphocytes: potential mediators of postischemic injury and neuroprotection.

Antigen-nonspecific inflammation appears to contribute to postischemic brain injury. Because there is a breach in the integrity of the blood-brain barrier after stroke, the immune system encounters novel central nervous system (CNS) antigens that allow for the development of a CNS antigen-specific autoimmune response. The nature of the immune response generated on antigen encounter is determined by the microenvironment at the site of antigen encounter. For instance, a systemic inflammatory response, such as that which would accompany an infection, could alter the microenvironment in such a way as to promote the initiation of deleterious autoimmunity. If patients who develop an infection in the immediate poststroke period are predisposed toward a CNS autoimmune response, it might help to explain why infection after stroke is associated with increased disability. We present data to support this hypothesis and to show that the breach in the blood-brain barrier can also be capitalized on to modulate the immune response to create a neuroprotective environment after stroke.