Chronic cranial windows for long term multimodal neurovascular imaging in mice

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.R21 EY030727 - NEI NIH HHS; R01 NS108472 - NINDS NIH HHS; R01 NS057198 - NINDS NIH HHS; K99 AG063762 - NIA NIH HHS; R01 DA050159 - NIDA NIH HHS; R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHSPublished versio

Migren Atağı Sırasında Serum Inflamazom Aktivasyonu Belirteçlerinin Analizi

Pathogenesis of migraine, a significant health issue of our time, has not yet been elucidated. Cortical spreading depression (CSD), associated with migraine aura has been shown in mice to activate neuronal inflammasomes and HMGB1 release and resultant trigeminovascular activation. Considering possibility of a similar process occurring in migraine, we aimed to investigate the venous HMGB1 level during headache attack. HMGB1 level was also studied in jugular venous blood of rats after CSD. In rats, following CSD initiated via two different methods, a significant increase in jugular HMGB1 compared to basal levels was detected after one hour and this increase persisted for three hours following CSD. No significant change in HMGB1 was detected in control group. These findings are consistent with previous clinical studies showing increased levels of cytokines in internal jugular venous blood of patients during migraine. Meanwhile, in migraine patients, when compared with those samples drawn 24 hours and at least 5 days after the end of attack, no significant change in HMGB1 level was found within the first three hours of attack with or without aura. When microvesicle pools were isolated from the serum samples, HMGB1 was detected within those pools, however no significant difference was found between headache and headache-free periods. In conclusion, this study has shown that in rats following CSD, HMGB1 released from brain may be detected in central venous blood within one hour, and we were unable to demonstrate a similar increase in HMGB1 level in peripheral venous blood of patients during migraine attack. This discrepancy between experimental and clinical findings might depend on patient factors or technical issues. In the next phase, if specific analysis of HMGB1 acutely released from brain becomes available, possibly with help of microvesicle pools, it might be probable to better demonstrate the role of HMGB1 and inflammasome activation in migraine pathogenesis, leading to development of reasonable novel therapeutic approaches.Günümüzün önemli sağlık sorunlarından biri olan migren hastalığının patogenezi halen bilinmemektedir. Migren aurasını oluşturan kortikal yayılan depresyonun (KYD), nöronal inflamazom aktivasyonuna ve HMGB1 salınımına bağlı olarak trigeminovasküler aktivasyona yol açtığı farede gösterilmiştir. Aynı sürecin insanda migren atağında da rol oynayabileceği fikrinden yola çıkılarak, bir inflamazom belirteci olan HMGB1'in, baş ağrısı atağı sırasında venöz kanda çalışılması amaçlanmıştır. Ayrıca sıçanlarda KYD sonrası juguler ven kanlarında da HMGB1'in değişimi incelenmiştir. Sıçanlarda iki farklı teknikle KYD başlatıldıktan sonra, bazal koşullara göre, juguler HMGB1 düzeyinin, birinci saatte anlamlı olarak arttığı ve takip eden üç saat boyunca bu artışın korunduğu görülmüştür. Kontrol grubunda ise HMGB1'de anlamlı değişim saptanmamıştır. Bulgular bu yönü ile migren atağı sırasında, internal juguler vende sitokinlerin artışını gösteren klinik çalışmalarla uyumludur. Öte yandan, hastalardan auralı veya aurasız migren atağının ilk üç saatinde alınan kan örneklerinde ise, atak bitiminden 24 saat sonra ve en az 5 gün sonra alınan kanlarla karşılaştırıldığında, HMGB1'de anlamlı değişim görülmemiştir. Aynı serumlardan mikrovezikül havuzları izole edildiğinde, bu havuzların içinde ölçülebilir düzeyde HMGB1 saptanmış, ancak migren atağı ve başağrısız dönem arasında yine anlamlı fark görülmemiştir. Sonuç olarak, bu çalışmada, sıçanlarda KYD sonrası beyinden salınan HMGB1'in santral venöz kan ölçümlerine 1 saat içerisinde yansıyabileceği görülmüş olup, bu artış migren hastalarından atak sırasında alınan periferik venöz kanlarda gösterilememiştir. Deneysel ve klinik bulgular arasındaki fark, hastalarla ilişkili veya teknik konulardan kaynaklanıyor olabilir. Yakın gelecekte beyinden salınan ve biyolojik aktivite gösteren HMGB1'in özgül analizinin, belki mikrovezikül havuzlarının da yardımıyla, mümkün olması halinde HMGB1'in ve inflamazom aktivasyonunun migren patogenezinde rolünü daha iyi ortaya konabilir ve migren tedavisine yönelik akılcı yeni hedeflerin oluşturulması sağlanabilir

Unraveling the interplay of neuroinflammatory signaling between parenchymal and meningeal cells in migraine headache

Abstract Background The initiation of migraine headaches and the involvement of neuroinflammatory signaling between parenchymal and meningeal cells remain unclear. Experimental evidence suggests that a cascade of inflammatory signaling originating from neurons may extend to the meninges, thereby inducing neurogenic inflammation and headache. This review explores the role of parenchymal inflammatory signaling in migraine headaches, drawing upon recent advancements. Body Studies in rodents have demonstrated that sterile meningeal inflammation can stimulate and sensitize meningeal nociceptors, culminating in headaches. The efficacy of relatively blood-brain barrier-impermeable anti-calcitonin gene-related peptide antibodies and triptans in treating migraine attacks, both with and without aura, supports the concept of migraine pain originating in meninges. Additionally, PET studies utilizing inflammation markers have revealed meningeal inflammatory activity in patients experiencing migraine with aura, particularly over the occipital cortex generating visual auras. The parenchymal neuroinflammatory signaling involving neurons, astrocytes, and microglia, which eventually extends to the meninges, can link non-homeostatic perturbations in the insensate brain to pain-sensitive meninges. Recent experimental research has brought deeper insight into parenchymal signaling mechanisms: Neuronal pannexin-1 channels act as stress sensors, initiating the inflammatory signaling by inflammasome formation and high-mobility group box-1 release in response to transient perturbations such as cortical spreading depolarization (CSD) or synaptic metabolic insufficiency caused by transcriptional changes induced by migraine triggers like sleep deprivation and stress. After a single CSD, astrocytes respond by upregulating the transcription of proinflammatory enzymes and mediators, while microglia are involved in restoring neuronal structural integrity; however, repeated CSDs may prompt microglia to adopt a pro-inflammatory state. Transcriptional changes from pro- to anti-inflammatory within 24 h may serve to dampen the inflammatory signaling. The extensive coverage of brain surface and perivascular areas by astrocyte endfeet suggests their role as an interface for transporting inflammatory mediators to the cerebrospinal fluid to contribute to meningeal nociception. Conclusion We propose that neuronal stress induced by CSD or synaptic activity-energy mismatch may initiate a parenchymal inflammatory signaling cascade, transmitted to the meninges, thereby triggering lasting headaches characteristic of migraine, with or without aura. This neuroinflammatory interplay between parenchymal and meningeal cells points to the potential for novel targets for migraine treatment and prophylaxis

Tumefactive Brain Demyelination Accompanying MADSAM Neuropathy

Multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy is characterized by asymmetric multifocal motor and sensory loss and conduction blocks in peripheral nerves. Peripheral demyelinating diseases may be accompanied by demyelination in central nervous system (CNS). In this report, a MADSAM patient with a solitary tumefactive demyelinating lesion in brain is presented. Neuroimaging due to a visual field defect revealed a right parietooccipital lesion, which was initially misdiagnosed as a tumor. Pathological examination showed that it was demyelinating in nature. Peripheral nervous symptoms of the patient developed two years later and she was then diagnosed with MADSAM. There was prominent clinical and electrophysiological response to steroid treatment. Tumefactive brain involvement was not previously reported for MADSAM neuropathy, although it was documented in a single case with typical chronic inflammatory demyelinating polyneuropathy (CIDP). CNS involvement should therefore be considered in MADSAM patients

KCl-induced cortical spreading depression waves more heterogeneously propagate than optogenetically-induced waves in lissencephalic brain: an analysis with optical flow tools

Although cortical spreading depolarizations (CSD) were originally assumed to be homogeneously and concentrically propagating waves, evidence obtained first in gyrencephalic brains and later in lissencephalic brains suggested a rather non-uniform propagation, shaped heterogeneously by factors like cortical region differences, vascular anatomy, wave recurrences and refractory periods. Understanding this heterogeneity is important to better evaluate the experimental models on the mechanistics of CSD and to make appropriate clinical estimations on neurological disorders like migraine, stroke, and traumatic brain injury. This study demonstrates the application of optical flow analysis tools for systematic and objective evaluation of spatiotemporal CSD propagation patterns in anesthetized mice and compares the propagation profile in different CSD induction models. Our findings confirm the asymmetric angular CSD propagation in lissencephalic brains and suggest a strong dependency on induction-method, such that continuous potassium chloride application leads to significantly higher angular propagation variability compared to optogenetically-induced CSDs

Thrombotic Distal Middle Cerebral Artery Occlusion Produced By Topical Fecl3 Application: A Novel Model Suitable For Intravital Microscopy And Thrombolysis Studies

Intravital or multiphoton microscopy and laser-speckle imaging have become popular because they allow live monitoring of several processes during cerebral ischemia. Available rodent models have limitations for these experiments; e. g., filament occlusion of the proximal middle cerebral artery (MCA) is difficult to perform under a microscope, whereas distal occlusion methods may damage the MCA and the peri-arterial cortex. We found that placement of a 10% FeCl3-soaked filter paper strip (0.3 x 1 mm(2)) on the duramater over the trunk of the distal MCA through a cranial window for 3 minutes induced intraarterial thrombus without damaging the peri-arterial cortex in the mouse. This caused a rapid regional cerebral blood flow decrease within 10 minutes and total occlusion of the MCA segment under the filter paper in 17 +/- 2 minutes, which resulted in a typical cortical infarct of 27 +/- 4 mm(3) at 24 hours and moderate sensorimotor deficits. There was no significant hemispheric swelling or hemorrhage or mortality at 24 hours. Reperfusion was obtained in half of the mice with tissue plasminogen activator, which allowed live monitoring of clot lysis along with restoration of tissue perfusion and MCA flow. In conclusion, this relatively simple and noninvasive stroke model is easy to perform under a microscope, making it suitable for live imaging and thrombolysis astudies. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1452-1460; doi:10.1038/jcbfm.2011.8; published online 16 February 2011WoSScopu

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an “inflammatory signaling cascade” in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation

Additional file 1 of Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes

Additional file 1: Figure S1. Gating strategy applied to flow cytometry. Forward and side scatter gating is used in flow cytometry analysis to identify the single beads based on the relative size and complexity (clumping) of the beads while removing debris and other events that are not of interest. The mean fluorescence intensity was used as a quantitative measure