Simultaneous multiplane imaging with reverberation multiphoton microscopy

Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compromise in performance. Specifically, multiple planes are acquired by near-instantaneous axial scanning while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, is well adapted for large-scale imaging in scattering media with low repetition-rate lasers, and can be implemented with conventional MPM as a simple add-on.Accepted manuscrip

Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS108472 - NINDS NIH HHSPublished versio

Methods and applications in vascular physiology : 2021

This Research Topic is a part of the Methods and Applications in Physiology series and aims at highlighting the latest experimental and computational methods in vascular physiology and their pharmacological, clinical and healthcare applications. The Research Topic includes methodological articles, describing either novel technical approaches or new applications of the conventional methods for measurement of vascular functions and parameters in health and pathological conditions

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

More Homogeneous Capillary Flow and Oxygenation in Deeper Cortical Layers Correlate with Increased Oxygen Extraction

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation

Baseline oxygen consumption decreases with cortical depth

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase—the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.publishedVersio

Glikojen Döngüsünün Bozulmasının Kortikal Yayılan Depolarizasyon Oluşumu ve Panneksin 1 Kanalları Üzerine Etkisinin Araştırılması

Migraine is an episodic neurological disorder that affects 10-20 % of general population. Cortical spreading depression (CSD) is proposed as the mechanism of migraine aura and the downstream trigeminal activation as the mechanism of migraine headache. Hunger and sleep deprivation are trigger factors for migraine. The mechanism underlying these triggers could be related to glycogen turnover since astrocytic glycogen stores can be used as a source of energy especially during intense glutamatergic neurotransmission as those events require the maintenance of low extracellular glutamate and K+ concentrations around synapses. Glycogen turnover is regulated by synthesis and degradation enzymes called glycogen synthase and glycogen phosphorylase, respectively as well as glucose concentration in astrocytes. We hypothesized that alteration of the glycogen synthase activity with extended wakefulness, inhibition of glycogen phosphorylase by DAB and restriction of glucose influx to astrocytes by phloretin could interfere with glycogen turnover. Our attempts that restrict the utilization of glycogen decreased CSD threshold and this decreased threshold recovered when the animal was superfused with energy substrates L-lactate and D-glucose. Recently, it was shown that pannexin1 hemi-channels are activated during CSD, high K+ and/or glutamate and low extracellular glucose levels. We also investigated the effect of impaired glycogen turnover on pannexin1 hemi-channels and found that intense somatosensory stimulus activates pannexin1 hemi-channels during impaired glycogen turnover. In conclusion, our data is consistent with our hypothesis that glycogen may be required for preventing CSD generation and conditions that compromise glycogen use such as extended wakefulness, DAB and intense synaptic activation could lead to pannexin channel activation and therefore migraine attacks.Migren toplumun %10-20?sini etkileyen, ataklarla seyreden nörolojik bir bozukluktur. Kortikal yayılan depolarizasyon (KYD) migren aurasının, KYD sonucu oluşan trigeminal aktivasyon ise migren ağrısının mekanizması olarak öne sürülmüştür. Uykusuzluk ve uzamış açlık migren için tetikleyici faktörlerdir. Bu tetikleyicilerin altında yatan mekanizma glikojen döngüsü ile ilgili olabilir; çünkü astrositlerdeki glikojen depoları KYD sırasında sinaps aralığındaki çok yüksek seviyelere ulaşan glutamat ve K+ seviyelerini düzenlemede destekleyici enerji kaynağı olarak kullanılmaktadır. Glikojen döngüsü, sırasıyla glikojenin sentezinde ve yıkımında rol alan enzimler olan glikojen sentaz ve glikojen fosforilaz aktivitelerinin yanı sıra astrosit içindeki glukoz derişiminden de etkilenmektedir. Bu çalışmada, glikojen sentaz aktivitesinin uzatılmış uyanıklık ile artırılması, glikojen fosforilazın DAB ile inhibisyonu ve astrositlere glukoz girişinin floretin ile engellenmesinin KYD oluşumu üzerine etkileri incelenmiştir. Bulgularımız bu fizyolojik ve farmakolojik yaklaşımların glikojen kullanımını aksatarak KYD eşiğini düşürdüğünü göstermektedir. Uzatılmış uyanıklık ve DAB ile düşürülen eşik, enerji substratları olan L-laktat ve D-glukoz süperfüzyonu ile geri çevrilmiştir. Yakın zamanda, panneksin1 kanallarının KYD, yüksek hücre dışı K+ ve/veya glutamat ve hücre dışında glukoz seviyelerinin düşmesi ile aktive olduğu gösterilmiştir. Bizim çalışmamızda, bozulmuş glikojen döngüsünün panneksin 1 kanalları üzerine etkisi incelenmiş ve glikojen döngüsünün aksadığı durumlarda yoğun somatosensöriyel uyarı ile panneksin 1 kanallarını aktive olduğu saptanmıştır. Sonuç olarak, verilerimiz, glikojenin KYD oluşumunun engellenmesi için gerekli olduğunu göstermekte ayrıca uzatılmış uyanıklık, DAB, yoğun sinaptik aktivite gibi glikojen kullanımının bozulduğu durumların, panneksin kanalı aktivasyonuna ve dolayısıyla migren ataklarına yol açabileceği hipotezini desteklemektedir

Determination of Weed Species, Distribution and Density in Potato Fields (Solanum tuberosum L.) in Niğde Province

Bu çalışma, Niğde ili ve İlçeleri patates ekim alanlarında sorun olan yabancı ot türlerinin yoğunluklarını ve rastlanma sıklıklarını belirlemek amacıyla 2016 yılında yürütülmüştür. Sürvey çalışmaları Niğde Merkez ve İlçelerinde (Altunhisar, Bor, Çamardı, Çiftlik, Ulukışla) 180 tarlada Mayıs-Temmuz aylarında gerçekleştirilmiştir. Yapılan sürvey çalışması sonucunda, 21 farklı familyaya ait 60 farklı yabancı ot türü tespit edilmiştir. Survey yapılan tarlalardaki m2'deki yoğunluklara göre en fazla sorun olan tür 4,24 bitki/m2 yoğunluk ile Kırmızı köklü tilki kuyruğu (Amaranthus retroflexus L.) olmuştur. Bu türü 2,81 bitki/m2 ile Sirken (Chenopodium album L.), 2,67 bitki/m2 ile Melez horozibiği (A. hybridus L.), 1,46 bitki/m2 ile Siyah it üzümü (Solanum nigrum L.), 1,45 bitki/m2 ile Yabani hardal (Sinapis arvensis L.), 1,45 bitki/m2 ile Tarla sarmaşığı (Convolvulus arvensis L.) ve 1,32 bitki/m2 ile Darıcan [Echinochloa crus-galli (L.)] izlemiştir. Rastlanma sıklığına göre ise %68 Sirken (C. album), %65 Kırmızı köklü tilki kuyruğu (A. retroflexus), %42 Tarla sarmaşığı (C. arvensis), %25 Melez horozibiği (A. hybridus L.), %24 Yabani hardal (S. arvensis), %19 Yatık horoz ibiği (A. blitoides L.), %15 Siyah it üzümü (S. nigrum ), %13 Kekre (Acroptilon repens L.), %12 Domuz pıtrağı (Xanthium strumarium L.) olarak belirlenmiştir.This study was carried out to determine the distribution and density of weed species which are problem on potato fields at around Niğde Province in 2016 year. Survey studies were made in potato cultivation field in Niğde center and its district (Altunhisar, Bor, Çamardı, Çiftlik, Ulukışla) between May and July on 180 potato fields. As a result of survey, 60 different weed species from 21 families were identified. The weed species with higher infestation rate and density according to average number of weeds m were found in this study as follows: Red pigweed (Amaranthus retroflexus L.,) with the density of 4.24 plants/m2. Followed by 2.81 plants/m2 with white goosefoot (Chenopodium album L), 2.67 plants/m2 with lying amaranth (Amaranthus hybridus L.), 1.46 plants/m2 with black nightshade (Solanum nigrum L.), 1.45 plants/m2 with white mustard (Sinapis arvensis L.), with field bindweed 1.45 plants/m2 with field bindweed (Convolvulus arvensis L.), 1.32 plants/m2 of barnyard grass (Echinochloa crus-galli). Weeds which are important in terms of frequency of occurrence is white goosefoot by 68%, red pigweed by 65%, field bindweed by 42%, lying amaranth by 25%, field mustard by 24%, mat amaranth by 19%, black nightshade by 15%, Russian knapweed by 13%, rough cocklebur by 12% were determined

Capillary Pericytes Express Α-Smooth Muscle Actin, Which Requires Prevention of Filamentous-Actin Depolymerization For Detection

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at −20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling., Blood vessels in animals’ bodies are highly organized. The large blood vessels from the heart branch to smaller vessels that are spread throughout the tissues. The smallest vessels, the capillaries, allow oxygen and nutrients to pass from the blood to nearby cells in tissues. Some capillaries, including those at the back of the eye (in the retina) and those in the brain, change their diameter in response to activity in the nervous system. This allows more or less oxygen and nutrients to be delivered to match these tissues’ demands. However, unlike for larger blood vessels, how capillaries constrict or dilate is debated., While large vessels are encircled by smooth muscle cells, capillaries are instead surrounded by muscle-like cells called pericytes, and some scientists have suggested that it is these cells that contract to narrow the diameter of a capillary or relax to widen it. However, other researchers have questioned this explanation. This is mostly because several laboratories could not detect the proteins that would be needed for contraction within these pericytes – the most notable of which is a protein called α-smooth muscle actin (or α-SMA for short)., Alarcon-Martinez, Yilmaz-Ozcan et al. hypothesized that the way samples are usually prepared for analysis was causing the α-SMA to be degraded before it could be detected. To test this hypothesis, they used different methods to fix and preserve capillaries and pericytes in samples taken from the retinas of mice. When the tissue samples were immediately frozen with ice-cold methanol instead of a more standard formaldehyde solution, α-SMA could be detected at much higher levels in the capillary pericytes. Treating samples with a toxin called phalloidin, which stabilizes filaments of actin, also made α-SMA more readily visible. When α-SMA was experimentally depleted from the mouse retinas, the capillary pericytes were more affected than the larger blood vessels. This finding supports the idea that the pericytes contain, and rely upon, only a small amount of α-SMA., Finding α-SMA in capillary pericytes may explain how these small blood vessels can change their diameter. Future experiments will clarify how these pericytes regulate blood flow at the level of individual capillaries, and may give insights into conditions such as stroke, which is caused by reduced blood flow to the brain.WoSScopusPubMe