Chronic Cranial Windows Research Articles

Young blood-mediated cerebromicrovascular rejuvenation through heterochronic parabiosis: enhancing blood-brain barrier integrity and capillarization in the aged mouse brain.

Age-related cerebromicrovascular changes, including blood-brain barrier (BBB) disruption and microvascular rarefaction, play a significant role in the development of vascular cognitive impairment (VCI) and neurodegenerative diseases. Utilizing the unique model of heterochronic parabiosis, which involves surgically joining young and old animals, we investigated the influence of systemic factors on these vascular changes. Our study employed heterochronic parabiosis to explore the effects of young and aged systemic environments on cerebromicrovascular aging in mice. We evaluated microvascular density and BBB integrity in parabiotic pairs equipped with chronic cranial windows, using intravital two-photon imaging techniques. Our results indicate that short-term exposure to young systemic factors leads to both functional and structural rejuvenation of cerebral microcirculation. Notably, we observed a marked decrease in capillary density and an increase in BBB permeability to fluorescent tracers in the cortices of aged mice undergoing isochronic parabiosis (20-month-old C57BL/6 mice [A-(A)]; 6 weeks of parabiosis), compared to young isochronic parabionts (6-month-old, [Y-(Y)]). However, aged heterochronic parabionts (A-(Y)) exposed to young blood exhibited a significant increase in cortical capillary density and restoration of BBB integrity. In contrast, young mice exposed to old blood from aged parabionts (Y-(A)) rapidly developed cerebromicrovascular aging traits, evidenced by reduced capillary density and increased BBB permeability. These findings underscore the profound impact of systemic factors in regulating cerebromicrovascular aging. The rejuvenation observed in the endothelium, following exposure to young blood, suggests the existence of anti-geronic elements that counteract microvascular aging. Conversely, pro-geronic factors in aged blood appear to accelerate cerebromicrovascular aging. Further research is needed to assess whether the rejuvenating effects of young blood factors could extend to other age-related cerebromicrovascular pathologies, such as microvascular amyloid deposition and increased microvascular fragility.

Neurovascular Uncoupling Is Linked to Microcirculatory Dysfunction in Regions Outside the Ischemic Core Following Ischemic Stroke.

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor β. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P<0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P=0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Combining in vivo two-photon microscopy with ex vivo pressure myography as a novel approach to assess neurovascular coupling

The brain’s vascular network maintains cerebral blood flow and crucially, responds neuronal demands by rapidly altering local perfusion. In vivo techniques using cranial windows can visualise this neurovascular communication and measure real-time blood flow changes. In turn, the ex vivo capillary-to-parenchymal-arteriole preparation (CaPA Prep) can examine the mechanisms of signalling between capillaries and arterioles in response to physiological or pharmacological stimuli, and study these in health and disease models. In order to promote the 3R’s of animal research, and reduce the number of animals required in studies of neurovascular function, we wished to examine whether vessels from mice which have undergone cranial window surgery are comparable to vessels from mice without cranial window surgery by using the ex vivo CaPA Prep.C57 mice underwent craniotomies to implant chronic cranial windows and were left to recover. In order to examine capillary-to-arteriole signalling, we utilised the CaPA Prep, where parenchymal arterioles (&lt;45μms) with attached capillary branches were isolated and pressurised. The capillary branch and arteriole are independently stimulated via a micropipette and the arteriole diameter is recorded. This approach enables study of multiple ion channels integral to neurovascular coupling to be assessed.Cranial window implantation did not significantly affect the function of cerebral vessels, compared to control C57 mice. Mice underwent craniotomies and were left to recover for up to 8 weeks before euthanization. Parenchymal arterioles with attached capillary branches were pressurised to 40mmHg. No significant difference in tone was observed between control and implanted animals (32.7 ± 4.7 vs 29.7 ± 2.57, N = 4-6, P= .56). We next examined the capillary-to-arteriolar KIR2.1 signalling mechanism via application of 10mM K+ to the capillary branch. Responses were not significantly different between control and implanted animals (68.1 ± 13.2 vs 75.2 ± 8.4, N = 4, P= .66) and KIR2.1 function via directly stimulating the arteriole was also not significantly different (74.7 ± 9.4 vs 60.7 ± 5.3, N = 4, P= .24). Arteriole responses to NS309 (3mM) and 60mM K+ were also not significantly different between groups (73.5 ± 11.51 vs 104.8 ± 19.6, N = 4-5, P= .23, 60.6 ± 5.5 vs 64.9 ± 5.5, N = 4-5, P= .59, respectively).Moreover, our preliminary data suggests that in vivo imaging in mice with cranial windows also has no effect on vascular function in relation to pressure induced tone, KIR2.1 capillary-to-arteriolar signalling and responses to NS309 and 60mMK+. Overall, our data suggests cranial window implantation and in vivo imaging preserves vascular function and these mice can be utilised for ex vivo analysis of vessel physiology. Minimising the number of animals avoids the need for additional animals for different techniques, and maximises the data obtained per animal. British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A versatile toolbox for studying cortical physiology in primates

SUMMARYLesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments.MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

In Vivo Imaging of the Microglial Landscape After Whole Brain Radiation Therapy

Whole brain radiation therapy (WBRT) is an important treatment for patients with multiple brain metastases, but can also cause cognitive deterioration. Microglia, the resident immune cells of the brain, promote a proinflammatory environment and likely contribute to cognitive decline after WBRT. To investigate the temporal dynamics of the microglial reaction in individual mice to WBRT, we developed a novel in vivo experimental model using cranial window implants and longitudinal imaging. Chronic cranial windows were surgically implanted over the somatosensory cortex of transgenic Cx3cr1-enhanced green fluorescent protein (EGFP)/+ C57BL/6 mice, where microglia were fluorescently tagged with EGFP. Cx3cr1-EGFP/+ mice were also crossed with Thy1-YFP mice to fluorescently dual label microglia and subsets of neurons throughout the brain. Three weeks after window implantation and recovery, computed tomography image guided WBRT was delivered (single dose 10 Gy using two 5 Gy parallel-opposed lateral beams). Radiation dosing was confirmed using radiochromic film. Then, in vivo 2-photon microscopy was used to longitudinally image the microglial landscape and microglial motility at 7 days and 16 days after irradiation in the same mice. Film dosimetry confirmed the average delivered dose per beam at midpoint was accurate within 2%, with no attenuation from the window frame. By 7 days after WBRT, significant changes in the microglial landscape were seen, characterized by apparent loss of microglial cells (20%) and significant rearrangements of microglial location with time after irradiation (36% of cells not found in original location). Using longitudinal in vivo 2-photon imaging, this study demonstrated the feasibility of imaging microglia-neuron interactions and defining how microglia react to WBRT in the same mouse. Having demonstrated utility of the model, this experimental paradigm can be used to investigate the dynamic changes of many different brain cell types and their interactions after WBRT and uncover the underlying cellular mechanisms of WBRT-induced cognitive decline.

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.

TMOD-33. THE ROLE OF BAFF-R SIGNALING IN THE GROWTH OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Abstract Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we demonstrate a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate a potential of BAFF-R as a novel treatment target for PCNSL.

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain.

SummaryIn vivo two-photon deep imaging with a broad field of view has revealed functional connectivity among brain regions. Here, we developed a novel observation method that utilizes a polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet with a thickness of ∼130 nm that exhibited a water retention effect and a hydrophilized adhesive surface. PEO-CYTOP nanosheets firmly adhered to brain surfaces, which suppressed bleeding from superficial veins. By taking advantage of the excellent optical properties of PEO-CYTOP nanosheets, we performed in vivo deep imaging in mouse brains at high resolution. Moreover, PEO-CYTOP nanosheets enabled to prepare large cranial windows, achieving in vivo imaging of neural structure and Ca2+ elevation in a large field of view. Furthermore, the PEO-CYTOP nanosheets functioned as a sealing material, even after the removal of the dura. These results indicate that this method would be suitable for the investigation of neural functions that are composed of interactions among multiple regions.

Chronic Imaging of Mouse Brain: From Optical Systems to Functional Ultrasound.

Utilization of functional ultrasound (fUS) in cerebral vascular imaging is gaining popularity among neuroscientists. In this article, we describe a chronic surgical preparation method that allows longitudinal studies and therefore is applicable to a wide range of studies, especially on aging, stroke, and neurodegenerative diseases. This method can also be used with awake mice; hence, the deleterious effects of anesthesia on neurovascular responses can be avoided. In addition to fUS imaging, this surgical preparation allows researchers to take advantage of common optical imaging methods to acquire complementary datasets to help increase the technical rigor of studies. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Surgical preparation of mouse chronic cranial windows using polymethylpentene Basic Protocol 2: Imaging of mice with chronic cranial windows.

The Role of BAFF-R Signaling in the Growth of Primary Central Nervous System Lymphoma.

Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we have created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we have demonstrated a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate the high potential of BAFF-R as a novel treatment target for PCNSL.

Meningeal Lymphangiogenesis and Enhanced Glymphatic Activity in Mice with Chronically Implanted EEG Electrodes.

Chronic electroencephalography (EEG) is a widely used tool for monitoring cortical electrical activity in experimental animals. Although chronic implants allow for high-quality, long-term recordings in preclinical studies, the electrodes are foreign objects and might therefore be expected to induce a local inflammatory response. We here analyzed the effects of chronic cranial electrode implantation on glymphatic fluid transport and in provoking structural changes in the meninges and cerebral cortex of male and female mice. Immunohistochemical analysis of brain tissue and dura revealed reactive gliosis in the cortex underlying the electrodes and extensive meningeal lymphangiogenesis in the surrounding dura. Meningeal lymphangiogenesis was also evident in mice prepared with the commonly used chronic cranial window. Glymphatic influx of a CSF tracer was significantly enhanced at 30 d postsurgery in both awake and ketamine-xylazine anesthetized mice with electrodes, supporting the concept that glymphatic influx and intracranial lymphatic drainage are interconnected. Altogether, the experimental results provide clear evidence that chronic implantation of EEG electrodes is associated with significant changes in the brain's fluid transport system. Future studies involving EEG recordings and chronic cranial windows must consider the physiological consequences of cranial implants, which include glial scarring, meningeal lymphangiogenesis, and increased glymphatic activity.SIGNIFICANCE STATEMENT This study shows that implantation of extradural electrodes provokes meningeal lymphangiogenesis, enhanced glymphatic influx of CSF, and reactive gliosis. The analysis based on CSF tracer injection in combination with immunohistochemistry showed that chronically implanted electroencephalography electrodes were surrounded by lymphatic sprouts originating from lymphatic vasculature along the dural sinuses and the middle meningeal artery. Likewise, chronic cranial windows provoked lymphatic sprouting. Tracer influx assessed in coronal slices was increased in agreement with previous reports identifying a close association between glymphatic activity and the meningeal lymphatic vasculature. Lymphangiogenesis in the meninges and altered glymphatic fluid transport after electrode implantation have not previously been described and adds new insights to the foreign body response of the CNS.

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.

In vivo characterization of spontaneous microhemorrhage formation in mice with cerebral amyloid angiopathy

The pathophysiology of microhemorrhages in the context of cerebral amyloid angiopathy (CAA) remains poorly understood. Here we used in vivo two-photon microscopy in aged APP/PS1 mice with mild-to-moderate CAA to assess the formation of microhemorrhages and their spatial relationship with vascular Aβ depositions in the surrounding microvascular network. Mice with chronic cranial windows were intravenously injected with fluorescent dextran to visualize the vessels and a fluorescently labeled anti-fibrin antibody to visualize microhemorrhages. Focal vessel irradiations resulted in extravascular fibrin-positive clots at individual rupture sites that remained visible for weeks. Spontaneous extravascular fibrin-positive clots were more often observed in 19-month-old transgenic APP/PS1 mice compared to their wild-type littermate controls (p = 0.039), after heparin administration. In the transgenic mice, these spontaneous leakage sites frequently occurred at arteriolar segments without CAA at bifurcations or vessel bends. These findings suggest that the presence of vascular Aβ per se does not directly predispose vessels to leak, but that complex flow dynamics within CAA-affected vascular networks likely play a role. Our in vivo approach for the detection of individual spontaneous leakage sites may be used in longitudinal studies aimed to assess structural and functional alterations at the single-vessel level leading up to microhemorrhage formation.

Micelle-Encapsulated Quantum Dot-Porphyrin Assemblies as in Vivo Two-Photon Oxygen Sensors.

Micelles have been employed to encapsulate the supramolecular assembly of quantum dots with palladium(II) porphyrins for the quantification of O2 levels in aqueous media and in vivo. Förster resonance energy transfer from the quantum dot (QD) to the palladium porphyrin provides a means for signal transduction under both one- and two-photon excitation. The palladium porphyrins are sensitive to O2 concentrations in the range of 0-160 Torr. The micelle-encapsulated QD-porphyrin assemblies have been employed for in vivo multiphoton imaging and lifetime-based oxygen measurements in mice with chronic dorsal skinfold chambers or cranial windows. Our results establish the utility of the QD-micelle approach for in vivo biological sensing applications.

Quantitative evaluation of SOCS-induced optical clearing efficiency of skull.

In vivo cortex optical imaging methods for visualization of both structural and functional architecture with high spatial-temporal resolution have shown tremendous advantages in the studies on neurons, glia and microvasculature. To overcome the strong scattering of skull above the cortex, several chronic cranial windows were proposed through craniotomy, but there are some problems. Here, an innovative skull optical clearing solution (SOCS) has been invented to make the skull transparent within 25 min, but SOCS-induced optical clearing efficacy of skull is to be evaluated. Based on the measurements of divergence of beam spot, collimated transmittance of skull, the efficiency of skull optical clearing has been further evaluated quantitatively by comparing with the Monte Carlo simulation. The results show that the light beam bandwidth is 5.2±0.3 mm through the initial skull, and reduces to 2.0±0.2 mm trough the treated skull with SOCS; and the calculated scattering coefficient almost decreases to one third after the treatment. The quantitative evaluation of SOCS-induced optical clearing efficacy of skull provides an important reference for performing transcranial cortical optical imaging or operation based on skull optical clearing technique.

Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology.

Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health.

In vivo imaging of axonal and dendritic structures in neonatal mouse cortex.

Time-lapse in vivo imaging of neuronal structures is critical for understanding the assembly of neural circuits during development. Imaging developing neurons in vivo can be performed with relative ease in lower vertebrates, but ideally, one would also like to image the developing mammalian brain. In vivo chronic imaging of mice is particularly desirable because of the availability of transgenic lines that model human neuropsychiatric disease or those that allow cell- or region-specific expression of fluorescent proteins (e.g., green fluorescent protein [GFP], channelrhodopsins, and genetically encoded calcium indicators). Unfortunately, although chronic imaging of neural structures in adult mice that express GFP is now commonplace, similar approaches in neonatal mice face several additional challenges. First, the small size of the animal complicates the cranial window surgery. Second, there is a tendency for dams to cannibalize pups with head caps. Third, the head cap can impede the normal growth of the skull in neonates, which can limit the duration of imaging. Here, we describe a method for implanting chronic glass-covered cranial windows in the skulls of early postnatal mice through which axonal and dendritic structures can be imaged in vivo over a period of hours or even days.

Improved Cerebral Blood Flow Measurement with Multiexposure Speckle Imaging

Laser speckle contrast imaging (LSCI) is a powerful tool for in vivo imaging of blood flow dynamics. Based on the blurring of the random interference pattern (‘speckle') produced at the camera by laser light reflecting off a surface, LSCI has been rapidly adopted because of its high spatial and temporal resolution coupled with the relative simplicity and low cost of assembling a LSCI instrument. Wide-field maps of cerebral blood flow acquired via LSCI have made significant contributions to our understanding of cerebral hemodynamics in the healthy and ischemic brain in animal models.1, 2, 3, 4 More recently, LSCI has been applied in studies of human patients.5, 6 While the high-resolution maps of relative changes in blood flow acquired during LSCI have been well validated, flow estimates from LSCI are affected by a number of parameters unrelated to blood flow. As such, the exact quantitative relationship between speckle contrast and blood flow velocity has not been precisely defined.7, 8 The sensitivity of LSCI to nonflow parameters is particularly problematic in long-term studies where nonflow parameters are more likely to vary between imaging sessions. Recently, traditional single-exposure LSCI was extended with a multiexposure speckle imaging (MESI) protocol that improved quantitative accuracy in microfluidic flow simulations and in vivo imaging in rodents.9, 10 Using improved mathematical models and instrumentation to acquire speckle contrast images at multiple, defined exposures, MESI accurately estimates flow changes associated during acute ischemic stroke, contrary to the underestimates of flow changes associated with traditional single-exposure LSCI.10 However, the utility of MESI in long-term, repeated-imaging studies has not been validated. In the current issue, Kazmi et al11 perform MESI in mice implanted with chronic cranial imaging windows. Multiexposure speckle imaging data are compared with quantitative measurements of absolute blood flow acquired via high-frame rate red blood cell photography (RBC tracking). In healthy mice and mice with targeted ischemic strokes imaged over multiple days, MESI consistently quantified blood flow more accurately than single-exposure LSCI. Notably, by testing multiple exposure times, Kazmi et al demonstrated that no single exposure time was optimal in all animals and imaging sessions. By incorporating multiple exposure times and improved mathematical modeling, MESI better accounted for variations in imaging conditions and more reliably quantified the blood flow during chronic imaging. Improved quantitative accuracy is demonstrated by reduced deviation between flow measurements derived from MESI and RBC tracking (∼10%) relative to single-exposure LSCI and RBC tracking (∼24%). While the reliability of quantitative accuracy between animal comparisons remains to be confirmed, MESI significantly improves the quantitative accuracy of LSCI blood flow measurements in both acute and chronic imaging paradigms. This improved quantitative accuracy extends LSCI beyond qualitative descriptions of changes in flow, an important contribution to preclinical and clinical studies of stroke hemodynamics and flow restoration therapies.

&lt;em&gt;In Vivo&lt;/em&gt; Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc). The transcription of Arc is rapidly and highly induced by intensified neuronal activity and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes.