Specific and Nonuniform Brain States during Cold Perception in Mice.

The quest to decode the complex supraspinal mechanisms that integrate cutaneous thermal information in the central system is still ongoing. The dorsal horn of the spinal cord is the first hub that encodes thermal input which is then transmitted to brain regions via the spinothalamic and thalamocortical pathways. So far, our knowledge about the strength of the interplay between the brain regions during thermal processing is limited. To address this question, we imaged the brains of adult awake male mice in resting state using functional ultrasound imaging during plantar exposure to constant and varying temperatures. Our study reveals for the first time the following: (1) a dichotomy in the response of the somatomotor-cingulate cortices and the hypothalamus, which was never described before, due to the lack of appropriate tools to study such regions with both good spatial and temporal resolutions. (2) We infer that cingulate areas may be involved in the affective responses to temperature changes. (3) Colder temperatures (ramped down) reinforce the disconnection between the somatomotor-cingulate and hypothalamus networks. (4) Finally, we also confirm the existence in the mouse brain of a brain mode characterized by low cognitive strength present more frequently at resting neutral temperature. The present study points toward the existence of a common hub between somatomotor and cingulate regions, whereas hypothalamus functions are related to a secondary network.

Backscattering amplitude in ultrasound localization microscopy.

In the last decade, Ultrafast ultrasound localisation microscopy has taken non-invasive deep vascular imaging down to the microscopic level. By imaging diluted suspensions of circulating microbubbles in the blood stream at kHz frame rate and localizing the center of their individual point spread function with a sub-resolution precision, it enabled to break the unvanquished trade-off between depth of imaging and resolution by microscopically mapping the microbubbles flux and velocities deep into tissue. However, ULM also suffers limitations. Many small vessels are not visible in the ULM images due to the noise level in areas dimly explored by the microbubbles. Moreover, as the vast majority of studies are performed using 2D imaging, quantification is limited to in-plane velocity or flux measurements which hinders the accurate velocity determination and quantification. Here we show that the backscattering amplitude of each individual microbubble can also be exploited to produce backscattering images of the vascularization with a higher sensitivity compared to conventional ULM images. By providing valuable information about the relative distance of the microbubble to the 2D imaging plane in the out-of-plane direction, backscattering ULM images introduces a physically relevant 3D rendering perception in the vascular maps. It also retrieves the missing information about the out-of-plane motion of microbubbles and provides a way to improve 3D flow and velocity quantification using 2D ULM. These results pave the way to improved visualization and quantification for 2D and 3D ULM.

Retinal functional ultrasound imaging (rfUS) for assessing neurovascular alterations: a pilot study on a rat model of dementia.

Fifty million people worldwide are affected by dementia, a heterogeneous neurodegenerative condition encompassing diseases such as Alzheimer's, vascular dementia, and Parkinson's. For them, cognitive decline is often the first marker of the pathology after irreversible brain damage has already occurred. Researchers now believe that structural and functional alterations of the brain vasculature could be early precursors of the diseases and are looking at how functional imaging could provide an early diagnosis years before irreversible clinical symptoms. In this preclinical pilot study, we proposed using functional ultrasound (fUS) on the retina to assess neurovascular alterations non-invasively, bypassing the skull limitation. We demonstrated for the first time the use of functional ultrasound in the retina and applied it to characterize the retinal hemodynamic response function in vivo in rats following a visual stimulus. We then demonstrated that retinal fUS could measure robust neurovascular coupling alterations between wild-type rats and TgF344-AD rat models of Alzheimer's disease. We observed an average relative increase in blood volume of 21% in the WT versus 37% for the TG group (p = 0.019). As a portable, non-invasive and inexpensive technique, rfUS is a promising functional screening tool in clinics for dementia years before symptoms.

Global dissociation of the posterior amygdala from the rest of the brain during REM sleep.

Rapid-eye-movement sleep (REMS) or paradoxical sleep is associated with intense neuronal activity, fluctuations in autonomic control, body paralysis and brain-wide hyperemia. The mechanisms and functions of these energy-demanding patterns remain elusive and a global picture of brain activation during REMS is currently missing. In the present work, we performed functional ultrasound imaging on rats over multiple coronal and sagittal brain sections during hundreds of spontaneous REMS episodes to provide the spatiotemporal dynamics of vascular activity in 259 brain regions spanning more than 2/3 of the total brain volume. We first demonstrate a dissociation between basal/midbrain and cortical structures, the first ones sustaining tonic activation during REMS while the others are activated in phasic bouts. Second, we isolated the vascular compartment in our recordings and identified arteries in the anterior part of the brain as strongly involved in the blood supply during REMS episodes. Finally, we report a peculiar activation pattern in the posterior amygdala, which is strikingly disconnected from the rest of the brain during most REMS episodes. This last finding suggests that the amygdala undergoes specific processing during REMS and may be linked to the regulation of emotions and the creation of dream content during this very state.

Functional ultrasound localization microscopy reveals brain-wide neurovascular activity on a microscopic scale.

The advent of neuroimaging has increased our understanding of brain function. While most brain-wide functional imaging modalities exploit neurovascular coupling to map brain activity at millimeter resolutions, the recording of functional responses at microscopic scale in mammals remains the privilege of invasive electrophysiological or optical approaches, but is mostly restricted to either the cortical surface or the vicinity of implanted sensors. Ultrasound localization microscopy (ULM) has achieved transcranial imaging of cerebrovascular flow, up to micrometre scales, by localizing intravenously injected microbubbles; however, the long acquisition time required to detect microbubbles within microscopic vessels has so far restricted ULM application mainly to microvasculature structural imaging. Here we show how ULM can be modified to quantify functional hyperemia dynamically during brain activation reaching a 6.5-µm spatial and 1-s temporal resolution in deep regions of the rat brain.

In vivo whole brain microvascular imaging in mice using transcranial 3D Ultrasound Localization Microscopy.

BACKGROUND: Non-invasive high-resolution imaging of the cerebral vascular anatomy and function is key for the study of intracranial aneurysms, stenosis, arteriovenous malformations, and stroke, but also neurological pathologies, such as degenerative diseases. Direct visualization of the microvascular networks in the whole brain remains however challenging in vivo. METHODS: In this work, we performed 3D ultrafast ultrasound localization microscopy (ULM) using a 2D ultrasound matrix array and mapped the whole-brain microvasculature and flow at microscopic resolution in C57Bl6 mice in vivo. FINDINGS: We demonstrated that the mouse brain vasculature can be imaged directly through the intact skull at a spatial resolution of 20 µm and over the whole brain depth and at high temporal resolution (750 volumes.s-1). Individual microbubbles were tracked to estimate the flow velocities that ranged from 2 mm.s-1 in arterioles and venules up to 100 mm.s-1 in large vessels. The vascular maps were registered automatically with the Allen atlas in order to extract quantitative vascular parameters such as local flow rates and velocities in regions of interest. INTERPRETATION: We show the potential of 3D ULM to provide new insights into whole-brain vascular flow in mice models at unprecedented vascular scale for an in vivo technique. This technology is highly translational and has the potential to become a major tool for the clinical investigation of the cerebral microcirculation. FUNDING: This study was supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013) / ERC Grant Agreement n° 311025 and by the Fondation Bettencourt-Schueller under the program "Physics for Medicine". We acknowledge the ART (Technological Research Accelerator) biomedical ultrasound program of INSERM.

Ultrafast Doppler imaging and ultrasound localization microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion.

Acute spinal cord injury (SCI) leads to severe damage to the microvascular network. The process of spontaneous repair is accompanied by formation of new blood vessels; their functionality, however, presumably very important for functional recovery, has never been clearly established, as most studies so far used fixed tissues. Here, combining ultrafast Doppler imaging and ultrasound localization microscopy (ULM) on the same animals, we proceeded at a detailed analysis of structural and functional vascular alterations associated with the establishment of chronic SCI, both at macroscopic and microscopic scales. Using a standardized animal model of SCI, our results demonstrate striking hemodynamic alterations in several subparts of the spinal cord: a reduced blood velocity in the lesion site, and an asymmetrical hypoperfusion caudal but not rostral to the lesion. In addition, the worsening of many evaluated parameters at later time points suggests that the neoformed vascular network is not yet fully operational, and reveals ULM as an efficient in vivo readout for spinal cord vascular alterations. Finally, we show statistical correlations between the diverse biomarkers of vascular dysfunction and SCI severity. The imaging modality developed here will allow evaluating recovery of vascular function over time in pre-clinical models of SCI. Also, used on SCI patients in combination with other quantitative markers of neural tissue damage, it may help classifying lesion severity and predict possible treatment outcomes in patients.

Ultrasound localization microscopy and functional ultrasound imaging reveal atypical features of the trigeminal ganglion vasculature.

The functional imaging within the trigeminal ganglion (TG) is highly challenging due to its small size and deep localization. This study combined a methodological framework able to dive into the rat trigeminal nociceptive system by jointly providing 1) imaging of the TG blood vasculature at microscopic resolution, and 2) the measurement of hemodynamic responses evoked by orofacial stimulations in anesthetized rats. Despite the small number of sensory neurons within the TG, functional ultrasound imaging was able to image and quantify a strong and highly localized hemodynamic response in the ipsilateral TG, evoked not only by mechanical or chemical stimulations of corneal nociceptive fibers, but also by cutaneous mechanical stimulations of the ophthalmic and maxillary orofacial regions using a von Frey hair. The in vivo quantitative imaging of the TG's vasculature using ultrasound localization microscopy combined with in toto labelling reveals particular features of the vascularization of the area containing the sensory neurons, that are likely the origin of this strong vaso-trigeminal response. This innovative imaging approach opens the path for future studies on the mechanisms underlying changes in trigeminal local blood flow and evoked hemodynamic responses, key mechanisms for the understanding and treatment of debilitating trigeminal pain conditions.

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

Large-scale functional ultrasound imaging of the spinal cord reveals in-depth spatiotemporal responses of spinal nociceptive circuits in both normal and inflammatory states.

Despite a century of research on the physiology/pathophysiology of the spinal cord in chronic pain condition, the properties of the spinal cord were rarely studied at the large-scale level from a neurovascular point of view. This is mostly due to the limited spatial and/or temporal resolution of the available techniques. Functional ultrasound imaging (fUS) is an emerging neuroimaging approach that allows, through the measurement of cerebral blood volume, the study of brain functional connectivity or functional activations with excellent spatial (100 µm) and temporal (1 msec) resolutions and a high sensitivity. The aim of this study was to increase our understanding of the spinal cord physiology through the study of the properties of spinal hemodynamic response to the natural or electrical stimulation of afferent fibers. Using a combination of fUS and ultrasound localization microscopy, the first step of this study was the fine description of the vascular structures in the rat spinal cord. Then, using either natural or electrical stimulations of different categories of afferent fibers (Aß, Aδ, and C fibers), we could define the characteristics of the typical hemodynamic response of the rat spinal cord experimentally. We showed that the responses are fiber-specific, located ipsilaterally in the dorsal horn, and that they follow the somatotopy of afferent fiber entries in the dorsal horn and that the C-fiber response is an N-methyl-D-aspartate receptor-dependent mechanism. Finally, fUS imaging of the mesoscopic hemodynamic response induced by natural tactile stimulations revealed a potentiated response in inflammatory condition, suggesting an enhanced response to allodynic stimulations.

Pharmaco-fUS: Quantification of pharmacologically-induced dynamic changes in brain perfusion and connectivity by functional ultrasound imaging in awake mice.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.

Ultrafast ultrasound imaging pattern analysis reveals distinctive dynamic brain states and potent sub-network alterations in arthritic animals.

Chronic pain pathologies, which are due to maladaptive changes in the peripheral and/or central nervous systems, are debilitating diseases that affect 20% of the European adult population. A better understanding of the mechanisms underlying this pathogenesis would facilitate the identification of novel therapeutic targets. Functional connectivity (FC) extracted from coherent low-frequency hemodynamic fluctuations among cerebral networks has recently brought light on a powerful approach to study large scale brain networks and their disruptions in neurological/psychiatric disorders. Analysis of FC is classically performed on averaged signals over time, but recently, the analysis of the dynamics of FC has also provided new promising information. Keeping in mind the limitations of animal models of persistent pain but also the powerful tool they represent to improve our understanding of the neurobiological basis of chronic pain pathogenicity, this study aimed at defining the alterations in functional connectivity, in a clinically relevant animal model of sustained inflammatory pain (Adjuvant-induced Arthritis) in rats by using functional ultrasound imaging, a neuroimaging technique with a unique spatiotemporal resolution (100 µm and 2 ms) and sensitivity. Our results show profound alterations of FC in arthritic animals, such as a subpart of the somatomotor (SM) network, occurring several weeks after the beginning of the disease. Also, we demonstrate for the first time that dynamic functional connectivity assessed by ultrasound can provide quantitative and robust information on the dynamic pattern that we define as brain states. While the main state consists of an overall synchrony of hemodynamic fluctuations in the SM network, arthritic animal spend statistically more time in two other states, where the fluctuations of the primary sensory cortex of the inflamed hind paws show asynchrony with the rest of the SM network. Finally, correlating FC changes with pain behavior in individual animals suggest links between FC alterations and either the cognitive or the emotional aspects of pain. Our study introduces fUS as a new translational tool for the enhanced understanding of the dynamic pain connectome and brain plasticity in a major preclinical model of chronic pain.

4D functional ultrasound imaging of whole-brain activity in rodents.

We extended the capabilities of functional ultrasound to whole-brain four-dimensional (4D) neuroimaging. Our multiplane-wave transmission scheme on matrix arrays at thousands of frames per second provides volumetric recordings of cerebral blood volume changes at high spatiotemporal resolution. We illustrated the approach in rats while providing multiple sensory stimuli, for 4D functional connectivity and during instantaneous tracking of epileptiform events.

In Vivo Imaging of Single Tumor Cells in Fast-Flowing Bloodstream Using Near-Infrared Quantum Dots and Time-Gated Imaging.

Whereas in vivo fluorescence imaging of cells immobilized within tissues provides a valuable tool to a broad range of biological studies, it still lacks the sensitivity required to visualize isolated cells circulating fast in the bloodstream due, in particular, to the autofluorescence from endogenous fluorophores. Time-gated imaging of near-infrared emitting ZnCuInSe/ZnS quantum dots (QDs) with fluorescence lifetimes in the range of 150-300 ns enables the efficient rejection of fast autofluorescence photons and the selection of QD fluorescence photons, thus significantly increasing sensitivity. We labeled model erythrocytes as well as lymphoma cells using these QDs coated with a stable zwitterionic polymer surface chemistry. After reinjection in the bloodstream, we were able to image and count individual QD-labeled cells circulating at mm·s-1 velocities in blood vessels.

Transcranial Functional Ultrasound Imaging in Freely Moving Awake Mice and Anesthetized Young Rats without Contrast Agent.

Functional ultrasound (fUS) imaging by ultrasensitive Doppler detection of blood volume was previously reported to measure adult rat brain activation and functional connectivity with unmatched spatiotemporal sampling (100 µm, 1 ms), but skull-induced attenuation of ultrasonic waves imposed skull surgery or contrast agent use. Also, fUS feasibility remains to be validated in mice, a major pre-clinical model organism. In the study described here, we performed full-depth ultrasensitive Doppler imaging and 3-D Doppler tomography of the entire mouse brain under anesthesia, non-invasively through the intact skull and skin, without contrast agents. Similar results were obtained in anesthetized young rats up to postnatal day 35, thus enabling longitudinal studies on postnatal brain development. Using a newly developed ultralight ultrasonic probe and an optimized ultrasonic sequence, we also performed minimally invasive full-transcranial fUS imaging of brain vasculature and whisker stimulation-induced barrel cortex activation in awake and freely moving mice, validating transcranial fUS for brain imaging, without anesthesia-induced bias, for behavioral studies.

Lentiviral vector-driven inhibition of 5-HT synthesis in B3 bulbo-spinal serotonergic projections - Consequences on nociception, inflammatory and neuropathic pain in rats.

Although it is well established that bulbo-spinal serotonergic projections contribute to pain control mechanisms, whether they exert anti- or pro-nociceptive modulations is still a matter of debate. In order to reappraise the role of 5-HT in descending controls, we used RNA interference to selectively inhibit 5-HT synthesis in B3 neurons and assess resulting changes in nociception. Rats were injected into the bulbar B3 group with a recombinant lentiviral vector, LV-shTPH2, encoding RNA interfering with tryptophan hydroxylase 2 expression. Together with the long term disappearance of this enzyme in the whole rostro-caudal extent of B3 group, 5-HT was markedly depleted selectively in the dorsal horn at all levels of the spinal cord. In contrast, immunolabeling of the 5-HT transporter was unaffected by LV-shTPH2 injection, indicating the preservation of serotonergic fibers integrity. Whereas mechanical and thermal nociceptive thresholds were unchanged by 5-HT depletion, marked reductions in intraplantar formalin (but not carrageenin)-evoked nocifensive responses, and, in contrast, significant increases in mechanical and thermal hyperalgesia evoked by sciatic nerve ligation were noted in LV-shTPH2-injected rats versus controls. Parallel changes in c-Fos immunolabeling within the dorsal horn confirmed that bulbo-spinal serotonergic projections modulate pain signaling under these various conditions. These results suggest that serotonergic neurons of the B3 group are only moderately concerned, if any, by acute nociception but exert modulatory influences under pain sensitizing conditions. The opposite changes in formalin injected- versus sciatic nerve ligated rats might be related to the implication of different receptors in 5-HT-mediated modulation of inflammatory versus neuropathic pain.

Orofacial Neuropathic Pain Leads to a Hyporesponsive Barrel Cortex with Enhanced Structural Synaptic Plasticity.

Chronic pain is a long-lasting debilitating condition that is particularly difficult to treat due to the lack of identified underlying mechanisms. Although several key contributing processes have been described at the level of the spinal cord, very few studies have investigated the supraspinal mechanisms underlying chronic pain. Using a combination of approaches (cortical intrinsic imaging, immunohistochemical and behavioural analysis), our study aimed to decipher the nature of functional and structural changes in a mouse model of orofacial neuropathic pain, focusing on cortical areas involved in various pain components. Our results show that chronic neuropathic orofacial pain is associated with decreased haemodynamic responsiveness to whisker stimulation in the barrel field cortex. This reduced functional activation is likely due to the increased basal neuronal activity (measured indirectly using cFos and phospho-ERK immunoreactivity) observed in several cortical areas, including the contralateral barrel field, motor and cingulate cortices. In the same animals, immunohistochemical analysis of markers for active pre- or postsynaptic elements (Piccolo and phospho-Cofilin, respectively) revealed an increased immunofluorescence in deep cortical layers of the contralateral barrel field, motor and cingulate cortices. These results suggest that long-lasting orofacial neuropathic pain is associated with exacerbated neuronal activity and synaptic plasticity at the cortical level.

Transcranial functional ultrasound imaging of the brain using microbubble-enhanced ultrasensitive Doppler.

Functional ultrasound (fUS) is a novel neuroimaging technique, based on high-sensitivity ultrafast Doppler imaging of cerebral blood volume, capable of measuring brain activation and connectivity in rodents with high spatiotemporal resolution (100µm, 1ms). However, the skull attenuates acoustic waves, so fUS in rats currently requires craniotomy or a thinned-skull window. Here we propose a non-invasive approach by enhancing the fUS signal with a contrast agent, inert gas microbubbles. Plane-wave illumination of the brain at high frame rate (500Hz compounded sequence with three tilted plane waves, PRF=1500Hz with a 128 element 15MHz linear transducer), yields highly-resolved neurovascular maps. We compared fUS imaging performance through the intact skull bone (transcranial fUS) versus a thinned-skull window in the same animal. First, we show that the vascular network of the adult rat brain can be imaged transcranially only after a bolus intravenous injection of microbubbles, which leads to a 9dB gain in the contrast-to-tissue ratio. Next, we demonstrate that functional increase in the blood volume of the primary sensory cortex after targeted electrical-evoked stimulations of the sciatic nerve is observable transcranially in presence of contrast agents, with high reproducibility (Pearson's coefficient ρ=0.7±0.1, p=0.85). Our work demonstrates that the combination of ultrafast Doppler imaging and injection of contrast agent allows non-invasive functional brain imaging through the intact skull bone in rats. These results should ease non-invasive longitudinal studies in rodents and open a promising perspective for the adoption of highly resolved fUS approaches for the adult human brain.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

Respective pharmacological features of neuropathic-like pain evoked by intrathecal BDNF versus sciatic nerve ligation in rats.

Numerous reported data support the idea that Brain Derived Neurotrophic Factor (BDNF) is critically involved in both depression and comorbid pain. The possible direct effect of BDNF on pain mechanisms was assessed here and compared with behavioral/neurobiological features of neuropathic pain caused by chronic constriction injury to the sciatic nerve (CCI-SN). Sprague-Dawley male rats were either injected intrathecally with BDNF (3.0 ng i.t.) or subjected to unilateral CCI-SN. Their respective responses to anti-hyperalgesic drugs were assessed using the Randall-Selitto test and both immunohistochemical and RT-qPCR approaches were used to investigate molecular/cellular mechanisms underlying hyperalgesia in both models. Long lasting hyperalgesia and allodynia were induced by i.t. BDNF in intact healthy rats like those found after CCI-SN. Acute treatment with the BDNF-TrkB receptor antagonist cyclotraxin B completely prevented i.t. BDNF-induced hyperalgesia and partially reversed this symptom in both BDNF-pretreated and CCI-SN lesioned rats. Acute administration of the anticonvulsant pregabalin, the NMDA receptor antagonist ketamine, the opioid analgesics morphine and tapentadol or the antidepressant agomelatine also transiently reversed hyperalgesia in both i.t. BDNF injected- and CCI-SN lesioned-rats. Marked induction of microglia activation markers (OX42, Iba1, P-p38), proinflammatory cytokine IL-6, NMDA receptor subunit NR2B and BDNF was found in spinal cord and/or dorsal root ganglia of CCI-SN rats. A long lasting spinal BDNF overexpression was also observed in BDNF i.t. rats, indicating an autocrine self-induction, with downstream long lasting TrkB-mediated neuropathic-like pain. Accordingly, TrkB blockade appeared as a relevant approach to alleviate not only i.t. BDNF- but also nerve lesion-evoked neuropathic pain.