Awake Rats Research Articles

Adapting Human-Based Transcutaneous Spinal Cord Stimulation to Develop a Clinically Relevant Animal Model.

Transcutaneous spinal cord stimulation (tSCS) as a neuromodulatory strategy has received great attention as a method to promote functional recovery after spinal cord injury (SCI). However, due to the noninvasive nature of tSCS, investigations have primarily focused on human applications. This leaves a critical need for the development of a suitable animal model to further our understanding of this therapeutic intervention in terms of functional and neuroanatomical plasticity and to optimize stimulation protocols. The objective of this study is to establish a new animal model of thoracolumbar tSCS that (1) can accurately recapitulate studies in healthy humans and (2) can receive a repeated and stable tSCS treatment after SCI with minimal restraint, while the electrode remains consistently positioned. We show that our model displays bilateral evoked potentials in multisegmental leg muscles characteristically comparable to humans. Our data also suggest that tSCS mainly activates dorsal root structures like in humans, thereby accounting for the different electrode-to-body-size ratio between the two species. Finally, a repeated tSCS treatment protocol in the awake rat after a complete spinal cord transection is feasible, tolerable, and safe, even with minimal body restraint. Additionally, repeated tSCS was capable of modulating motor output after SCI, providing an avenue to further investigate stimulation-based neuroplasticity and optimize treatment.

Brain-wide ongoing activity is responsible for significant cross-trial BOLD variability.

A notorious issue of task-based functional magnetic resonance imaging (fMRI) is its large cross-trial variability. To quantitatively characterize this variability, the blood oxygenation level-dependent (BOLD) signal can be modeled as a linear summation of a stimulation-relevant and an ongoing (i.e. stimulation-irrelevant) component. However, systematic investigation on the spatiotemporal features of the ongoing BOLD component and how these features affect the BOLD response is still lacking. Here we measured fMRI responses to light onsets and light offsets in awake rats. The neuronal response was simultaneously recorded with calcium-based fiber photometry. We established that between-region BOLD signals were highly correlated brain-wide at zero time lag, including regions that did not respond to visual stimulation, suggesting that the ongoing activity co-fluctuates across the brain. Removing this ongoing activity reduced cross-trial variability of the BOLD response by ~30% and increased its coherence with the Ca2+ signal. Additionally, the negative ongoing BOLD activity sometimes dominated over the stimulation-driven response and contributed to the post-stimulation BOLD undershoot. These results suggest that brain-wide ongoing activity is responsible for significant cross-trial BOLD variability, and this component can be reliably quantified and removed to improve the reliability of fMRI response. Importantly, this method can be generalized to virtually all fMRI experiments without changing stimulation paradigms.

Antioxidant therapy reverses sympathetic dysfunction, oxidative stress, and hypertension in male hyperadipose rats

AimsThe rostral ventrolateral medulla (RVLM) is the main sympathetic output of the central nervous system to control blood pressure. Reportedly, reactive oxygen species (ROS) can increase arterial pressure, leading to hypertension. As ROS increase the sympathetic tone in RVLM and obese animals present grater oxidative stress, it would be important to note this relationship. Main methodsTherefore, we evaluated the systemic and central effects (in the RVLM) of vitamin C (vit C, an antioxidant) on the redox balance and cardiovascular and autonomic profiles in hyperadipose male rats. We also evaluated the neurotransmission by L-glutamate (L-glu) and vit C in the RVLM of awake hyperadipose rats. Key findingsOur study confirmed that hyperadipose rats were hypertensive and tachycardic, presented increased sympathetic and decreased parasympathetic modulation of the heart, and had increased plasma lipoperoxidation compared with the control rats (CTR). Oral vitamin C treatment reverted cardiovascular, autonomic, and plasma redox dysfunction. Hyperadipose rats presented a higher blood pressure increase after L-glu microinjection and a lower response to vit C in the RVLM compared with the CTR group. Biochemical analysis of redox balance in RVLM punches showed that hyperadipose rats have increased NBT and T-BARS, and after treatment with vit C, the oxidative profile decreased. The antioxidative activity of vit C reduced the amount of ROS in the RVLM area that might have resulted in lowered blood pressure and sympathetic modulation. SignificanceOur data suggest central and peripheral benefits of vit C treatment on cardiovascular, autonomic, and oxidative dysfunctions in hyperadipose animals.

Rates of pyruvate carboxylase, glutamate and GABA neurotransmitter cycling, and glucose oxidation in multiple brain regions of the awake rat using a combination of [2-13C]/[1-13C]glucose infusion and 1H-[13C]NMR ex vivo

Anaplerosis occurs predominately in astroglia through the action of pyruvate carboxylase (PC). The rate of PC (Vpc) has been reported for cerebral cortex (or whole brain) of awake humans and anesthetized rodents, but regional brain rates remain largely unknown and, hence, were subjected to investigation in the current study. Awake male rats were infused with either [2-13C]glucose or [1-13C]glucose (n = 27/30) for 8, 15, 30, 60 or 120 min, followed by rapid euthanasia with focused-beam microwave irradiation to the brain. Blood plasma and extracts of cerebellum, hippocampus, striatum, and cerebral cortex were analyzed by 1H-[13C]-NMR to establish 13C-enrichment time courses for glutamate-C4,C3,C2, glutamine-C4,C3, GABA-C2,C3,C4 and aspartate-C2,C3. Metabolic rates were determined by fitting a three-compartment metabolic model (glutamatergic and GABAergic neurons and astroglia) to the eighteen time courses. Vpc varied by 44% across brain regions, being lowest in the cerebellum (0.087 ± 0.004 µmol/g/min) and highest in striatum (0.125 ± 0.009) with intermediate values in cerebral cortex (0.106 ± 0.005) and hippocampus (0.114 ± 0.005). Vpc constituted 13–19% of the oxidative glucose consumption rate. Combination of cerebral cortical data with literature values revealed a positive correlation between Vpc and the rates of glutamate/glutamine-cycling and oxidative glucose consumption, respectively, consistent with earlier observations.

Light sedation with short habituation time for large-scale functional magnetic resonance imaging studies in rats.

Traditionally, preclinical resting state functional magnetic resonance imaging (fMRI) studies have been performed in anesthetized animals. Nevertheless, as anesthesia affects the functional connectivity (FC) in the brain, there has been a growing interest in imaging in the awake state. Obviously, awake imaging requires resource‐ and time‐consuming habituation prior to data acquisition to reduce the stress and motion of the animals. Light sedation has been a less widely exploited alternative for awake imaging, requiring shorter habituation times, while still reducing the effect of anesthesia. Here, we imaged 102 rats under light sedation and 10 awake animals to conduct an FC analysis. We established an automated data‐processing pipeline suitable for both groups. Additionally, the same pipeline was used on data obtained from an openly available awake rat database (289 measurements in 90 rats). The FC pattern in the light sedation measurements closely resembled the corresponding patterns in both onsite and offsite awake datasets. However, fewer datasets had to be excluded due to movement in rats with light sedation. The temporal analysis of FC in the lightly sedated group indicated a lingering effect of anesthesia that stabilized after the first 5 min. In summary, our results indicate that the light sedation protocol is a valid alternative for large‐scale studies where awake protocols may become prohibitively resource‐demanding, as it provides similar results to awake imaging, preserves more scans, and requires shorter habituation times. The large amount of fMRI data obtained in this work are openly available for further analyses.

Early Repetitive Transcranial Magnetic Stimulation Exerts Neuroprotective Effects and Improves Motor Functions in Hemiparkinsonian Rats.

Repetitive transcranial magnetic stimulation (rTMS) is a popular noninvasive technique for modulating motor cortical plasticity and has therapeutic potential for the treatment of Parkinson's disease (PD). However, the therapeutic benefits and related mechanisms of rTMS in PD are still uncertain. Accordingly, preclinical animal research is helpful for enabling translational research to explore an effective therapeutic strategy and for better understanding the underlying mechanisms. Therefore, the current study was designed to identify the therapeutic effects of rTMS on hemiparkinsonian rats. A hemiparkinsonian rat model, induced by unilateral injection of 6-hydroxydopamine (6-OHDA), was applied to evaluate the therapeutic potential of rTMS in motor functions and neuroprotective effect of dopaminergic neurons. Following early and long-term rTMS intervention with an intermittent theta burst stimulation (iTBS) paradigm (starting 24 h post-6-OHDA lesion, 1 session/day, 7 days/week, for a total of 4 weeks) in awake hemiparkinsonian rats, the effects of rTMS on the performance in detailed functional behavioral tests, including video-based gait analysis, the bar test for akinesia, apomorphine-induced rotational analysis, and tests of the degeneration level of dopaminergic neurons, were identified. We found that four weeks of rTMS intervention significantly reduced the aggravation of PD-related symptoms post-6-OHDA lesion. Immunohistochemically, the results showed that tyrosine hydroxylase- (TH-) positive neurons in the substantia nigra pars compacta (SNpc) and fibers in the striatum were significantly preserved in the rTMS treatment group. These findings suggest that early and long-term rTMS with the iTBS paradigm exerts neuroprotective effects and mitigates motor impairments in a hemiparkinsonian rat model. These results further highlight the potential therapeutic effects of rTMS and confirm that long-term rTMS treatment might have clinical relevance and usefulness as an additional treatment approach in individuals with PD.

Gradual Restraint Habituation for Awake Functional Magnetic Resonance Imaging Combined With a Sparse Imaging Paradigm Reduces Motion Artifacts and Stress Levels in Rodents.

Functional magnetic resonance imaging, as a non-invasive technique, offers unique opportunities to assess brain function and connectivity under a broad range of applications, ranging from passive sensory stimulation to high-level cognitive abilities, in awake animals. This approach is confounded, however, by the fact that physical restraint and loud unpredictable acoustic noise must inevitably accompany fMRI recordings. These factors induce marked stress in rodents, and stress-related elevations of corticosterone levels are known to alter information processing and cognition in the rodent. Here, we propose a habituation strategy that spans specific stages of adaptation to restraint, MRI noise, and confinement stress in awake rats and circumvents the need for surgical head restraint. This habituation protocol results in stress levels during awake fMRI that do not differ from pre-handling levels and enables stable image acquisition with very low motion artifacts. For this, rats were gradually trained over a period of three weeks and eighteen training sessions. Stress levels were assessed by analysis of fecal corticosterone metabolite levels and breathing rates. We observed significant drops in stress levels to below pre-handling levels at the end of the habituation procedure. During fMRI in awake rats, after the conclusion of habituation and using a non-invasive head-fixation device, breathing was stable and head motion artifacts were minimal. A task-based fMRI experiment, using acoustic stimulation, conducted 2 days after the end of habituation, resulted in precise whole brain mapping of BOLD signals in the brain, with clear delineation of the expected auditory-related structures. The active discrimination by the animals of the acoustic stimuli from the backdrop of scanner noise was corroborated by significant increases in BOLD signals in the thalamus and reticular formation. Taken together, these data show that effective habituation to awake fMRI can be achieved by gradual and incremental acclimatization to the experimental conditions. Subsequent BOLD recordings, even during superimposed acoustic stimulation, reflect low stress-levels, low motion and a corresponding high-quality image acquisition. Furthermore, BOLD signals obtained during fMRI indicate that effective habituation facilitates selective attention to sensory stimuli that can in turn support the discrimination of cognitive processes in the absence of stress confounds.

Targeting the motor cortex to restore walking after incomplete spinal cord injury.

Targeting the motor cortex to restore walking after incomplete spinal cord injury.

Neurokinin receptor mechanisms in forebrain medial septum modulate nociception in the formalin model of inflammatory pain

The present study has explored the hypothesis that neurokinin1 receptors (NK1Rs) in medial septum (MS) modulate nociception evoked on hind paw injection of formalin. Indeed, the NK1Rs in MS are localized on cholinergic neurons which have been implicated in nociception. In anaesthetized rat, microinjection of L-733,060, an antagonist at NK1Rs, into MS antagonized the suppression of CA1 population spike (PS) evoked on peripheral injection of formalin or on intraseptal microinjection of substance P (SP), an agonist at NK1Rs. The CA1 PS reflects the synaptic excitability of pyramidal cells in the region. Furthermore, microinjection of L-733,060 into MS, but not LS, attenuated formalin-induced theta activation in both anaesthetized and awake rat, where theta reflects an oscillatory information processing by hippocampal neurons. The effects of L-733,060 on microinjection into MS were nociceptive selective as the antagonist did not block septo-hippocampal response to direct MS stimulation by the cholinergic receptor agonist, carbachol, in anaesthetized animal or on exploration in awake animal. Interestingly, microinjection of L-733,060 into both MS and LS attenuated formalin-induced nociceptive flinches. Collectively, the foregoing novel findings highlight that transmission at NK1R provide an affective valence to septo-hippocampal information processing and that peptidergic transmission in the septum modulates nociceptive behaviours.

Do We Swallow the Waste From Our Brain?

Ferumoxytol, an iron oxide nanoparticle, was infused into the lateral cerebroventricle of awake rats to follow its movement and clearance from the brain using magnetic resonance imaging. Within minutes the contrast agent could be observed accumulating in the subarachnoid space, nasal cavity, nasal pharynx, and soft palate at the back of the throat. In a subsequent study fluorescent quantum dots were infused into the brain of rats and within 15 min could be observed in the esophagus using microscopy. These imaging studies clearly show that these large nanoparticle tracers (∼20 nm in diameter) leave the brain through the nasal cavity and end up in the gut. There are numerous studies going back decades reporting the clearance of tracers put directly into the brain. While these studies show the slow accumulation of trace in the blood and lymphatics, they report only accounting for less than 50% of what was originally put in the brain.

Assessment of Brain Functional Activity Using a Miniaturized Head-Mounted Scanning Photoacoustic Imaging System in Awake and Freely Moving Rats.

Understanding the relationship between brain function and natural behavior remains a significant challenge in neuroscience because there are very few convincing imaging/recording tools available for the evaluation of awake and freely moving animals. Here, we employed a miniaturized head-mounted scanning photoacoustic imaging (hmPAI) system to image real-time cortical dynamics. A compact photoacoustic (PA) probe based on four in-house optical fiber pads and a single custom-made 48-MHz focused ultrasound transducer was designed to enable focused dark-field PA imaging, and miniature linear motors were included to enable two-dimensional (2D) scanning. The total dimensions and weight of the proposed hmPAI system are only approximately 50 × 64 × 48 mm and 58.7 g (excluding cables). Our ex vivo phantom experimental tests revealed that a spatial resolution of approximately 0.225 mm could be achieved at a depth of 9 mm. Our in vivo results further revealed that the diameters of cortical vessels draining into the superior sagittal sinus (SSS) could be clearly imaged and continuously observed in both anesthetized rats and awake, freely moving rats. Statistical analysis showed that the full width at half maximum (FWHM) of the PA A-line signals (relative to the blood vessel diameter) was significantly increased in the selected SSS-drained cortical vessels of awake rats (0.58 ± 0.17 mm) compared with those of anesthetized rats (0.31 ± 0.09 mm) (p < 0.01, paired t-test). In addition, the number of pixels in PA B-scan images (relative to the cerebral blood volume (CBV)) was also significantly increased in the selected SSS-drained blood vessels of awake rats (107.66 ± 23.02 pixels) compared with those of anesthetized rats (81.99 ± 21.52 pixels) (p < 0.01, paired t-test). This outcome may result from a more active brain in awake rats than in anesthetized rats, which caused cerebral blood vessels to transport more blood to meet the increased nutrient demand of the tissue, resulting in an obvious increase in blood vessel volume. This hmPAI system was further validated for utility in the brains of awake and freely moving rats, showing that their natural behavior was unimpaired during vascular imaging, thereby providing novel opportunities for studies of behavior, cognition, and preclinical models of brain diseases.

Metabolism of hyperpolarised [1-13 C]pyruvate in awake and anaesthetised rat brains.

The use of hyperpolarised 13 C pyruvate for nononcological neurological applications has not been widespread so far, possibly due to delivery issues limiting the visibility of metabolites. First proof-of-concept results have indicated that metabolism can be detected in human brain, and this may supersede the results obtained in preclinical settings. One major difference between the experimental setups is that preclinical MRI/MRS routinely uses anaesthesia, which alters both haemodynamics and metabolism. Here, we used hyperpolarised [1-13 C]pyruvate to compare brain metabolism in awake rats and under isoflurane, urethane or medetomidine anaesthesia. Spectroscopic [1-13 C]pyruvate time courses measured sequentially showed that pyruvate-to-bicarbonate and pyruvate-to-lactate labelling rates were lower in isoflurane animals than awake animals. An increased bicarbonate-to-lactate ratio was observed in the medetomidine group compared with other groups. The study shows that hyperpolarised [1-13 C]pyruvate experiments can be performed in awake rats, thus avoiding anaesthesia-related issues. The results suggest that haemodynamics probably dominate the observed pyruvate-to-metabolite labelling rates and area-under-time course ratios of referenced to pyruvate. On the other hand, the results obtained with medetomidine suggest that the ratios are also modulated by the underlying cerebral metabolism. However, the ratios between intracellular metabolites were unchanged in awake compared with isoflurane-anaesthetised rats.

Sound-Evoked Responses in the Vestibulo-Ocular Reflex Pathways of Rats.

Vestibular evoked myogenic potentials (VEMP) have been used to assess otolith function in clinics worldwide. However, there are accumulating evidence suggesting that the clinically used sound stimuli activate not only the otolith afferents, but also the canal afferents, indicating canal contributions to the VEMPs. To better understand the neural mechanisms underlying the VEMPs and develop discriminative VEMP protocols, we further examined sound-evoked responses of the vestibular nucleus neurons and the abducens neurons, which have the interneurons and motoneurons of the vestibulo-ocular reflex (VOR) pathways. Air-conducted clicks (50–80 dB SL re ABR threshold, 0.1 ms duration) or tone bursts (60–80 dB SL, 125–4,000 Hz, 8 ms plateau, 1 ms rise/fall) were delivered to the ears of Sprague-Dawley or Long-Evans rats. Among 425 vestibular nucleus neurons recorded in anesthetized rats and 18 abducens neurons recorded in awake rats, sound activated 35.9% of the vestibular neurons that increased discharge rates for ipsilateral head rotation (Type I neuron), 15.7% of the vestibular neurons that increased discharge rates for contralateral head rotation (Type II neuron), 57.2% of the vestibular neurons that did not change discharge rates during head rotation (non-canal neuron), and 38.9% of the abducens neurons. Sound sensitive vestibular nucleus neurons and abducens neurons exhibited characteristic tuning curves that reflected convergence of canal and otolith inputs in the VOR pathways. Tone bursts also evoked well-defined eye movements that increased with tone intensity and duration and exhibited peak frequency of ∼1,500 Hz. For the left eye, tone bursts evoked upward/rightward eye movements for ipsilateral stimulation, and downward/leftward eye movements for contralateral stimulation. These results demonstrate that sound stimulation results in activation of the canal and otolith VOR pathways that can be measured by eye tracking devices to develop discriminative tests of vestibular function in animal models and in humans.

An epidural stimulating interface unveils the intrinsic modulation of electrically motor evoked potentials in behaving rats.

In intact and spinal-injured anesthetized animals, stimulation levels that did not induce any visible muscle twitches were used to elicit motor evoked potentials (MEPs) of varying amplitude, reflecting the temporal and amplitude dynamics of the background excitability of spinal networks. To characterize the physiological excitability states of neuronal networks driving movement, we designed five experiments in awake rats chronically implanted with an epidural stimulating interface, with and without a spinal cord injury (SCI). First, an uninjured rat at rest underwent a series of single electrical pulses at sub-motor threshold intensity, which generated responses that were continuously recorded from flexor and extensor hindlimb muscles, showing an intrinsic patterned modulation of MEPs. Responses were recruited by increasing strengths of stimulation, and the amplitudes were moderately correlated between flexors and extensors. Next, after SCI, four awake rats at rest showed electrically induced MEPs, varying largely in amplitude, of both flexors and extensors that were mainly synchronously modulated. After full anesthesia, MEP amplitudes were largely reduced, although stimulation still generated random baseline changes, unveiling an intrinsic stochastic modulation. The present five cases demonstrate a methodology that can be feasibly replicated in a broader group of awake and behaving rats to further define experimental treatments involving neuroplasticity. Besides validating a new technology for a neural stimulating interface, the present data support the broader message that there is intrinsic patterned and stochastic modulation of baseline excitability reflecting the dynamics of physiological states of spinal networks.NEW & NOTEWORTHY Chronic implants of a new epidural stimulating interface trace dynamics of spinal excitability in awake rats, before and after injury. Motor evoked potentials induced by trains of pulses at sub-motor threshold intensity were continuously modulated in amplitude. Oscillatory patterns of amplitude modulation reduced with increasing strengths of stimulation and were replaced by an intrinsic stochastic tone under anesthesia. Variability of baseline excitability is a fundamental feature of spinal networks, affecting their responses to external input.

3D microelectrode cluster and stimulation paradigm yield powerful analgesia without noticeable adverse effects.

The lack of satisfactory treatment for persistent pain profoundly impairs the quality of life for many patients. Stimulation of brainstem pain control systems can trigger powerful analgesia, but their complex network organization frequently prevents separation of analgesia from side effects. To overcome this long-standing challenge, we developed a biocompatible gelatin-embedded cluster of ultrathin microelectrodes that enables fine-tuned, high-definition three-dimensional stimulation in periaqueductal gray/dorsal raphe nucleus in awake rats. Analgesia was assessed from both motor reactions and intracortical signals, corresponding to pain-related signals in humans. We could select an individual-specific subset of microelectrodes in each animal that reliably provided strong pain inhibition during normal and hyperalgesia conditions, without noticeable behavioral side effects. Gait, spontaneous cortical activity at rest, and cortical tactile responses were minimally affected, indicating a highly selective action. In conclusion, our developed biocompatible microelectrode cluster and stimulation paradigm reliably enabled powerful, fine-tuned, and selective analgesia without noticeable side effects.

Mild repetitive head impacts alter perivascular flow in the midbrain dopaminergic system in awake rats.

Head injury is a known risk factor for Parkinson’s disease. Disruption in the perivascular clearance of metabolic waste and unwanted proteins is thought to be a contributing factor to disease progression. We hypothesized that repetitive mild head impacts, without evidence of structural brain damage, would increase microgliosis and AQP4 expression and depolarization and alter perivascular flow in the midbrain dopaminergic system. Adult male rats were subjected to sham, or two mild head impacts separated by 48 h. Three weeks later, fully awake rats were imaged using dynamic, contrast-enhanced MRI to follow the distribution of intraventricular gadobenate dimeglumine contrast agent. Images were registered to and analysed using a 3D MRI rat atlas providing site-specific data on 171 different brain areas. Following imaging, rats were tested for cognitive function using the Barnes maze assay. Histological analyses of tyrosine hydroxylase, microglia activation and AQP4 expression and polarization were performed on a parallel cohort of head impacted rats at 20 days post insult to coordinate with the time of imaging. There was no change in the global flux of contrast agent between sham and head impacted rats. The midbrain dopaminergic system showed a significant decrease in the influx of contrast agent as compared to sham controls together with a significant increase in microgliosis, AQP4 expression and depolarization. There were no deficits in cognitive function. The histology showed a significant level of neuroinflammation in the midbrain dopaminergic system 3 weeks post mild repetitive head impact but no loss in tyrosine hydroxylase. MRI revealed no structural brain damage emphasizing the potential serious consequences of mild head impacts on sustained brain neuroinflammation in this area critical to the pathophysiology of Parkinson’s.

Bone loss after severe spinal cord injury coincides with reduced bone formation and precedes bone blood flow deficits.

Diminished bone perfusion develops in response to disuse and has been proposed as a mechanism underlying bone loss. Bone blood flow (BF) has not been investigated within the unique context of severe contusion spinal cord injury (SCI), a condition that produces neurogenic bone loss that is precipitated by disuse and other physiological consequences of central nervous system injury. Herein, 4-mo-old male Sprague-Dawley rats received T9 laminectomy (SHAM) or laminectomy with severe contusion SCI (n = 20/group). Time course assessments of hindlimb bone microstructure and bone perfusion were performed in vivo at 1- and 2-wk postsurgery via microcomputed tomography (microCT) and intracardiac microsphere infusion, respectively, and bone turnover indices were determined via histomorphometry. Both groups exhibited cancellous bone loss beginning in the initial postsurgical week, with cancellous and cortical bone deficits progressing only in SCI thereafter. Trabecular bone deterioration coincided with uncoupled bone turnover after SCI, as indicated by signs of ongoing osteoclast-mediated bone resorption and a near-complete absence of osteoblasts and cancellous bone formation. Bone BF was not different between groups at 1 wk, when both groups displayed bone loss. In comparison, femur and tibia perfusion was 30%-40% lower in SCI versus SHAM at 2 wk, with the most pronounced regional BF deficits occurring at the distal femur. Significant associations existed between distal femur BF and cancellous and cortical bone loss indices. Our data provide the first direct evidence indicating that bone BF deficits develop in response to SCI and temporally coincide with suppressed bone formation and with cancellous and cortical bone deterioration.NEW & NOTEWORTHY We provide the first direct evidence indicating femur and tibia blood flow (BF) deficits exist in conscious (awake) rats after severe contusion spinal cord injury (SCI), with the distal femur displaying the largest BF deficits. Reduced bone perfusion temporally coincided with unopposed bone resorption, as indicated by ongoing osteoclast-mediated bone resorption and a near absence of surface-level bone formation indices, which resulted in severe cancellous and cortical microstructural deterioration after SCI.

Investigation of metabolic kinetics in different brain regions of awake rats using the [1H-13C]-NMR technique

Energy metabolism and neurotransmission are necessary for sustaining normal life activities. Hence, neurological or psychiatric disorders are always associated with changes in neurotransmitters and energy metabolic states in the brain. Most studies have only focused on the most important neurotransmitters, particularly GABA and Glu, however, other metabolites such as NAA and aspartate which are also very important for cerebral function are rarely investigated. In this study, most of the metabolic kinetics information of different brain regions was investigated in awake rats using the [1H-13C]-NMR technique. Briefly, rats (n = 8) were infused [1-13C] glucose through the tail vein for two minutes. After 20 min of glucose metabolism, the animals were sacrificed and the brain tissue was extracted and treated. Utilizing the 1H observed/13C-edited nuclear magnetic resonance (POCE-NMR), the enrichment of neurochemicals was detected which reflected the metabolic changes in different brain regions and the metabolic connections between neurons and glial cells in the brain. The results suggest that the distribution of every metabolite differed from every brain region and the metabolic rate of NAA was relatively low at 8.64 ± 2.37 μmol/g/h. In addition, there were some correlations between several 13C enriched metabolites, such as Glu4-Gln4 (p = 0.062), Glu4-GABA2 (p < 0.01), Glx2-Glx3 (p < 0.001), Asp3-NAA3 (p < 0.001). This correlativity reflects the signal transmission between astrocytes and neurons, as well as the potential interaction between energy metabolism and neurotransmission. In conclusion, the current study systematically demonstrated the metabolic kinetics in the brain which shed light on brain functions and the mechanisms of various pathophysiological states.

Animal studies in clinical MRI scanners: A custom setup for combined fMRI and deep-brain stimulation in awake rats

BackgroundIn humans, functional magnetic resonance imaging (fMRI) cannot be used to its full potential to study the effects of deep-brain stimulation (DBS) on the brain due to safety reasons. Application of DBS in small animals is an alternative, but was hampered by technical limitations thus far. New MethodWe present a novel setup that extends the range of available applications by studying animals in a clinical scanner. We used a 3 T-MRI scanner with a custom-designed receiver coil and a restrainer to measure brain activity in awake rats. DBS electrodes made of silver were used to minimize electromagnetic artifacts. Before scanning, rats were habituated to the restrainer. ResultsUsing our novel setup, we observed minor DBS-electrode artifacts, which did not interfere with brain-activity measurements significantly. Movement artifacts were also minimal and were not further reduced by restrainer habituation. Bilateral DBS in the dorsal part of the ventral striatum (dVS) resulted in detectable increases in brain activity around the electrodes tips. Comparison with Existing MethodsThis novel setup offers a low-cost alternative to dedicated small-animal scanners. Moreover, it can be implemented in widely available clinical 3 T scanners. Although spatial and temporal resolution was lower than what is achieved in anesthetized rats in high-field small-animal scanners, we obtained scans in awake animals, thus, testing the effects of bilateral DBS of the dVS in a more physiological state. ConclusionsWith this new technical setup, the neurobiological mechanism of action of DBS can be explored in awake, restrained rats in a clinical 3 T-MRI scanner.

Chronic, closed‐loop, cervical epidural stimulation elicits diaphragm motor plasticity after spinal cord injury

Cervical spinal cord injury (cSCI) accounts for over half of all traumatic SCI (NSCISC, 2020). Unfortunately, respiratory insufficiency due to cSCI is the leading cause of mortality and morbidity after SCI. Electrical epidural stimulation (EES) can partially restore volitional locomotor function after SCI in humans while high-frequency EES activates respiratory muscles (DiMarco and Kowalski, 2009) and may lead to at least short-term plasticity (Gonzalez-Rothi et al., 2017). We've shown that our model of cervical closed-loop epidural stimulation (CL-ES) in awake, freely-behaving rats with C2 hemisection (C2HS) elicits short term plasticity in diaphragm motor output (Malone et al., FASEB J 2020). Here we show that this expression of plasticity may be bimodal in that there are “responders” and “non-responders” just as that seen in people with SCI (Tan et al., 2020; Lamy and Boakye, 2013). Rats were implanted with CL-ES electrodes at C4 and bilateral diaphragm EMG electrodes to record respiratory muscle activity in awake rats. Four groups were studied: C2HS + CL-ES (n=8); C2HS + no stim (n=6); Sham + CL-ES (n=6); Sham + no stim (n=6). As expected, increasing current (range: 50–750 μA; inspiratory-triggered) increased the magnitude of evoked potentials. Further, four days of chronic (12-20 hours/day) CL-ES elicited facilitation of ipsilesional evoked responses in 6 of 8 animals with C2HS. Data were calculated as maximum percent change from baseline (-/+) of the stimulus triggered averages at the current with the largest dispersion during sweeps. CL-ES in C2HS rats elicited facilitation in max percent change in stimulus triggered average: 1) peak amplitude (pA; 879% ± 455%) vs. Sham + CL-ES and Sham + no stim (109% ± 76%; p=0.04 and 92% ± 68% p=0.05, respectively) and 2) area under the curve (AUC; 555% ± 142%) vs. groups without stimulation (C2HS + no stim: 99% ± 52%, p=0.05; Sham + no stim: 73% ± 60%, p=0.038). The lack of robust facilitation in Sham + CL-ES rats suggests the possibility of an intact inhibitory modulatory network in healthy rats. These results are preliminary and highly variable, thus we can only speculate about mechanisms giving rise to this form of respiratory neuroplasticity. Ongoing RTqPCR experiments utilizing correlative measures of spinal neurotrophic factor expression (e.g. brain derived neurotrophic factor) and siRNA knock-down of relevant receptors on phrenic motor neurons (Dale et al., 2017) may give more insight. We hypothesize that facilitation of these evoked potentials may depend on activation of neurotrophic factor receptors within the phrenic motor network. This work demonstrates for the first time that CL-ES can elicit respiratory motor plasticity after cSCI, demonstrating potential for a neuroceutical therapy to address breathing impairment in individuals with spinal cord injury. Data presented as mean ± SEM.