Spectral and Spatial Dependence of Diffuse Optical Signals in Response to Peripheral Nerve Stimulation

Using non-invasive, near-infrared spectroscopy we have previously reported optical signals measured at or around peripheral nerves in response to their stimulation. Such optical signals featured amplitudes on the order of 0.1% and peaked about 100 ms after peripheral nerve stimulation in human subjects. Here, we report a study of the spatial and spectral dependence of the optical signals induced by stimulation of the human median and sural nerves, and observe that these optical signals are: (1) unlikely due to either dilation or constriction of blood vessels, (2) not associated with capillary bed hemoglobin, (3) likely due to blood vessel(s) displacement, and (4) unlikely due to fiber-skin optical coupling effects. We conclude that the most probable origin of the optical response to peripheral nerve stimulation is from displacement of blood vessels within the optically probed volume, as a result of muscle twitch in adjacent areas.National Institutes of Health (R01-NS059933); U.S. Army Medical Acquisition Activity (W81XWH-07-2-0011

Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naive rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naive rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level

Acute anti-allodynic action of gabapentin in dorsal horn and primary somatosensory cortex: Correlation of behavioural and physiological data

Neuropathic pain is a debilitating consequence of neuronal injury or disease. Although first line treatments include the alpha-2-delta (a2d)-ligands, pregabalin and gabapentin (GBP), the mechanism of their anti-allodynic action is poorly understood. One specific paradox is that GBP relieves signs of neuropathic pain in animal models within 30min of an intraperitoneal (IP) injection yet its actions in vitro on spinal dorsal horn or primary afferent neurons take hours to develop. We found, using confocal Ca2þ imaging, that substantia gelatinosa neurons obtained ex vivo from rats subjected to sciatic chronic constriction injury (CCI) were more excitable than controls. We confirmed that GBP (100 mg/kg) attenuated mechanical allodynia in animals subject to CCI within 30min of IP injection.Substantia gelatinosa neurons obtained ex vivo from these animals no longer displayed CCI-induced increased excitability. Electrophysiological analysis of substantia gelatinosa neurons ex vivo suggest that rapidly developing in vivo anti-allodynic effects of GBP i) are mediated intracellularly, ii) involve actions on the neurotransmitter release machinery and iii) depend on decreased excitatory synaptic drive to excitatory neurons without major actions on inhibitory neurons or on intrinsic neuronal excitability. Experiments using in vivo Ca2þ imaging showed that 100 mg/kg GBP also suppressed the response of the S1 somatosensory cortex of CCI rats, but not that of control rats, to vibrotactile stimulation. Since the level of a2d1 protein is increased in primary afferent fibres after sciatic CCI, we suggest this dictates the rate of GBP action; rapidly developing actions can only be seen when a2d1 levels are elevated

Autonomic Nervous System and Neurocardiac Physiopathology

The autonomic nervous system regulates multiple physiological functions; how distinct neurons in peripheral autonomic and intrathoracic ganglia communicate remains to be established. Increasing focus is being paid to functionality of the neurocardiac axis and crosstalk between the intrinsic nervous system and diverse organ systems. Current findings indicate that progression of cardiovascular disease comprises peripheral and central aspects of the cardiac nervous system hierarchy. Indeed, autonomic neuronal dysfunction is known to participate in arrhythmogenesis and sudden cardiac death; diverse interventions (pharmacological, non-pharmacological) that affect neuronal remodeling in the heart following injury caused by cardiovascular disease (congestive heart failure, etc.) or acute myocardial infarction are being investigated. Herein we examine recent findings from clinical and animal studies on the role of the intrinsic cardiac nervous system on regulation of myocardial perfusion and the consequences of cardiac injury. We also discuss different interventions that target the autonomic nervous system, stimulate neuronal remodeling and adaptation, and thereby optimize patient outcomes

Molecular mechanisms of nociception and pain

My thesis uses in vivo calcium imaging to investigate the cell and molecular mechanisms of two unusual pain states: congenital analgesia and cold allodynia. Genetic deletion of voltage-gated sodium channel NaV1.7 in mice and humans leads to profound pain insensitivity. Paradoxically, peripherally-targeted pharmacological antagonists of NaV1.7 fail to relieve pain in the clinic. To determine the mechanism of analgesia in NaV1.7 null mutants, I used optical, electrophysiological and behavioural methods to investigate the effect of peripheral NaV1.7 deletion on nociceptor function. Surprisingly, both calcium imaging and extracellular recording of NaV1.7-deficient sensory neurons in vivo found limited deficits in the response to noxious stimuli. Synaptic transmission from nociceptor central terminals in the spinal cord was however compromised following NaV1.7 deletion. Importantly, both synaptic deficits and behavioural analgesia were reversed by blocking central opioid receptors. Collectively, these data account for the failure of peripherally-targeted NaV1.7 blockers and point to a central mechanism of analgesia in NaV1.7 null mutants that requires opioid receptors. Chronic pain patients suffering from cold allodynia experience normally innocuous cooling as excruciating pain, but the cells and molecules driving cold allodynia remain elusive. I used in vivo calcium imaging to investigate how the activity of cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, peripheral nerve injury and ciguatera poisoning. In neuropathic mice exhibiting cold allodynia, a subset of cold-insensitive, large-diameter, peptidergic nociceptors became responsive to cooling. Diptheria toxin-mediated ablation of these silent cold-sensing neurons decreased neuropathic cold hypersensitivity. Voltage-gated potassium channels KV1.1 and KV1.2 were highly expressed in silent cold-sensing neurons and pharmacological inhibition of these channels rapidly induced cold responsiveness in cold-insensitive neurons. Taken together, I reveal that silent-cold sensing neurons contribute to cold allodynia in neuropathic pain and identify KV1 channel downregulation as a driver of de novo cold sensitivity, in vivo

Analysis of Tissue Sparing and Circuit Regeneration in Spinal Cord Injuries Treated with Biomaterials and Gene Therapy

Each year, the U.S. sees nearly 17,700 new cases of spinal cord injuries (SCIs). Despite intense rehabilitation, patients with SCIs most often suffer lifelong physical consequences and substantial increases in medical expenses per individual. While immobilization and surgery can be used for immediate stabilization of the injury, no clinical methods exist to address the subsequent inflammation and lack of tissue regeneration that further contribute to the motor and sensory deficits seen after an SCI. This dissertation aimed to understand how biomaterials and gene therapy treatment affect SCIs. In a mouse SCI model, where a left C5 hemisection results in loss of function of the left arm, a poly(lactide-co-glycolide) (PLG) scaffold or “bridge” can be implanted in place of the resected tissues. The bridge can be loaded with lentivirus for local delivery of gene therapy that can aid in control of the post-SCI microenvironment. Using lentiviral interleukin-10 (IL10), we found that IL10 animals significantly outperform animals that received a control lentivirus on a ladder beam test at 2- and 12- weeks post-injury (wpi). Closer examination of components of the forelimb motor circuitry suggest IL10 animals had increased sparing of lower motor neurons and neuromuscular junctions. Electrophysiological studies at 2 wpi showed that control injured animals had electromyogram recordings that were significantly dampened when compared to IL10 and control uninjured animals, thereby confirming that the motor circuitry remained more intact with IL10 treatment. These results, which were consistent in both male and female mice, are the first to show that IL10 spares motor circuitry directly responsible for enhanced muscle function. We then tested a combination therapy of lentiviral IL10 and brain-derived neurotrophic factor (BDNF), followed by examination of tissue sparing and regeneration. At 2 wpi, histological and electrophysiological analyses show that the tissue sparing effects of IL10 alone are only slightly enhanced by the addition of BDNF. By 12 wpi, most innervation differences among the treatment conditions disappeared, though electrophysiological examination suggests that IL10 may prevent some of the injury-associated shifts in muscle composition that result in increased fatigability in control injured animals. Within the spinal cord, we found that IL10 alone and IL10+BDNF cause a similar increase in axon growth across the injury site. 3D imaging using Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue Hydrogel (CLARITY) shows these axons do completely traverse the injury site, while electrophysiological studies suggest these axons are able to carry action potentials. These results are the first to show that regenerated axons can be electrophysiologically active. Taken together, these studies suggest early immunomodulation can have long-lasting benefits through tissue sparing, and that regenerated axons have the potential to transduce signals across an injury site. These findings provide novel insights into how the pathophysiology following an SCI can be altered using biomaterials and gene therapy. Future studies will involve identifying the synaptic targets of regenerated axons and determining how the formation of new circuits can influence motor function.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163028/1/chenjess_1.pd

Dynamics of Synaptic Transmission in the CNS: Contribution of Neuron-glia Interactions

The last 30 years have seen a growing appreciation of the importance for CNS functioning of the internal state of neural tissues, which is exquisitely reflective of the immediate-to-long-term history of the preceding neural activity experienced by those tissues. This dissertation comprises three research projects that together address different, but related aspects of dynamics of the state of neural tissues, with a focus on the roles played by astroglia and GABAergic synaptic transmission. The first two projects study the relationship between stimulus-evoked glial and neuronal activities within local networks of the dorsal horn of the spinal cord and sensitivity of GABAergic actions to the state of local glia and prior sensory stimulation, whereas the third project investigates the sensitivity of GABAergic actions to prior sensory stimulation in the neocortex. Project 1: Origins of Optical Intrinsic Signal and its significance. In rat spinal cord slice, repetitive electrical stimulation of the dorsal root at an intensity that activates C-fibers evokes a slow-to-develop and prolonged (30-50 s) change in light transmittance (OISDR) in the superficial part of the ipsilateral dorsal horn (DHs). Inhibition of astrocyte metabolism by bath-applied fluoroacetate and glutamine (FAc+Gln), or interference with glial and neuronal K+ transport by 4-aminopyridine (4-AP) lead to dissociation of the OISDR and the postsynaptic DHs response to a single-pulse dorsal root stimulus (P-PSPDR). The OISDR decreases under FAc+Gln, whereas the P-PSPDR remains unaltered; under 4-AP, the P-PSPDR increases, but the OISDR decreases. In contrast, both the OISDR and P-PSPDR increase when K+o is elevated. These observations indicate that the OISDR mainly reflects cell volume and light scattering changes associated with DHs astrocyte uptake of K+ and glutamate (GLU). In slices from subjects that received an intracutaneous injection of formalin 3-5 days earlier, both the OISDR and the response of the DHs to local application of K+ or GLU are profoundly reduced, and the normally exquisite sensitivity of the DHs to elevated K+o is decreased. Considered collectively, the observations raise the possibility that impaired regulation of DHs K+o and GLUo may contribute to initiation and maintenance of the CNS pain circuit and sensorimotor abnormalities that develop following intracutaneous formalin injection. Project 2: Effects of alteration of glia on neuronal plasticity. Transient (20min) exposure of the spinal cord slice to fluorocitrate (FC; a reversible inhibitor of glial energy production via the TCA cycle) is shown to be accompanied by a protracted decrease of the superficial dorsal horn (DHs) optical response to repetitive electrical stimulation of the ipsilateral dorsal root, and by a similarly protracted increase in the postsynaptic response of the DHs to single-pulse stimulation of the attached dorsal root (LTPFC). It also is shown that LTPFC does not occur in the presence of d-aminophosphopentanoic acid (APV), becomes progressively smaller as [K+]o in the perfusion solution is decreased from 3.0 mM (normal) to 0.0 mM, and is reduced or eliminated by bath application of 1 mM bicuculline. Somal whole-cell patch recordings were carried out to evaluate the effects of FC on the response of DHs neurons to puffer-applied GABA. The observations reveal that transient exposure of the slice to FC is reliably accompanied by a prolonged (>1 hr) depolarizing shift of the equilibrium potential for the DHs neuron transmembrane ionic currents evoked by GABA (average EGABApreFC: -75 mV ; EGABApostFC: -50 mV). Considered collectively, the findings demonstrate that LTPFC involves (1) elevation of [K+]o in the DHs, (2) NMDA receptor activation, and (3) conversion of the effect of GABA on DHs neurons from inhibition to excitation. It is proposed that a transient impairment of astrocyte energy production via the TCA cycle can trigger the cascade of dorsal horn mechanisms that underlies hyperalgesia and persistent pain. Project 3: Contribution of GABA to cerebral cortical dynamics. Imaging of the optical intrinsic signal (OIS), evoked in the rat sensorimotor cortical slice by 1s-long 20Hz electrical stimulation applied to locus at the layer VI/white matter junction, was used to delineate a column-shaped cortical region responding to a local thalamocortical input drive, and whole-cell patch clamp recordings were obtained from layer II-III pyramidal neurons residing in that region. Puffs of pressure-ejected GABA were released from a micropipette in a close vicinity of the recorded neuron's soma before and also immediately after conditioning electrical stimulation. Prior to conditioning stimulation, GABA puffs hyperpolarized the recorded neurons, whereas for ~15s subsequent to conditioning stimulation GABA puffs depolarized the same neurons. Two-photon Cl- imaging in cortical slices taken from CLM1 Clomeleon mice revealed that conditioning stimulation transiently elevates [Cl-]i in the stimulated cortical column; this increase is blocked by SR95531 (gabazine), a selective GABAA receptor antagonist. Next, two-photon Ca2+ imaging revealed that isoguvacine (GABAA receptor agonist) increases Ca2+ influx into neurons in the stimulated cortical column. Finally, OIS imaging in the presence of GABA antagonist bicuculline suggests that the depolarizing action of GABA is confined to the center of the stimulated cortical region, while at its margins GABA remains hyperpolarizing. Taken together, these findings suggest that synaptically released GABA can be either inhibitory or excitatory, depending on the activity state of the local network. Such activity dependence of GABA action can be expected to funnel stimulus-evoked activity in a cortical area into the central, most strongly driven cortical columns

Speckle variance optical coherence tomography of the rodent spinal cord: in vivo feasibility

Optical coherence tomography (OCT) has the combined advantage of high temporal (µsec) and spatial (<10µm) resolution. These features make it an attractive tool to study the dynamic relationship between neural activity and the surrounding blood vessels in the spinal cord, a topic that is poorly understood. Here we present work that aims to optimize an in vivo OCT imaging model of the rodent spinal cord. In this study we image the microvascular networks of both rats and mice using speckle variance OCT. This is the first report of depth resolved imaging of the in vivo spinal cord using an entirely endogenous contrast mechanism

Enhanced pre-synaptic glutamate release in deep-dorsal horn contributes to calcium channel alpha-2-delta-1 protein-mediated spinal sensitization and behavioral hypersensitivity

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavα2δ1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavα2δ1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavα2δ1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavα2δ1 transgenic mice. Our data indicated that overexpression of Cavα2δ1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavα2δ1-mediated spinal sensitization in which elevated Cavα2δ1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavα2δ1 expression