Discontinuity third harmonic generation microscopy for label-free imaging and quantification of intraepidermal nerve fibers.

Label-free imaging methodologies for nerve fibers rely on spatial signal continuity to identify fibers and fail to image free intraepidermal nerve endings (FINEs). Here, we present an imaging methodology-called discontinuity third harmonic generation (THG) microscopy (dTHGM)-that detects three-dimensional discontinuities in THG signals as the contrast. We describe the mechanism and design of dTHGM and apply it to reveal the bead-string characteristics of unmyelinated FINEs. We confirmed the label-free capability of dTHGM through a comparison study with the PGP9.5 immunohistochemical staining slides and a longitudinal spared nerve injury study. An intraepidermal nerve fiber (IENF) index based on a discontinuous-dot-connecting algorithm was developed to facilitate clinical applications of dTHGM. A preliminary clinical study confirmed that the IENF index was highly correlated with skin-biopsy-based IENF density (Pearson's correlation coefficient R = 0.98) and could achieve differential identification of small-fiber neuropathy (p = 0.0102) in patients with diabetic peripheral neuropathy.

A Dual-Mode Multifunctional Pulsed Radio-Frequency Stimulator for Trigeminal Neuralgia Relief and its Animal Model.

This work proposed a programmable pulsed radio-frequency (PRF) stimulator for trigeminal neuralgia (TN) relief on demand. The implantable stimulator is a miniaturized micro-system which integrates a wireless interface circuit, a sensor interface circuit, a PRF pattern generation circuit and a logic controller. The multifunctional stimulator capable of delivering current/voltage stimulation provides the choice of the biphasic sinusoidal, square and patterned waveform for PRF treatment researches. The external handheld device can wirelessly transmit the parameters of frequency, amplitude, pulse duration and repetition rate of the pulse train to the implanted stimulator. While stimulating, the temperature sensor can monitor the operating temperature. The feedback signal is transmitted in medical implanted communication system (MICS). The micro-system is fabricated in a 0.35 µm CMOS process with a chip size of 3.1 × 2.7 mm2. The fabricated chip was mounted on a 2.6 × 2.1 cm2 test board for studying the in vivo efficacy of pain relief by PRF. Animal studies of PRF stimulation and commonly-used medication for trigeminal neuralgia are also demonstrated and the presented results prove that PRF stimulation has greater effectiveness on trigeminal neuralgia relief comparing to the medication. The effectiveness period lasts at least 14 days. The results of neural recording show that the PRF stimulation of trigeminal ganglion (TG) attenuated neuron activities without being severely damaged. Pathology also revealed no lesion found on the stimulated area.

Nociceptive transient receptor potential canonical 7 (TRPC7) mediates aging-associated tumorigenesis induced by ultraviolet B.

Aging, cancer, and longevity have been linked to intracellular Ca2+ signaling and nociceptive transient receptor potential (TRP) channels. We found that TRP canonical 7 (TRPC7) is a nociceptive mechanoreceptor and that TRPC7 channels specifically mediate the initiation of ultraviolet B (UVB)-induced skin aging and tumor development due to p53 gene family mutations. Within 30 min after UVB irradiation, TRPC7 mediated UVB-induced Ca2+ influx and the subsequent production of reactive oxygen species in skin cells. Notably, this function was unique to TRPC7 and was not observed for other TRP channels. In TRPC7 knockout mice, we did not observe the significant UVB-associated pathology seen in wild-type mice, including epidermal thickening, abnormal keratinocyte differentiation, and DNA damage response activation. TRPC7 knockout mice also had significantly fewer UVB-induced cancerous tumors than did wild-type mice, and UVB-induced p53 gene family mutations were prevented in TRPC7 knockout mice. These results indicate that TRPC7 activity is pivotal in the initiation of UVB-induced skin aging and tumorigenesis and that the reduction in TRPC7 activity suppresses the UVB-induced aging process and tumor development. Our findings support that TRPC7 is a potential tumor initiator gene and that it causes cell aging and genomic instability, followed by a change in the activity of proto-oncogenes and tumor suppressor genes to promote tumorigenesis.

Longitudinal FDG-PET scan study of brain changes in mice with cancer-induced bone pain and after morphine analgesia.

Morphine is the most commonly used drug for treating physical and psychological suffering caused by advanced cancer. Although morphine is known to elicit multiple supraspinal analgesic effects, its behavioral correlates with respect to the whole-brain metabolic activity during cancer-induced bone pain have not been elucidated. We injected 4T1 mouse breast cancer cells into the left femur bone marrow cavity of BALB/c mice. All mice developed limb use deficits, mechanical allodynia, and hypersensitivity to cold, which were effectively suppressed with morphine. Serial 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) was performed for each mouse before cancer induction (0 day), after cancer-induced bone pain was established (14 days), and during effective morphine treatment (16 days). The longitudinal FDG-PET imaging analysis demonstrated that cancer-induced bone pain increased glucose uptake in the insular cortex and hypothalamus and decreased the activity of the retrosplenial cortex. Morphine reversed the activation of the insular cortex and hypothalamus. Furthermore, morphine activated the amygdala and rostral ventromedial medulla and suppressed the activity of anterior cingulate cortex. Our findings of hypothalamic and insular cortical activation support the hypothesis that cancer-induced bone pain has strong inflammatory and affective components in freely moving animals. Morphine may provide descending inhibitory and facilitatory actions in the treatment of cancer-induced bone pain in a clinical setting.

Differential Expression of Phosphorylated ERK and c-Fos of Limbic Cortices Activities in Response to Tactile Allodynia of Neuropathic Rats.

Neuropathic pain is due to lesion or dysfunction of the somatosensory system. Treating patients with neuropathic pain is difficult because the underlying mechanisms are understood limitedly, especially at the supraspinal level. In this study, we used two kinds of molecular markers to investigate the neuronal activity changes in the anterior cingulate cortex, insular cortex (IC), and medial prefrontal cortex (mPFC) of the neuropathic rats under tactile allodynia. We used spared nerve injury of the sciatic nerve (SNI) as the neuropathic pain model. Two weeks after SNI surgery, we applied repetitive allodynic stimulation to the conscious rats. After stimulation, the rats were sacrificed, and the immunohistochemistry of phosphorylated extracellular signal-regulated kinase (pERK) and c-Fos was performed. Quantification of immunoreactive cells was carried out by stereological method. For pERK study, the expression of pERK was significantly increased in the mPFC and IC of the SNI rats. For c-Fos study, only mPFC had elevated expression of c-Fos in the SNI rats. The analgesic, gabapentin, reversed the mechanical hyper-sensitivity and the augmented expression of limbic pERK and c-Fos in the SNI rats. Immunofluorescent staining revealed the expression of pERK or c-Fos was restricted to neurons, not glia cells. Our results demonstrated that tactile allodynia represented differential expression of pERK and c-Fos in the limbic cortices of the neuropathic rats.

Functional assessment of glioma pathogenesis by in vivo multi-parametric magnetic resonance imaging and in vitro analyses.

Gliomas are aggressive brain tumors with poor prognosis. In this study, we report a novel approach combining both in vivo multi-parametric MRI and in vitro cell culture assessments to evaluate the pathogenic development of gliomas. Osteopontin (OPN), a pleiotropic factor, has been implicated in the formation and progression of various human cancers, including gliomas, through its functions in regulating cell proliferation, survival, angiogenesis, and migration. Using rat C6 glioma model, the combined approach successfully monitors the acquisition and decrease of cancer hallmarks. We show that knockdown of the expression of OPN reduces C6 cell proliferation, survival, viability and clonogenicity in vitro, and reduces tumor burden and prolongs animal survival in syngeneic rats. OPN depletion is associated with reduced tumor growth, decreased angiogenesis, and an increase of tumor-associated metabolites, as revealed by T2-weighted images, diffusion-weighted images, K(trans) maps, and 1H-MRS, respectively. These strategies allow us to define an important role of OPN in conferring cancer hallmarks, which can be further applied to assess the functional roles of other candidate genes in glioma. In particular, the non-invasive multi-parametric MRI measurement of cancer hallmarks related to proliferation, angiogenesis and altered metabolism may serve as a useful tool for diagnosis and for patient management.

Nocifensive behavior-related laser heat-evoked component in the rostral agranular insular cortex revealed using morphine analgesia.

The rostral agranular insular cortex (RAIC), an opioid-responsive site, is essential for modulating nociception in rats. Our previous studies have shown that morphine suppressed long latency laser heat-evoked nociceptive responses in the primary somatosensory cortex (SmI). By contrast, morphine significantly attenuated both short and long latency responses in the anterior cingulate cortex (ACC). The present study assessed the effect of morphine on laser heat-evoked responses in the RAIC. Laser heat irradiation applied to the rat forepaws at graded levels was used as a specific noxious stimulus. In the RAIC, the first part of the long latency component (140-250ms) of the laser heat-evoked response was enhanced by intraperitoneal morphine (5mg/kg). When the laser heat-evoked cortical responses were examined for trials showing strong nocifensive movement (paw licking), moderate nocifensive movement (paw lifting), and no nocifensive movement, a 140-250ms period enhancement was observed in the RAIC only for the paw lifting movement. This enhancement was absent in the SmI. Thus, our data suggest that the RAIC has a pain-related behavior-dependent neuronal component. Furthermore, the RAIC, ACC, and SmI are differentially modulated by morphine analgesia.

Promoted Growth of Brain Tumor by the Transplantation of Neural Stem/Progenitor Cells Facilitated by CXCL12.

The targeted migration of neural stem/progenitor cells (NSPCs) is a prerequisite for the use of stem cell therapy in the treatment of pathologies. This migration is regulated mainly by C-X-C motif chemokine 12 (CXCL12). Therefore, promotion of the migratory responses of grafted cells by upregulating CXCL12 signaling has been proposed as a strategy for improving the efficacy of such cell therapies. However, the effects of this strategy on brain tumors have not yet been examined in vivo. The aim of the present study was thus to elucidate the effects of grafted rat green fluorescent protein (GFP)-labeled NSPCs (GFP-NSPCs) with CXCL12 enhancement on a model of spontaneous rat brain tumor induced by N-ethyl-N-nitrosourea. T2-weighted magnetic resonance imaging was applied to determine the changes in tumor volume and morphology over time. Postmortem histology was performed to confirm the tumor pathology, expression levels of CXCL12 and C-X-C chemokine receptor type 4, and the fate of GFP-NSPCs. The results showed that the tumor volume and hypointense areas of T2-weighted images were both significantly increased in animals treated with combined NSPC transplantation and CXCL12 induction, but not in control animals or in those with tumors that received only one of the treatments. GFP-NSPCs appear to migrate toward tumors with CXCL12 enhancement and differentiate uniquely into a neuronal lineage. These findings suggest that CXCL12 is an effective chemoattractant that facilitates exogenous NSPC migration toward brain tumors and that CXCL12 and NSPC can act synergistically to promote tumor progression with severe hemorrhage.

Acid-induced hyperalgesia and anxio-depressive comorbidity in rats.

Fibromyalgia is a prevalent disorder characterized by chronic widespread pain (CWP) and complex comorbid symptoms. A CWP model is developed through repeated unilateral intramuscular injections of acid saline resulting in bilateral mechanical hyperalgesia in rats. The present study aims to evaluate whether both anxious and depressive comorbidities exist in this acid-induced pain model, similarly to patients with CWP syndromes. The anxiety-like behaviors were evaluated using the open field and elevated plus maze tests, and depression-like behaviors were measured by the forced swimming, sucrose consumption, and sucrose preference tests. The pain group receiving acidic saline displayed significantly lower paw withdrawal thresholds for 4weeks than animals in the vehicle group after repetitive intramuscular injections. The pain group showed a significantly shorter duration of exploring the central zone of the open field and the open arms of the elevated plus maze compared to the vehicle group. The pain group had a significantly lower preference for and consumption of the hedonic sucrose. Moreover, rats with chronic pain showed significantly longer immobility than the vehicle group in the forced swimming test. The results indicate that psychiatric behaviors are exacerbated in the CWP model. This study provides evidence for the validity of the acid-induced pain model analogous to patients with CWP syndromes.

Comparison of fMRI BOLD response patterns by electrical stimulation of the ventroposterior complex and medial thalamus of the rat.

The objective of this study was to compare the functional connectivity of the lateral and medial thalamocortical pain pathways by investigating the blood oxygen level-dependent (BOLD) activation patterns in the forebrain elicited by direct electrical stimulation of the ventroposterior (VP) and medial (MT) thalamus. An MRI-compatible stimulation electrode was implanted in the VP or MT of α-chloralose-anesthetized rats. Electrical stimulation was applied to the VP or MT at various intensities (50 µA to 300 µA) and frequencies (1 Hz to 12 Hz). BOLD responses were analyzed in the ipsilateral forelimb region of the primary somatosensory cortex (iS1FL) after VP stimulation and in the ipsilateral cingulate cortex (iCC) after MT stimulation. When stimulating the VP, the strongest activation occurred at 3 Hz. The stimulation intensity threshold was 50 µA and the response rapidly peaked at 100 µA. When stimulating the MT, The optimal frequency for stimulation was 9 Hz or 12 Hz, the stimulation intensity threshold was 100 µA and we observed a graded increase in the BOLD response following the application of higher intensity stimuli. We also evaluated c-Fos expression following the application of a 200-µA stimulus. Ventroposterior thalamic stimulation elicited c-Fos-positivity in few cells in the iS1FL and caudate putamen (iCPu). Medial thalamic stimulation, however, produced numerous c-Fos-positive cells in the iCC and iCPu. The differential BOLD responses and c-Fos expressions elicited by VP and MT stimulation indicate differences in stimulus-response properties of the medial and lateral thalamic pain pathways.

New nerve regeneration strategy combining laminin-coated chitosan conduits and stem cell therapy.

Nerve regeneration remains a difficult challenge due to the lack of safe and efficient matrix support. We designed a laminin (LN)-modified chitosan multi-walled nerve conduit combined with bone marrow stem cell (BMSC) grating to bridge a 10 mm long gap in the sciatic nerve of Sprague-Dawley rats. The repair outcome was monitored during 16 weeks after surgery. Successful grafting of LN onto the chitosan film, confirmed by immunolocalization, significantly improved cell adhesion. In vivo study showed that newly formed nerve cells covered the interior of the conduit to connect the nerve gap successfully in all groups. The rats implanted with the conduit combined with BMSCs showed the best results, in terms of nerve regrowth, muscle mass of gastrocnemius, function recovery and tract tracing. Neuroanatomical horseradish peroxidase tracer analysis of motor neurons in the lumbar spinal cord indicated that the amount and signal intensity were significantly improved. Furthermore, BMSCs suppressed neuronal cell death and promoted regeneration by suppressing the inflammatory and fibrotic response induced by chitosan after long-term implantation. In summary, this study suggests that LN-modified chitosan multi-walled nerve conduit combined with BMSCs is an efficient and safe conduit matrix for nerve regeneration.

Neonatal whisker clipping alters behavior, neuronal structure and neural activity in adult rats.

Early experience plays critical roles during the development of sensory systems. For example, neonatal surgical manipulations of the whiskers in rodents lead to altered neural activity and behaviors later in life. However, while surgical procedures damage the sensory pathway; it is hard to examine the impact of whisker deprivation on adult animals. To address this issue, we performed a neonatal whisker clipping (WC0-3) paradigm, a non-invasive procedure, from the day of birth (P0) to postnatal day (P) 3, and examined behavioral performances in their adult age. With fully regrown whiskers, the WC0-3 rats exhibited shorter crossable distance than controls in a gap-crossing task, suggesting a defect in their whisker-specific tactile function. In their somatosensory cortex, the layer IV spiny stellate neurons had reduced dendritic complexity and spine density. After exploration in a novel environment, the expression of an activity-dependent immediate early gene, c-fos, increased dramatically in the somatosensory cortex. However, in WC0-3 rats, the number of c-Fos positive cells was less than those in control rats, indicating a fault in transducing sensory-related neural activity between cortical layers in WC0-3 rats. Together, our results demonstrate the roles of early tactile experience on the establishment of layer-specific excitatory connection in the barrel cortex. Early sensory insufficiency would leave long-lasting functional deficits in the sensory system.

Ca(v)3.2 T-type Ca2+ channel-dependent activation of ERK in paraventricular thalamus modulates acid-induced chronic muscle pain.

Treatments for chronic musculoskeletal pain, such as lower back pain, fibromyalgia, and myofascial pain syndrome, remain inadequate because of our poor understanding of the mechanisms that underlie these conditions. Although T-type Ca2+ channels (T-channels) have been implicated in peripheral and central pain sensory pathways, their role in chronic musculoskeletal pain is still unclear. Here, we show that acid-induced chronic mechanical hyperalgesia develops in Ca(v)3.1-deficient and wild-type but not in Ca(v)3.2-deficient male and female mice. We also show that T-channels are required for the initiation, but not maintenance, of acid-induced chronic muscle pain. Blocking T-channels using ethosuximide prevented chronic mechanical hyperalgesia in wild-type mice when administered intraperitoneally or intracerebroventricularly, but not intramuscularly or intrathecally. Furthermore, we found an acid-induced, Ca(v)3.2 T-channel-dependent activation of ERK (extracellular signal-regulated kinase) in the anterior nucleus of paraventricular thalamus (PVA), and prevention of the ERK activation abolished the chronic mechanical hyperalgesia. Our findings suggest that Ca(v)3.2 T-channel-dependent activation of ERK in PVA is required for the development of acid-induced chronic mechanical hyperalgesia.

Tumor necrosis factor-alpha-elicited stimulation of gamma-secretase is mediated by c-Jun N-terminal kinase-dependent phosphorylation of presenilin and nicastrin.

Gamma-secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1, and Pen-2, and it catalyzes the final proteolytic step in the processing of amyloid precursor protein to generate amyloid-beta. Our previous results showed that tumor necrosis factor-alpha (TNF-alpha) can potently stimulate gamma-secretase activity through a c-Jun N-terminal kinase (JNK)-dependent pathway. Here, we demonstrate that TNF-alpha triggers JNK-dependent serine/threonine phosphorylation of PS1 and NCT to stimulate gamma-secretase activity. Blocking of JNK activity with a potent JNK inhibitor (SP600125) reduces TNF-alpha-triggered phosphorylation of PS1 and NCT. Consistent with this, we show that activated JNKs can be copurified with gamma-secretase complexes and that active recombinant JNK2 can promote the phosphorylation of PS1 and NCT in vitro. Using site-directed mutagenesis and a synthetic peptide, we clearly show that the Ser(319)Thr(320) motif in PS1 is an important JNK phosphorylation site that is critical for the TNF-alpha-elicited regulation of gamma-secretase. This JNK phosphorylation of PS1 at Ser(319)Thr(320) enhances the stability of the PS1 C-terminal fragment that is necessary for gamma-secretase activity. Together, our findings strongly suggest that JNK is a critical intracellular mediator of TNF-alpha-elicited regulation of gamma-secretase and governs the pivotal step in the assembly of functional gamma-secretase.